WO2024030959A1 - Procédés de différenciation de cardiomyocytes - Google Patents

Procédés de différenciation de cardiomyocytes Download PDF

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Publication number
WO2024030959A1
WO2024030959A1 PCT/US2023/071516 US2023071516W WO2024030959A1 WO 2024030959 A1 WO2024030959 A1 WO 2024030959A1 US 2023071516 W US2023071516 W US 2023071516W WO 2024030959 A1 WO2024030959 A1 WO 2024030959A1
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cells
population
aggregate
day
incubation
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PCT/US2023/071516
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English (en)
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Soren Lukas JOHNSON
Steven James KATTMAN
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Sana Biotechnology, Inc.
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Publication of WO2024030959A1 publication Critical patent/WO2024030959A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0657—Cardiomyocytes; Heart cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/40—Regulators of development
    • C12N2501/415—Wnt; Frizzeled
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/72—Transferases (EC 2.)
    • C12N2501/727—Kinases (EC 2.7.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells

Definitions

  • the present disclosure relates in some aspects to methods for differentiating cardiomyocytes from pluripotent stem cells, e.g., induced pluripotent stem cells, that involves dissociation and re-aggregation of cell aggregates, as well as to populations of cardiomyocytes, compositions thereof, and methods of treatment and uses thereof, such as for treating a subject with a heart condition or disease.
  • pluripotent stem cells e.g., induced pluripotent stem cells
  • a method of differentiating cardiomyocytes from pluripotent stem cells comprising: a) performing a first incubation comprising culturing a population of pluripotent stem cells under conditions to form a first aggregate, wherein the first incubation is initiated on day 0; b) contacting the first aggregate with a dissociating agent to form a population of dissociated cells; c) performing a second incubation comprising culturing the population of dissociated cells under conditions to aggregate the dissociated cells into a second aggregate; and (d) performing a third incubation comprising culturing the second aggregate under conditions to differentiate the population of cells in the second aggregate into a population of cardiomyocytes.
  • the pluripotent stem cells are selected from the group consisting of induced pluripotent stem cells, embryonic stem cells, bone marrow-mesenchymal stem cells, cardiac tissue stem cells, and adipose tissue stem cells. In some of any of such embodiments, the pluripotent stem cells are induced pluripotent stem cells.
  • the population of pluripotent stem cells has a viable cell concentration of between or between about 1 x 10 5 and 3 x 10 7 cells/mL, 2 x 10 5 and 2 x 10 7 cells/mL, 3 x 10 5 and 1 x 10 7 cells/mL, 4 x 10 5 and 9 x 10 6 cells/mL, 5 x 10 5 and 8 x 10 6 cells/mL, 6 x 10 5 and 7 x 10 6 cells/mL, 7 x 10 5 and 6 x 10 6 cells/mL, 8 x 10 5 and 5 x 10 6 cells/mL, 9 x 10 5 and 4 x 10 6 cells/mL, 9 x 10 5 and 3 x 10 6 cells/mL, 1 x 10 6 and 2 x 10 6 cells/mL, 1.1 x 10 6 and 1.9 x 10 6 cells/mL, 1.2 x 10 6 and 1.8 x 10 6 cells/mL, or 1.25 x 10 6 and 1.75
  • the population of pluripotent stem cells has a viable cell concentration of or of about 1.0 x 10 6 cells/mL, 1.05 x 10 6 cells/mL, 1.1 x 10 6 cells/mL, 1.15 x 10 6 cells/mL, 1.2 x 10 6 cells/mL, 1.25 x 10 6 cells/mL, 1.3 x 10 6 cells/mL, 1.35 x 10 6 cells/mL, 1.4 x 10 6 cells/mL, 1.45 x 10 6 cells/mL, 1.5 x 10 6 cells/mL, 1.55 x 10 6 cells/mL, 1.6 x 10 6 cells/mL, 1.65 x 10 6 cells/mL, 1.7 x 10 6 cells/mL, 1.75 x 10 6 cells/mL, 1.8 x 10 6 cells/mL, 1.85 x 10 6 cells/mL, 1.9 x 10 6 cells/mL, 1.95 x 10 6 cells/mL, 2.0 x 10 7 cells/m
  • the population of pluripotent stem cells has a viable cell concentration of or of about 1.4 x 10 6 cells/mL, 1.45 x 10 6 cells/mL, 1.5 x 10 6 cells/mL, 1.55 x 10 6 cells/mL, 1.6 x 10 6 cells/mL, 1.65 x 10 6 cells/mL, 1.7 x 10 6 cells/mL, 1.75 x 10 6 cells/mL, or 1.8 x 10 6 cells/mL.
  • the first incubation comprises culturing the population of pluripotent stem cells in a media comprising an inhibitor of glycogen synthase kinase 3 (GSK3)/activator of Wnt/p-catenin signaling.
  • the inhibitor of GSK3 /activator of Wnt/p-catenin signaling is an inhibitor of GSK-3a and/or GSK-3p.
  • the inhibitor of GSK-3a and/or GSK-3P is an inhibitor of GSK-3a and GSK-3p.
  • the inhibitor of GSK-3a and/or GSK-3P is CHIR99021.
  • the concentration of CHIR99021 is between about 5 pM and about 7 pM. In some of any of such embodiments, the concentration of CHIR99021 is about 5, 5.5, 6, 6.5, or 7 pM. In some of any of such embodiments, the concentration of CHIR99021 is between about 5
  • the first incubation comprises culturing the population of pluripotent stem cells in the media comprising the inhibitor of GSK-3a and/or GSK-3P on day 0. In some of any of such embodiments, the first incubation comprises culturing the population of pluripotent stem cells in the media comprising the inhibitor of GSK-3a and/or GSK-3P on day 0.
  • the first incubation comprises culturing the population of pluripotent stem cells in a media comprising an inhibitor of Wnt/p- catenin signaling.
  • the inhibitor of Wnt/p-catenin signaling is selected from the group consisting of WIKI4, NSC668036, iCRT3, iCRT5, iCRT14, IWP-2, XAV-939, ICG-001, LGK-974, OMP-18R5, FJ9, IWR-l-endo, KY02111, PFK115-584, Wnt-059, DKK1, FH-535, Box5, and Peptide Pen-N3.
  • the inhibitor of Wnt/p-catenin signaling is WIKI4.
  • the first incubation comprises culturing the population of pluripotent stem cells in the media comprising the inhibitor of Wnt/p-catenin signaling on or about day 2.
  • the culturing the population of pluripotent stem cells in the media comprising the inhibitor of Wnt/p-catenin signaling begins approximately 36 to 48 hours after the initiation of the first incubation.
  • the culturing the population of pluripotent stem cells in the media comprising the inhibitor of Wnt/p-catenin signaling begins approximately 40 hours after the initiation of the first incubation.
  • the first incubation occurs in suspension.
  • the contacting the first aggregate with the dissociating agent occurs when the first aggregate is between or between about 300 and about 700 pm in diameter. In some of any of such embodiments, the first aggregate is between or between about 350 and 650 pm, 400 and 650 pm, 450 and 650 pm, 450 and 600 pm, 500 and 650 pm, 550 and 650 pm, or 550 and 600 pm in diameter. In some of any of such embodiments, the first aggregate is between or between about 500 and about 650 pm in diameter. In some of any of such embodiments, the first aggregate is between or between about 550 and about 600 gm in diameter.
  • the contacting the first aggregate with the dissociating agent occurs at a time when the first aggregate is between or between about 300 and about 700 m in diameter. In some of any of such embodiments, the contacting the first aggregate with the dissociating agent occurs at a time when the first aggregate is between or between about 350 and 650 gm, 400 and 650 gm, 450 and 650 gm, 450 and 600 gm, 500 and 650 gm, 550 and 650 pm, or 550 and 600 pun in diameter.
  • the contacting the first aggregate with the dissociating agent occurs at a time when the first aggregate is between or between about 500 and about 650 pun in diameter. In some embodiments, the contacting the first aggregate with a dissociating agent occurs at a time when the first aggregate is between or between about 550 and about 600 pun in diameter.
  • the contacting the first aggregate with the dissociating agent occurs at a time when the first aggregate is between or between about 300 and about 700 pun in diameter. In some of any of such embodiments, the contacting the first aggregate with the dissociating agent occurs at a time when the first aggregate is between or between about 350 and 650 pun, 400 and 650 pun, 450 and 650 pun, 450 and 600 pun, 500 and 650 pun, 550 and 650 pun, or 550 and 600 pun in diameter. In some of any of such embodiments, the contacting the first aggregate with the dissociating agent occurs at a time when the first aggregate is between or between about 500 and about 650 pun in diameter. In some of any of such embodiments, the contacting the first aggregate with a dissociating agent occurs at a time when the first aggregate is between or between about 550 and about 600 pun in diameter.
  • the time is on or about any one of days 2 to 6. In some embodiments, the time is on or about day 2 or day 3. In some of any of such embodiments, the time is on or about any one of days 3 to 6. In some of any of such embodiments, the time is on or about any one of days 4 to 6. In some of any of such embodiments, the time is on or about day 4. In some of any of such embodiments, the contacting occurs on or about any one of days 2 to 6. In some of any of such embodiments, the contacting occurs on or about any one of days 3 to 6. In some of any of such embodiments, the contacting occurs on or about any one of days 4 to 6. In some of any of such embodiments, the contacting occurs on or about day 4.
  • the dissociating agent is or comprises a cleavage enzyme.
  • the cleavage enzyme is a protease.
  • the protease is a recombinant enzyme that cleaves a peptide bond on the C- terminal side of a lysine or arginine residue.
  • the protease is an endopeptidase.
  • the endopeptidase is trypsin.
  • the contacting occurs for a duration of about 15 minutes to about 2 hours. In some of any of such embodiments, the contacting occurs for a duration of about 30 minutes to about 90 minutes, about 30 minutes to about 75 minutes, about 40 minutes to about 60 minutes, about 40 minutes to about 55 minutes, about 40 minutes to about 50 minutes, about 45 minutes to 55 minutes, or about 45 minutes to about 50 minutes. In some of any of such embodiments, the contacting occurs for a duration of about 40 to about 55 minutes.
  • the contacting occurs for a duration that is sufficient to result in a population of dissociated cells.
  • the method further comprises, during and/or after the contacting, triturating the population of dissociated cells.
  • the population of dissociated cells has a viable cell density of between or between about 1 x 10 5 and 3 x 10 7 cells/mL, 2 x 10 5 and 2 x 10 7 cells/mL, 3 x 10 5 and 1 x 10 7 cells/mL, 4 x 10 5 and 9 x 10 6 cells/mL, 5 x 10 5 and 8 x 10 6 cells/mL, 6 x 10 5 and 7 x 10 6 cells/mL, 7 x 10 5 and 6 x 10 6 cells/mL, 8 x 10 5 and 5 x 10 6 cells/mL, 9 x 10 5 and 4 x 10 6 cells/mL, 9 x 10 5 and 3 x 10 6 cells/mL, 1 x 10 6 and 2 x 10 6 cells/mL, 1.1 x 10 6 and 1.9 x 10 6 cells/mL, 1.2 x 10 6 and 1.8 x 10 6 cells/mL, or 1.25 x 10 6 and 1.75 x
  • the contacting occurs in suspension.
  • the second incubation comprises culturing the population of dissociated cells on one or more of any of about days 2-7. In some of any of such embodiments, the second incubation comprises culturing the population of dissociated cells on one day or two consecutive days from among days 2-7. In some of any of such embodiments: the second incubation comprises culturing the population of dissociated cells on or about day 2, 3, 4, 5, 6, or 7; and/or the second incubation comprises culturing the population of dissociated cells on or about days 2 and 3, on or about days 3 and 4, on or about days 4 and 5, on or about days 5 and 6, or on or about days 6 and 7. In some of any of such embodiments, the second incubation comprises culturing the population of dissociated cells on or about days 4 and 5. [0024] In some of any of such embodiments, the second incubation occurs in suspension.
  • the second aggregate that is formed is between or between about 25 and 200 pm in diameter.
  • the second aggregate that is formed is between or between about 25 and 200, 25 and 175, 25 and 150, 25 and 125, 30 and 200, 30 and 175, 30 and 150, 30 and 125, 35 and 200, 35 and 175, 35 and 150, 35 and 125, 40 and 200, 40 and 175, 40 and 150, 40 and 125, 45 and 200, 45 and 175, 45 and 150, 45 and 125, 50 and 200, 50 and 175, 50 and 150, 50 and 125, 55 and 200, 55 and 175, 55 and 150, 55 and 125, 60 and 200, 60 and 175, 60 and 150, 60 and 125, 65 and 200, 65 and 175, 65 and 150, 65 and 125, 70 and 200, 70 and 175, 70 and 150, 70 and 125, 75 and 200, 75 and 175, 75 and 150, or 75 and 125 pm in diameter.
  • the second aggregate that is formed on the second day of the second incubation and/or at or about 24 hours after contacting: (a) has a diameter that is less than 50% of the diameter of the first aggregate immediately prior to being contacted with the dissociating agent; and/or (b) has a diameter that is between or between about 5-50% of the diameter of the first aggregate immediately prior to being contacted with the dissociating agent; and/or (c) has a diameter that is at or about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the diameter of the first aggregate immediately prior to being contacted with the dissociating agent; and/or (d) has a diameter that is between or between about 5-25% of the diameter of the first aggregate immediately prior to being contacted with the dissociating agent.
  • the second aggregate on the second day of the second incubation and/or at or about 24 hours after the contacting, has a diameter that is between or between about 5-25% of the diameter of the first aggregate immediately prior to being contacted with the dissociating agent.
  • the second aggregate has a diameter that is between or between about 25 and 200, 25 and 175, 25 and 150, 25 and 125, 30 and 200, 30 and 175, 30 and 150, 30 and 125, 35 and 200, 35 and 175, 35 and 150, 35 and 125, 40 and 200, 40 and 175, 40 and 150, 40 and 125, 45 and 200, 45 and 175, 45 and 150, 45 and 125, 50 and 200, 50 and 175, 50 and 150, 50 and 125, 55 and 200, 55 and 175, 55 and 150, 55 and 125, 60 and 200, 60 and 175, 60 and 150, 60 and 125, 65 and 200, 65 and 175, 65 and 150, 65 and 125, 70 and 200, 70 and 175, 70 and 150, 70 and 125, 75 and 200, 75 and 175, 75 and 150, or 75 and 125
  • the day following the day of the contacting with the dissociating agent is day 3, 4, 5, 6, or 7. In some of any of such embodiments, the day following the day of the contacting with the dissociating agent is day 5.
  • the second aggregate on the second day of the second incubation, has a diameter that is between or between about 25 and 200, 25 and 175, 25 and 150, 25 and 125, 30 and 200, 30 and 175, 30 and 150, 30 and 125, 35 and 200, 35 and 175, 35 and 150, 35 and 125, 40 and 200, 40 and 175, 40 and 150, 40 and 125, 45 and 200, 45 and 175, 45 and 150, 45 and 125, 50 and 200, 50 and 175, 50 and 150, 50 and 125, 55 and 200, 55 and 175, 55 and 150, 55 and 125, 60 and 200, 60 and 175, 60 and 150, 60 and 125, 65 and 200, 65 and 175, 65 and 150, 65 and 125, 70 and 200, 70 and 175, 70 and 150, 70 and 125, 75 and 200, 75 and 175, 75 and 150, or 75 and 125 m.
  • the second day of the second incubation is day 3, 4, 5, 6, or 7. In some of the second day of the second incubation is day 3, 4, 5, 6, or 7. In
  • the second aggregate has a diameter that is between or between about 25 and 300, 25 and 275, 25 and 250, 40 and 300, 40 and 275, 40 and 250, 50 and 300, 50 and 275, 50 and 250, 60 and 300, 60 and 275, 60 and 250, 70 and 300, 70 and 275, 70 and 250, 80 and 300, 80 and 275, or 80 and 250 pm.
  • the second day following the day of the contacting with the dissociating agent and/or the second day following the initiation of the second incubation and/or on the first day of the third incubation is or is about day 4, 5, 6, 7, or 8. In some embodiments, the second day following the day of the contacting with the dissociating agent and/or the second day following the initiation of the second incubation and/or on the first day of the third incubation is or is about day 6.
  • the second aggregate has a diameter that is between or between about 25 and 200, 25 and 175, 25 and 150, 25 and 125, 30 and 200, 30 and 175, 30 and 150, 30 and 125, 35 and 200, 35 and 175, 35 and 150, 35 and 125, 40 and 200, 40 and 175, 40 and 150, 40 and 125, 45 and 200, 45 and 175, 45 and 150, 45 and 125, 50 and 200, 50 and 175, 50 and 150, 50 and 125, 55 and 200, 55 and 175, 55 and 150, 55 and 125, 60 and 200, 60 and 175, 60 and 150, 60 and 125, 65 and 200, 65 and 175, 65 and 150, 65 and 125, 70 and 200, 70 and 175, 70 and 150, 70 and 125, 75 and 200, 75 and 175, 75 and 150, or 75 and 125
  • the second aggregate has a diameter that is between or between about 25 and 200, 25 and 175, 25 and 150, 25 and 125, 30 and 200, 30 and 175, 30 and 150, 30 and 125, 35 and 200, 35 and 175, 35 and 150, 35 and 125, 40 and 200, 40 and 175, 40 and 150, 40 and 125, 45 and 200, 45 and 175, 45 and 150, 45 and 125, 50 and 200, 50 and 175, 50 and 150, 50 and 125, 55 and 200, 55 and 175, 55 and 150, 55 and 125, 60 and 200, 60 and 175, 60 and 150, 60 and 125, 65 and 200, 65 and 175, 65 and 150, 65 and 125, 70 and 200, 70 and 175, 70 and 150, 70 and 125, 75 and 200, 75 and 175, 75 and 150, or 75 and 125 m, on day 5.
  • the second aggregate has a diameter that is between or between about 25 and 300, 25 and 275, 25 and 250, 40 and 300, 40 and 275, 40 and 250, 50 and 300, 50 and 275, 50 and 250, 60 and 300, 60 and 275, 60 and 250, 70 and 300, 70 and 275, 70 and 250, 80 and 300, 80 and 275, or 80 and 250 pm on the second day following the initiation of the second incubation and/or on the first day of the third incubation.
  • the second aggregate has a diameter that is between or between about 25 and 300, 25 and 275, 25 and 250, 40 and 300, 40 and 275, 40 and 250, 50 and 300, 50 and 275, 50 and 250, 60 and 300, 60 and 275, 60 and 250, 70 and 300, 70 and 275, 70 and 250, 80 and 300, 80 and 275, or 80 and 250 pun on day 6.
  • the second aggregate has a diameter that is between or between about 25 and 400, 25 and 350, 25 and 300, 25 and 275, 25 and 250, 40 and 400, 40 and 350, 40 and 300, 40 and 275, 40 and 250, 50 and 400, 60 and 350, 50 and 300, 50 and 275, 50 and 250, 60 and 400, 60 and 350, 60 and 300, 60 and 275, 60 and 250, 70 and 400, 70 and 350, 70 and 300, 70 and 275, 70 and 250, 80 and 400, 80 and 350, 80 and 300, 80 and 275, or 80 and 250 pun on the third day following the initiation of the second incubation and/or on the second day of the third incubation.
  • the second aggregate has a diameter that is between or between about 25 and 400, 25 and 350, 25 and 300, 25 and 275, 25 and 250, 40 and 400, 40 and 350, 40 and 300, 40 and 275, 40 and 250, 50 and 400, 60 and 350, 50 and 300, 50 and 275, 50 and 250, 60 and 400, 60 and 350, 60 and 300, 60 and 275, 60 and 250, 70 and 400, 70 and 350, 70 and 300, 70 and 275, 70 and 250, 80 and 400, 80 and 350, 80 and 300, 80 and 275, or 80 and 250 pun on day 7.
  • the second aggregate has a diameter that is between or between about 25 and 400, 25 and 350, 25 and 300, 25 and 275, 25 and 250, 40 and 400, 40 and 350, 40 and 300, 40 and 275, 40 and 250, 50 and 400, 60 and 350, 50 and 300, 50 and 275, 50 and 250, 60 and 400, 60 and 350, 60 and 300, 60 and 275, 60 and 250, 70 and 400, 70 and 350, 70 and 300, 70 and 275, 70 and 250, 80 and 400, 80 and 350, 80 and 300, 80 and 275, or 80 and 250 m on the fourth day following the initiation of the second incubation and/or on the third day of the third incubation.
  • the second aggregate has a diameter that is between or between about 25 and 400, 25 and 350, 25 and 300, 25 and 275, 25 and 250, 40 and 400, 40 and 350, 40 and 300, 40 and 275, 40 and 250, 50 and 400, 60 and 350, 50 and 300, 50 and 275, 50 and 250, 60 and 400, 60 and 350, 60 and 300, 60 and 275, 60 and 250, 70 and 400, 70 and 350, 70 and 300, 70 and 275, 70 and 250, 80 and 400, 80 and 350, 80 and 300, 80 and 275, or 80 and 250 pm on day 8.
  • the first aggregate comprises a frequency of CD56+/PDGFRa+ cells that is or is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95%.
  • the first aggregate comprises a frequency of CD56+/PDGFRa+ cells that is or is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95%.
  • the first aggregate comprises a frequency of CD56+/PDGFRa+ cells that is or is about 80%, 81%, 92%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
  • the contacting occurs on or about day 4.
  • the contacting occurs in suspension.
  • the third incubation comprises culturing the second aggregate in a media comprising glucose, and in a media comprising sodium lactate.
  • the culturing the second aggregate in the media comprising glucose comprises culturing the second aggregate in the media comprising glucose on one or more of any of about days 4-22.
  • the culturing the second aggregate in the media comprising glucose comprises culturing the second aggregate in the media comprising glucose on or about days 6-10 and 12-22; and the culturing the second aggregate in the media comprising sodium lactate comprises culturing the second aggregate in the media comprising sodium lactate on or about days 10-12; or (b) the culturing the second aggregate in the media comprising glucose comprises culturing the second aggregate in the media comprising glucose on or about days 4-8 and 12-22; and the culturing the second aggregate in the media comprising sodium lactate comprises culturing the second aggregate in the media comprising sodium lactate on or about days 8-12; or (c) the culturing the second aggregate in the media comprising glucose comprises culturing the second aggregate in the media comprising glucose on or about days 5-9 and 13-22; and the culturing the second aggregate in the media comprising sodium lactate comprises culturing the second aggregate in the media comprising sodium lactate on or about days
  • the media comprising sodium lactate lacks glucose.
  • culturing the second aggregate in the media comprising sodium lactate comprises culturing the second aggregate in the media comprising sodium lactate on one or more of any of about days 9-13.
  • culturing the second aggregate in the media comprising sodium lactate comprises culturing the second aggregate in the media comprising sodium lactate on one or more of days 10-12.
  • culturing the second aggregate in the media comprising sodium lactate comprises culturing the second aggregate in the media comprising sodium lactate on or about days 10-12.
  • the third incubation begins on or about any one of days 4 to 8 and continues until the population of cardiomyocytes are harvested. In some of any of such embodiments, the third incubation begins on or about day 4 or day 5. In some of any of such embodiments, the third incubation begins on or about any one of days 6 to 8 and continues until the population of cardiomyocytes are harvested. In some of any of such embodiments, the third incubation begins on or about day 6 or day 7. In some of any of such embodiments, the third incubation begins on or about day 6. [0051] In some of any of such embodiments, the third incubation occurs in suspension.
  • the method further comprises harvesting the population of cardiomyocytes.
  • the population of cardiomyocytes is harvested on or about day 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24. In some of any of such embodiments, the population of cardiomyocytes is harvested on or about day 20, 21, 22, 23, or 24. In some of any of such embodiments, the population of cardiomyocytes is harvested on or about day 22.
  • the method further comprises cry opreserving the harvested population of cardiomyocytes.
  • the population of cardiomyocytes comprises a frequency of NKX2.5+/cTNT+ cells that is or is at least 75%, 76%, 77%, 78%, 79% 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on one or more of any of days 8-12 and/or on the sixth day following the day of the contacting with the dissociating agent.
  • the population of cardiomyocytes comprises a frequency of NKX2.5+/cTNT+ cells that is or is at least 75%, 76%, 77%, 78%, 79% 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on one or more of any of days 9-11 and/or on the sixth day following the day of the contacting with the dissociating agent.
  • the population of cardiomyocytes comprises a frequency of NKX2.5+/cTNT+ cells that is or is at least 75%, 76%, 77%, 78%, 79% 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 10 and/or on the sixth day following the day of the contacting with the dissociating agent.
  • the frequency of NKX2.5+/cTNT+ cells is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on one or more of any of days 8-12 and/or on the sixth day following the day of the contacting with the dissociating agent.
  • the frequency of NKX2.5+/cTNT+ cells is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on one or more of any of days 9-11 and/or on the sixth day following the day of the contacting with the dissociating agent.
  • the frequency of NKX2.5+/cTNT+ cells is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 10 and/or on the sixth day following the day of the contacting with the dissociating agent. In some of any of such embodiments, the frequency of NKX2.5+/cTNT+ cells is or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on one or more of any of days 8-12 and/or on the sixth day following the day of the contacting with the dissociating agent.
  • the frequency of NKX2.5+/cTNT+ cells is or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on one or more of any of days 9-11 and/or on the sixth day following the day of the contacting with the dissociating agent. In some of any of such embodiments, the frequency of NKX2.5+/cTNT+ cells is or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 10 and/or on the sixth day following the day of the contacting with the dissociating agent.
  • the frequency of NKX2.5+/cTNT+ cells is or is at least 85% on or about day 10 and/or on the sixth day following the day of the contacting with the dissociating agent. In some of any of such embodiments, the frequency of NKX2.5+/cTNT+ cells is or is at least 86% on or about day 10 and/or on the sixth day following the day of the contacting with the dissociating agent. In some of any of such embodiments, the frequency of NKX2.5+/cTNT+ cells is or is at least 87% on or about day 10 and/or on the sixth day following the day of the contacting with the dissociating agent.
  • the frequency of NKX2.5+/cTNT+ cells is or is at least 88% on or about day 10 and/or on the sixth day following the day of the contacting with the dissociating agent. In some of any of such embodiments, the frequency of NKX2.5+/cTNT+ cells is or is at least 89% on or about day 10 and/or on the sixth day following the day of the contacting with the dissociating agent. In some of any of such embodiments, the frequency of NKX2.5+/cTNT+ cells is or is at least 90% on or about day 10 and/or on the sixth day following the day of the contacting with the dissociating agent.
  • the frequency of NKX2.5+/cTNT+ cells is or is at least 91% on or about day 10 and/or on the sixth day following the day of the contacting with the dissociating agent. In some of any of such embodiments, the frequency of NKX2.5+/cTNT+ cells is or is at least 92% on or about day 10 and/or on the sixth day following the day of the contacting with the dissociating agent. In some of any of such embodiments, the frequency of NKX2.5+/cTNT+ cells is or is at least 93% on or about day 10 and/or on the sixth day following the day of the contacting with the dissociating agent.
  • the frequency of NKX2.5+/cTNT+ cells is or is at least 94% on or about day 10 and/or on the sixth day following the day of the contacting with the dissociating agent. In some of any of such embodiments, the frequency of NKX2.5+/cTNT+ cells is or is at least 95% on or about day 10 and/or on the sixth day following the day of the contacting with the dissociating agent. In some of any of such embodiments, the frequency of NKX2.5+/cTNT+ cells is or is at least 96% on or about day 10 and/or on the sixth day following the day of the contacting with the dissociating agent.
  • the frequency of NKX2.5+/cTNT+ cells is or is at least 97% on or about day 10 and/or on the sixth day following the day of the contacting with the dissociating agent. In some of any of such embodiments, the frequency of NKX2.5+/cTNT+ cells is or is at least 98% on or about day 10 and/or on the sixth day following the day of the contacting with the dissociating agent.
  • the population of cardiomyocytes in the second aggregate comprises a frequency of mature cardiomyocytes that is higher as compared to a reference population of cardiomyocytes in an aggregate comprising cells that were not previously dissociated.
  • the reference population of cardiomyocytes is differentiated under the same or substantially the same conditions as the population of cardiomyocytes in the second aggregate except for b) and c).
  • the reference population of cardiomyocytes is differentiated under conditions that do not comprise contacting the aggregate with a dissociating agent to form a population of dissociated cells.
  • the reference population of cardiomyocytes is differentiated under the same or substantially the same conditions as the population of cardiomyocytes in the second aggregate except that it does not comprise contacting the aggregate with a dissociating agent to form a population of dissociated cells.
  • the frequency of mature cardiomyocytes is based on a frequency of the presence of one or more mature cardiomyocyte markers in the population of cardiomyocytes in the second aggregate.
  • the one or more cardiomyocyte markers comprises one or more markers selected from the group consisting of NKX2.5, cTNT, ACTN2, TNNI1, TNNI3, MYH6, MYH7, MYL2, and MYL7.
  • the one or more cardiomyocyte markers comprise MYH6 and MYL7.
  • the one or more cardiomyocyte markers comprise NKX2.5, cTNT, MYH6, and MYL7.
  • the one or more cardiomyocyte markers comprise NKX2.5 and cTNT.
  • the population of cardiomyocytes comprise one or more modifications that (a) reduce expression of one or more of CACNA1 G, HCN4, and SLC8A1 and/or (b) increase expression of one or more of KCNJ2, TRDN, SRL, HRC, and CASQ2. In some of any of such embodiments, the population of cardiomyocytes comprise one or more modifications that (a) reduce expression of CACNA1 G, HCN4, and SLC8A1 and/or (b) increase expression of KCNJ2.
  • the population of cardiomyocytes comprise one or more modifications that (a) reduce expression of one or more of CACNA1H, HCN4, and SLC8A1 and/or (b) increase expression of one or more of KCNJ2, TRDN, SRL, HRC, and CASQ2. In some of any of such embodiments, the population of cardiomyocytes comprise one or more modifications that (a) reduce expression of CACNA1H, HCN4, and SLC8A1 and (b) increase expression of KCNJ2. In some of any of such embodiments, the population of cardiomyocytes comprise one or more modifications that reduce expression of one or more of CACNA1G, HCN4, and SLC8A1.
  • the population of cardiomyocytes comprise one or more modifications that increase expression of one or more of KCNJ2, TRDN, SRL, HRC, and CASQ2. In some of any of such embodiments, the population of cardiomyocytes comprise one or more modifications that reduce expression of CACNA1G, HCN4, and SLC8A1. In some of any of such embodiments, the population of cardiomyocytes comprise one or more modifications that increase expression of KCNJ2. In some of any of such embodiments, the population of cardiomyocytes comprise one or more modifications that reduce expression of one or more of CACNA1H, HCN4, and SLC8A1. In some of any of such embodiments, the population of cardiomyocytes comprise one or more modifications that increase expression of one or more of KCNJ2, TRDN, SRL, HRC, and CASQ2.
  • the population of cardiomyocytes comprise one or more modifications that: (i) increase expression of one or more tolerogenic factors; and/or (ii) reduce expression of one or more major histocompatibility complex (MHC) class I molecules and/or MHC class II molecules, relative to a population of cardiomyocytes that do not comprise the one or more modifications.
  • the population of cardiomyocytes comprise one or more modifications that increase expression of one or more tolerogenic factors.
  • the population of cardiomyocytes comprise one or more modifications that reduce expression of one or more major histocompatibility complex (MHC) class I molecules and/or MHC class II molecules, relative to a population of cardiomyocytes that do not comprise the one or more modifications.
  • the population of cardiomyocytes comprise one or more modifications that (a) reduce expression of one or more of CACNA1G, HCN4, and SLC8A1 and/or (b) increase expression of one or more of KCNJ2, TRDN, SRL, HRC, and CASQ2.
  • the population of cardiomyocytes comprise one or more modifications that (a) reduce expression of CACNA1G, HCN4, and SLC8AL, and/or (b) increase expression of KCNJ2.
  • the population of cardiomyocytes comprise one or more modifications that (a) reduce expression of one or more of CACNA1H, HCN4, and SLC8A1 and/or (b) increase expression of one or more of KCNJ2, TRDN, SRL, HRC, and CASQ2.
  • the population of cardiomyocytes comprise one or more modifications that (a) reduce expression of CACNA1 H, HCN4, and SLC8A1-, and (b) increase expression of KCNJ2.
  • Also provided herein is a population of cardiomyocytes produced by any of the methods described herein, such as any of the methods described in Section I.
  • Also provided herein is a population of cardiomyocytes differentiating from pluripotent stem cells, wherein the population has a frequency of the presence of one or more cardiomyocyte markers that is or is at least 75%, 76%, 77%, 78%, 79% 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about any one of days 8 to 22, and wherein the differentiating is initiated on day 0.
  • Also provided herein is a population of cardiomyocytes differentiated from pluripotent stem cells, wherein the population has a frequency of the presence of one or more cardiomyocyte markers that is or is at least 75%, 76%, 77%, 78%, 79% 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about any one of days 8 to 22, and wherein the differentiation is initiated on day 0.
  • the one or more cardiomyocyte markers comprises one or more markers selected from the group consisting of NKX2.5, cTNT, ACTN2, TNNI1, TNNI3, MYH6, MYH7, MYL2, and MYL7.
  • the one or more cardiomyocyte markers comprise MYH6 and MYL7.
  • the one or more cardiomyocyte markers comprise NKX2.5, cTNT, MYH6, and MYL7.
  • the one or more cardiomyocyte markers comprise NKX2.5 and cTNT.
  • the population has a frequency of NKX2.5+/cTNT+ cells that is or is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 8, 9, 10, 11, or 12.
  • the population has a frequency of NKX2.5+/cTNT+ cells that is or is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 9, 10, or 11.
  • the population has a frequency of NKX2.5+/cTNT+ cells that is or is at least 85% on or about day 9, 10, or 11.
  • the population has a frequency of MYH6+/MYL7+ cells that is or is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 8, 9, 10, 11, or 12.
  • the population has a frequency of NKX2.5+/cTNT+/MYH6+/MYL7+ cells that is or is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 8, 9, 10, 11, or 12.
  • the population has a frequency of NKX2.5+/cTNT+ cells that is or is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 20, 21, or 22.
  • the population has a frequency of NKX2.5+/cTNT+ cells that is or is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 22.
  • the population has a frequency of MYH6+/MYL7+ cells that is or is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 20, 21, or 22.
  • the population has a frequency of NKX2.5+/cTNT+/MYH6+/MYL7+ cells that is or is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 20, 21, or 22.
  • the differentiating is initiated on the first day that the pluripotent stem cells are cultured in a media comprising an inhibitor of glycogen synthase kinase 3 (GSK3)/activator of Wnt/p-catenin signaling.
  • GSK3 glycogen synthase kinase 3
  • the population has a frequency of NKX2.5+/cTNT+ cells, or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 9, 10, or 11.
  • the population has a frequency of NKX2.5+/cTNT+ cells, or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 10.
  • the population has a frequency of NKX2.5+/cTNT+ cells, or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 8, 9, 10, 11, or 12.
  • the population has a frequency of NKX2.5+/cTNT+ cells, or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 9, 10, or 11.
  • the population has a frequency of NKX2.5+/cTNT+ cells, or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 10.
  • the population has a frequency of NKX2.5+/cTNT+ cells, or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 8, 9, 10, 11, or 12.
  • the population has a frequency of NKX2.5+/cTNT+ cells, or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 9, 10, or 11.
  • the population has a frequency of NKX2.5+/cTNT+ cells, or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 10.
  • the population has a frequency of NKX2.5+/cTNT+ cells that is or is at least 85% on or about day 10.
  • the population has a frequency of NKX2.5+/cTNT+ cells that is or is at least 90% on or about day 10.
  • the pluripotent stem cells are selected from the group consisting of induced pluripotent stem cells, embryonic stem cells, bone marrow- mesenchymal stem cells, cardiac tissue stem cells, and adipose tissue stem cells.
  • the pluripotent stem cells are induced pluripotent stem cells.
  • the population of cardiomyocytes was contacted with a dissociating agent on or about day 2, 3, 4, 5, or 6. In some of any of such embodiments, the population of cardiomyocytes was contacted with a dissociating agent on or about day 2, 3, or 4. In some of any of such embodiments, the population of cardiomyocytes was contacted with a dissociating agent on or about day 3. In some of any of such embodiments, the population of cardiomyocytes was contacted with a dissociating agent on or about day 4.
  • the dissociating agent is or comprises a cleavage enzyme.
  • the cleavage enzyme is a protease.
  • the protease is a recombinant enzyme that cleaves a peptide bond on the C- terminal side of a lysine or arginine residue.
  • the protease is an endopeptidase. In some embodiments, the endopeptidase is trypsin.
  • the population of cardiomyocytes is at or about any one of days 8-22. In some of any of such embodiments, the population of cardiomyocytes is at or about day 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22. In some of any of such embodiments, the population of cardiomyocytes is at or about day 8, 9, 10, 11, 12, 13, 14, or 15. In some of any of such embodiments, the population of cardiomyocytes is at or about day 8, 9, 10, 11, or 12. In some of any of such embodiments, the population of cardiomyocytes is at or about day 9, 10, or 11. In some of any of such embodiments, the population of cardiomyocytes is at or about day 10.
  • the population of cardiomyocytes has a frequency of NKX2.5+/cTNT+ cells, or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%.
  • the population of cardiomyocytes has a frequency of NKX2.5+/cTNT+ cells, or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%.
  • the population of cardiomyocytes has a frequency of NKX2.5+/cTNT+ cells, or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%.
  • the population of cardiomyocytes has a frequency of NKX2.5+/cTNT+ cells, or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%.
  • a mixed population of cells comprising a first aggregate, a population of dissociated cells, and a second aggregate, wherein the first aggregate, the population of dissociated cells, and the second aggregate are formed by a method comprising: a) performing a first incubation comprising culturing a population of pluripotent stem cells under conditions to form a first aggregate, wherein the first incubation is initiated on day 0; b) contacting the first aggregate with a dissociating agent to form a population of dissociated cells; and c) performing a second incubation comprising culturing the population of dissociated cells under conditions to aggregate the dissociated cells into a second aggregate.
  • a mixed population of cells comprising a population of dissociated cells, a second aggregate, and a population of cardiomyocytes, wherein the first aggregate, the population of dissociated cells, and the second aggregate are formed by a method comprising: a) performing a first incubation comprising culturing a population of pluripotent stem cells under conditions to form a first aggregate, wherein the first incubation is initiated on day 0; b) contacting the first aggregate with a dissociating agent to form a population of dissociated cells; c) performing a second incubation comprising culturing the population of dissociated cells under conditions to aggregate the dissociated cells into a second aggregate; and d) performing a third incubation comprising culturing the second aggregate under conditions to differentiate the population of cells in the second aggregate into a population of cardiomyocytes.
  • a mixed population of cells comprising a population of dissociated cells and a second aggregate, wherein the population of dissociated cells and the second aggregate are formed by a method comprising: a) performing a first incubation comprising culturing a population of pluripotent stem cells under conditions to form a first aggregate, wherein the first incubation is initiated on day 0; b) contacting the first aggregate with a dissociating agent to form a population of dissociated cells; c) performing a second incubation comprising culturing the population of dissociated cells under conditions to aggregate the dissociated cells into a second aggregate; and d) performing a third incubation comprising culturing the second aggregate under conditions to differentiate the population of cells in the second aggregate into a population of cardiomyocytes.
  • a mixed population of cells comprising a population of cells dissociated from a second aggregate, wherein the population of cells dissociated from the second aggregate is formed by a method comprising: a) performing a first incubation comprising culturing a population of pluripotent stem cells under conditions to form a first aggregate, wherein the first incubation is initiated on day 0; b) contacting the first aggregate with a dissociating agent to form a population of dissociated cells; c) performing a second incubation comprising culturing the population of dissociated cells under conditions to aggregate the dissociated cells into a second aggregate; and d) performing a third incubation comprising culturing the second aggregate under conditions to differentiate the population of cells in the second aggregate into a population of cardiomyocytes, wherein the third incubation results in a population of cells dissociated from the second aggregate.
  • a mixed population of cells comprising a first aggregate and a population of dissociated cells, wherein the first aggregate and the population of dissociated cells are formed by a method comprising: a) performing a first incubation comprising culturing a population of pluripotent stem cells under conditions to form a first aggregate, wherein the first incubation is initiated on day 0; and b) contacting the first aggregate with a dissociating agent to form a population of dissociated cells.
  • a mixed population of cells comprising a second aggregate and a population of cells dissociated from the second aggregate, wherein the second aggregate and the population of cells dissociated from the second aggregate are formed by a method comprising: a) performing a first incubation comprising culturing a population of pluripotent stem cells under conditions to form a first aggregate, wherein the first incubation is initiated on day 0; b) contacting the first aggregate with a dissociating agent to form a population of dissociated cells; c) performing a second incubation comprising culturing the population of dissociated cells under conditions to aggregate the dissociated cells into a second aggregate; and d) performing a third incubation comprising culturing the second aggregate under conditions to differentiate the population of cells in the second aggregate into a population of cardiomyocytes, wherein the third incubation results in a population of cells dissociated from the second aggregate.
  • the mixed population of cells was harvested on or about day 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24.
  • composition comprising any of the populations of cardiomyocytes described herein.
  • composition comprising any of the mixed populations of cells described herein.
  • composition comprising a second aggregate, wherein the second aggregate is formed by a method comprising: a) performing a first incubation comprising culturing a population of pluripotent stem cells under conditions to form a first aggregate, wherein the first incubation is initiated on day 0; b) contacting the first aggregate with a dissociating agent to form a population of dissociated cells; and c) performing a second incubation comprising culturing the population of dissociated cells under conditions to aggregate the dissociated cells into a second aggregate.
  • the method is performed in suspension. In some of any of such embodiments, the contacting occurs on or about day, 2, 3, 4, 5, or 6. In some of any of such embodiments, the contacting occurs on or about day 4.
  • the dissociating agent is or comprises a cleavage enzyme.
  • the cleavage enzyme is a protease.
  • the protease is a recombinant enzyme that cleaves a peptide bond on the C- terminal side of a lysine or arginine residue.
  • the protease is an endopeptidase. In some embodiments, the endopeptidase is trypsin.
  • the contacting occurs for a duration of about 15 minutes to about 2 hours.
  • the contacting occurs for a duration of about 30 minutes to about 90 minutes, about 30 minutes to about 75 minutes, about 40 minutes to about 60 minutes, about 40 minutes to about 55 minutes, about 40 minutes to about 50 minutes, about 45 minutes to 55 minutes, or about 45 minutes to about 50 minutes. In some embodiments, the contacting occurs for a duration of about 40 to about 55 minutes.
  • the contacting occurs for a duration that is sufficient to result in a population of dissociated cells.
  • the method further comprises, during and/or after the contacting, triturating the population of dissociated cells.
  • the population of dissociated cells has a viable cell density of between or between about 1 x 10 5 and 3 x 10 7 cells/mL, 2 x 10 5 and 2 x 10 7 cells/mL, 3 x 10 5 and 1 x 10 7 cells/mL, 4 x 10 5 and 9 x 10 6 cells/mL, 5 x 10 5 and 8 x 10 6 cells/mL, 6 x 10 5 and 7 x 10 6 cells/mL, 7 x 10 5 and 6 x 10 6 cells/mL, 8 x 10 5 and 5 x 10 6 cells/mL, 9 x 10 5 and 4 x 10 6 cells/mL, 9 x 10 5 and 3 x 10 6 cells/mL, 1 x 10 6 and 2 x 10 6 cells/mL, 1.1 x 10 6 and 1.9 x 10 6 cells/mL, 1.2 x 10 6 and 1.8 x 10 6 cells/mL, or 1.25 x 10 6 and 1.75 x
  • the second aggregate is cryopreserved.
  • the second aggregate was harvested on or about day 2, 3, 4, 5, 6, or 7.
  • the second aggregate was harvested on or about day 4 or day 5.
  • the method further comprises: d) performing a third incubation comprising culturing the second aggregate under conditions to differentiate the population of cells in the second aggregate into a population of cardiomyocytes.
  • the population has a frequency of NKX2.5+/cTNT+ cells, or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 9, 10, or 11.
  • the population has a frequency of NKX2.5+/cTNT+ cells, or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 10.
  • the population has a frequency of NKX2.5+/cTNT+ cells, or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 8, 9, 10, 11, or 12.
  • the population has a frequency of NKX2.5+/cTNT+ cells, or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 10.
  • the second aggregate is cryopreserved.
  • the second aggregate was harvested on or about day 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22. In some of any of such embodiments, the second aggregate is cryopreserved on or about day 6, 7, 8, 9, 10, 11, or 12.
  • the composition comprises between about 5 x 10 8 and about 1 x 10 10 cardiomyocytes, inclusive of each. In some of any of such embodiments, the composition comprises between about 1 x 10 9 and about 5 x 10 9 cardiomyocytes, inclusive of each.
  • the composition comprises a pharmaceutically acceptable carrier.
  • a container comprising any of the composition described herein.
  • cardiac cell therapy comprising any of the populations of cardiomyocytes described herein.
  • cardiac cell therapy comprising any of the mixed populations of cardiomyocytes described herein.
  • a cardiac cell therapy comprising any of the compositions described herein.
  • a method of treatment comprising administering any of the cardiac cell therapies described herein to a subject.
  • a method of treatment comprising administering any of the populations described herein or any of the mixed populations described herein to a subject.
  • Also provided herein is a method of treatment, comprising administering a cardiac cell therapy comprising any of the populations of cardiomyocytes described herein to a subject.
  • Also provided herein is a method of treatment, comprising administering a cardiac cell therapy comprising any of the mixed populations described herein to a subject.
  • a method of treatment comprising administering a cardiac cell therapy to a subject, wherein the cardiac cell therapy comprises a population of cardiomyocytes, wherein the population of cardio myocytes is produced by a differentiation method comprising: a) performing a first incubation comprising culturing a population of pluripotent stem cells under conditions to form a first aggregate, wherein the first incubation is initiated on day 0; b) contacting the first aggregate with a dissociating agent to form a population of dissociated cells; c) performing a second incubation comprising culturing the population of dissociated cells under conditions to aggregate the dissociated cells into a second aggregate; and (d) performing a third incubation comprising culturing the second aggregate under conditions to differentiate the population of cells in the second aggregate into a population of cardiomyocytes.
  • the differentiation method is performed in suspension.
  • the contacting occurs on or about day, 2, 3, 4, 5, or 6. In some of any of such embodiments, the contacting occurs on or about day 4.
  • the dissociating agent is or comprises a cleavage enzyme.
  • the cleavage enzyme is a protease.
  • the protease is a recombinant enzyme that cleaves a peptide bond on the C- terminal side of a lysine or arginine residue.
  • the protease is an endopeptidase.
  • the endopeptidase is trypsin.
  • the contacting occurs for a duration of about 15 minutes to about 2 hours. In some of any of such embodiments, the contacting occurs for a duration of about 30 minutes to about 90 minutes, about 30 minutes to about 75 minutes, about 40 minutes to about 60 minutes, about 40 minutes to about 55 minutes, about 40 minutes to about 50 minutes, about 45 minutes to 55 minutes, or about 45 minutes to about 50 minutes. In some of any of such embodiments, the contacting occurs for a duration of about 40 to about 55 minutes. [0113] In some of any of such embodiments, the contacting occurs for a duration that is sufficient to result in a population of dissociated cells.
  • the differentiation method further comprises, during and/or after the contacting, triturating the population of dissociated cells.
  • the population of dissociated cells has a viable cell density of between or between about 1 x 10 5 and 3 x 10 7 cells/mL, 2 x 10 5 and 2 x 10 7 cells/mL, 3 x 10 5 and 1 x 10 7 cells/mL, 4 x 10 5 and 9 x 10 6 cells/mL, 5 x 10 5 and 8 x 10 6 cells/mL, 6 x 10 5 and 7 x 10 6 cells/mL, 7 x 10 5 and 6 x 10 6 cells/mL, 8 x 10 5 and 5 x 10 6 cells/mL, 9 x 10 5 and 4 x 10 6 cells/mL, 9 x 10 5 and 3 x 10 6 cells/mL, 1 x 10 6 and 2 x 10 6 cells/mL, 1.1 x 10 6 and 1.9 x 10 6 cells/mL, 1.2 x 10 6 and 1.8 x 10 6 cells/mL, or 1.25 x 10 6 and 1.75 x
  • the population has a frequency of NKX2.5+/cTNT+ cells, or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 9, 10, or 11.
  • the population has a frequency of NKX2.5+/cTNT+ cells, or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 10.
  • the population has a frequency of NKX2.5+/cTNT+ cells, or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 8, 9, 10, 11, or 12.
  • the population has a frequency of NKX2.5+/cTNT+ cells, or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 10.
  • the second aggregate was harvested on or about day 20, 21, or 22. In some of any of such embodiments, the second aggregate was harvested on or about day 20. In some of any of such embodiments, the second aggregate was harvested on or about day 21. In some of any of such embodiments, the second aggregate was harvested on or about day 22.
  • the cardiac cell therapy is administered as a suspension of cardiomyocytes or as an engineered tissue graft comprising cardiomyocytes and a matrix.
  • administration of the cardiac cell therapy comprises delivery into a subject’s heart tissue, optionally by intravenous injection, intraarterial injection, intracoronary injection, intramuscular injection, intraperitoneal injection, intramyocardial injection, trans-endocardial injection, trans-epicardial injection, and/or infusion.
  • the cardiac cell therapy comprises between about 5 x 10 8 and 1 x IO 10 engineered cardiomyocytes, inclusive of each. In some of any of such embodiments, the cardiac cell therapy comprises between about 1 x 10 9 and about 5 x 10 9 engineered cardiomyocytes, inclusive of each.
  • the cardiac cell therapy comprises a pharmaceutically acceptable carrier.
  • the subject has a heart disease or condition.
  • the heart disease or condition is pediatric cardiomyopathy, age-related cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, chronic ischemic cardiomyopathy, peripartum cardiomyopathy, inflammatory cardiomyopathy, other cardiomyopathy, myocarditis, myocardial infarction, myocardial ischemic reperfusion injury, ventricular dysfunction, heart failure, congestive heart failure, coronary artery disease, end stage heart disease, atherosclerosis, ischemia, hypertension, restenosis, angina pectoris, rheumatic heart, arterial inflammation, or cardiovascular disease.
  • the heart disease or condition is myocardial infarction.
  • the subject is a human.
  • compositions described herein in the manufacture of a medicament for treating a subject in need of a cardiac cell therapy.
  • the subject in need of a cardiac cell therapy has a heart disease or condition.
  • the heart disease or condition is pediatric cardiomyopathy, age-related cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, chronic ischemic cardiomyopathy, peripartum cardiomyopathy, inflammatory cardiomyopathy, other cardiomyopathy, myocarditis, myocardial infarction, myocardial ischemic reperfusion injury, ventricular dysfunction, heart failure, congestive heart failure, coronary artery disease, end stage heart disease, atherosclerosis, ischemia, hypertension, restenosis, angina pectoris, rheumatic heart, arterial inflammation, or cardiovascular disease.
  • the heart disease or condition is myocardial infarction.
  • the subject is a human.
  • any of the compositions described herein for use in treating a subject in need of a cardiac cell therapy has a heart disease or condition.
  • the heart disease or condition is pediatric cardiomyopathy, age-related cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, chronic ischemic cardiomyopathy, peripartum cardiomyopathy, inflammatory cardiomyopathy, other cardiomyopathy, myocarditis, myocardial infarction, myocardial ischemic reperfusion injury, ventricular dysfunction, heart failure, congestive heart failure, coronary artery disease, end stage heart disease, atherosclerosis, ischemia, hypertension, restenosis, angina pectoris, rheumatic heart, arterial inflammation, or cardiovascular disease.
  • the heart disease or condition is myocardial infarction.
  • the subject is a human.
  • FIG. 1 depicts a dot plot of varying concentrations of at 5pM and 6 pM in vessels 1, 2, and 3 on days DD4, DD10, and DD22 for evaluating dissociation and aggregation.
  • Results show a range of 85-95% CD56/PDGFRa ++ cells from Vessels V1-V6 following treatment with varying concentrations of at 5pM and 6 pM on DD4 prior to dissociation.
  • Non-dissociated vessels were between about 57-75 % cTNT/NKX2.5 ++ cells on DD10, while dissociated vessels were between about 91-98% ++ cTNT/NKX2.5 ++ cells on DD10.
  • FIG. 2A-2B shows the effect and cell viability on directed differentiation of CHIR concentrations at 5pM (FIG.2A) and 6 pM (FIG. 2B) on days DD4, DD10 and DD22.
  • FIG. 3A-3B shows the effect and cell viability on directed differentiation of CHIR concentrations at 5pM and 6 pM on days DD10 (FIG. 3A) and DD22 (FIG. 3B) for cells differentiated with or without dissociation and re- aggregation on DD4.
  • Cells harvested on DD22 that were dissociated and re-aggregated on DD4 showed a significant percentage of CTNT/NKX2.5 ++ cells at 93-96%.
  • FIG. 4A-4B show the average cluster size (FIG. 4A) and estimated cell diameter (FIG. 4B) at varying differentiation days following treatment with CHIR at 5 and 6 pM concentrations. Re-aggregated clusters grew larger from 6 uM vessel (-300 pm by DD8) compared to 5 uM vessel (-80 pm).
  • FIG. 5A depicts a dot plot of cells that were or were not dissociated and reaggregated on DD4 based on the markers CD56 and PDGFRa on DD3 (upper) or cTNT and NKX2.5 on DD10 (lower).
  • FIG. 5B depicts clusters of cells on DD10 that were (right) or were not (left) dissociated and re-aggregated on DD4.
  • FIG. 5C depicts graphs showing viable cell count, estimated cell size, and cluster size of cells of cells that were (right) or were not (left) dissociated and re-aggregated on DD4, on various days from DDO to DD10.
  • methods of differentiating cardiomyocytes from pluripotent stem cells that comprise, inter alia, dissociating a first aggregate of cells by contacting the first aggregate with a dissociating agent, followed by culturing the dissociated cells to form a second aggregate and to differentiate into a population of cardiomyocytes.
  • compositions comprising a population of cardiomyocytes, compositions comprising an aggregate, e.g., a second aggregate, compositions comprising a population of dissociated cells, cardiac cell therapies comprising a population of cardiomyocytes, cardiac cell therapies comprising a population of dissociated cells, cardiac cell therapies comprising an aggregate, e.g., a second aggregate, and cardiac cell therapies comprising any of the compositions described herein.
  • kits, containers, methods of treatment, and uses thereof are also provided herein.
  • a method of differentiating cardiomyocytes from pluripotent stem cells comprising: a) performing a first incubation comprising culturing a population of pluripotent stem cells under conditions to form a first aggregate, wherein the first incubation is initiated on day 0; b) contacting the first aggregate with a dissociating agent to form a population of dissociated cells; c) performing a second incubation comprising culturing the population of dissociated cells under conditions to aggregate the dissociated cells into a second aggregate; and (d) performing a third incubation comprising culturing the second aggregate under conditions to differentiate the population of cells in the second aggregate into a population of cardiomyocytes.
  • Also provided herein is a method of differentiating cardiomyocytes from pluripotent stem cells, the method comprising: a) performing a first incubation comprising culturing a population of pluripotent stem cells under conditions to form a first aggregate, wherein the first incubation is initiated on day 0; b) contacting the first aggregate with a dissociating agent on or about day 3, day 4, or day 5, to form a population of dissociated cells; c) performing a second incubation comprising culturing the population of dissociated cells under conditions to aggregate the dissociated cells into a second aggregate; and (d) performing a third incubation comprising culturing the second aggregate under conditions to differentiate the population of cells in the second aggregate into a population of cardiomyocytes.
  • a population of cardiomyocytes produced by any of the methods described herein.
  • Also provided herein is a population of cardiomyocytes differentiating from pluripotent stem cells, wherein the population has a frequency of the presence of one or more cardiomyocyte markers that is or is at least 75%, 76%, 77%, 78%, 79% 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about any one of days 8 to 22, and wherein the differentiating is initiated on day 0.
  • Also provided herein is a population of cardiomyocytes differentiated from pluripotent stem cells, wherein the population has a frequency of the presence of one or more cardiomyocyte markers that is or is at least 75%, 76%, 77%, 78%, 79% 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about any one of days 8 to 22, and wherein the differentiation is initiated on day 0.
  • Also provided herein is a population of cardiomyocytes differentiated from pluripotent stem cells, wherein the population has a frequency of the presence of one or more cardiomyocyte markers that is or is at least 85% on or about day 10, and wherein the differentiation is initiated on day 0.
  • Also provided herein is a mixed population of cells comprising one or more of any of the populations of cells and/or aggregates described herein.
  • composition comprising any population of cardiomyocytes described herein.
  • composition comprising a second aggregate, wherein the second aggregate is formed by a method comprising: a) performing a first incubation comprising culturing a population of pluripotent stem cells under conditions to form a first aggregate, wherein the first incubation is initiated on day 0; b) contacting the first aggregate with a dissociating agent to form a population of dissociated cells; and c) performing a second incubation comprising culturing the population of dissociated cells under conditions to aggregate the dissociated cells into a second aggregate.
  • a container comprising any of the compositions described herein.
  • cardiac cell therapy comprising any of the populations of cardiomyocytes described herein.
  • a cardiac cell therapy comprising any of the compositions described herein.
  • a cardiac cell therapy comprising one or more of any of the populations of cells and/or aggregates described herein.
  • Also provided herein is a method of treatment, comprising administering any of the cardiac cell therapies described herein to a subject.
  • Also provided herein is a method of treatment, comprising administering a cardiac cell therapy comprising any of the populations of cardiomyocytes described herein to a subject.
  • a method of treatment comprising administering a cardiac cell therapy to a subject, wherein the cardiac cell therapy comprises a population of cardiomyocytes, wherein the population of cardio myocytes is produced by a differentiation method comprising: a) performing a first incubation comprising culturing a population of pluripotent stem cells under conditions to form a first aggregate, wherein the first incubation is initiated on day 0; b) contacting the first aggregate with a dissociating agent to form a population of dissociated cells; c) performing a second incubation comprising culturing the population of dissociated cells under conditions to aggregate the dissociated cells into a second aggregate; and (d) performing a third incubation comprising culturing the second aggregate under conditions to differentiate the population of cells in the second aggregate into a population of cardiomyocytes.
  • compositions described herein in the manufacture of a medicament for treating a subject in need of a cardiac cell therapy.
  • compositions described herein for use in treating a subject in need of a cardiac cell therapy.
  • the methods provided herein can provide for improved differentiation potential, improved consistency of cardiomyocyte differentiation, improved harvesting and cryopreservation of cardiomyocytes and precursors thereof, and the ability to cryopreserve and use dissociated aggregates for cell therapy, e.g., cardiac cell therapy.
  • This in turn, can provide for improved populations, compositions, methods of treatment and uses, such as in the treatment of a disease or condition, e.g., a heart disease or condition.
  • a method for differentiating cardiomyocytes from pluripotent stem cells comprising contacting an aggregate formed by culturing a population of pluripotent stem cells with a dissociating agent to form a population of dissociated cells that are then cultured under conditions to aggregate the dissociated cells and differentiate them into a population of cardiomyocytes. Also provided herein are populations, compositions, and uses involving cardiomyocytes or precursors thereof or aggregates comprising such populations of cells generated using any of the methods described herein.
  • Cardiomyocyte differentiation from pluripotent stem cells typically begins with induction using an inhibitor of glycogen synthase kinase 3 (GSK3)/activator of Wnt/p-catenin signaling, such as CHIR99021, and mesoderm formation, followed by cardiac commitment.
  • GSK3 glycogen synthase kinase 3
  • CHIR99021 mesoderm formation
  • the methods, populations, compositions, and uses provided herein address these and other concerns and limitations of other existing methods by, e.g., providing advantages over other known methods for differentiating pluripotent stem cells into mature cardiomyocytes.
  • the methods provided herein can provide for improved differentiation potential, improved consistency of cardiomyocyte differentiation, improved harvesting and cryopreservation of cardiomyocytes and precursors thereof, and the ability to cryopreserve and use dissociated aggregates for cell therapy, e.g., cardiac cell therapy.
  • a method of differentiating cardiomyocytes from pluripotent stem cells comprising: a) performing a first incubation comprising culturing a population of pluripotent stem cells under conditions to form a first aggregate, wherein the first incubation is initiated on day 0; b) contacting the first aggregate with a dissociating agent to form a population of dissociated cells; c) performing a second incubation comprising culturing the population of dissociated cells under conditions to aggregate the dissociated cells into a second aggregate; and (d) performing a third incubation comprising culturing the second aggregate under conditions to differentiate the population of cells in the second aggregate into a population of cardiomyocytes.
  • the method is performed in suspension.
  • the first incubation, the contacting, the second incubation, and the third incubation are each performed in suspension.
  • one or more of the first incubation, the contacting, the second incubation, and the third incubation are each performed in suspension.
  • the population of pluripotent stem cells is a population of any pluripotent stem cells, e.g., any pluripotent stem cells that are capable of differentiating into cardiomyocytes.
  • the cardiomyocytes of the cardiac cell therapies provided herein are derived from pluripotent stem cells.
  • the pluripotent stem cells are selected from the group consisting of induced pluripotent stem cells, embryonic stem cells, bone marrow-mesenchymal stem cells, cardiac tissue stem cells, and adipose tissue stem cells.
  • the pluripotent stem cells are human pluripotent stem cells or are human-derived pluripotent stem cells.
  • the pluripotent stem cells are induced pluripotent stem cells (iPSCs).
  • the iPSCs are derived from a donor, such as a human donor..
  • a population of iPSCs can be generated using any available method.
  • a variety of different methods of generating pluripotent stem cells (generally referred to as iPSCs; miPSCs for murine cells, or hiPSCs for human cells) are known.
  • the original induction was done from mouse embryonic or adult fibroblasts using the viral introduction of four transcription factors, Oct3/4, Sox2, c-Myc and Klf4; see Takahashi and Yamanaka Cell 126:663-676 (2006), hereby incorporated by reference in its entirety and specifically for the techniques outlined therein.
  • iPSCs are generated by the transient expression of one or more “reprogramming factors” in the host cell, usually introduced using episomal vectors. Under these conditions, small amounts of the cells are induced to become iPSCs (in general, the efficiency of this step is low, as no selection markers are used). Once the cells are “reprogrammed”, and become pluripotent, they lose the episomal vector(s) and produce the factors using the endogenous genes. This loss of the episomal vector(s) results in cells that are called “zero footprint” cells. This is desirable as the fewer genetic modifications (particularly in the genome of the host cell), the better. Thus, it is preferred that the resulting hiPSCs have no permanent genetic modifications.
  • the number of reprogramming factors that can be used or are used can vary. Commonly, when fewer reprogramming factors are used, the efficiency of the transformation of the cells to a pluripotent state goes down, as well as the “pluripotency”, e.g. fewer reprogramming factors may result in cells that are not fully pluripotent but may only be able to differentiate into fewer cell types.
  • a single reprogramming factor, OCT4, is used.
  • two reprogramming factors, OCT4 and KLF4, are used.
  • three reprogramming factors, OCT4, KLF4 and SOX2, are used.
  • four reprogramming factors, OCT4, KLF4, SOX2 and c-Myc are used.
  • 5, 6 or 7 reprogramming factors can be used selected from SOKMNLT; SOX2, OCT4, (POU5F1), KLF4, MYC, NANOG, LIN28, and SV40L T antigen.
  • these reprogramming factor genes are provided on episomal vectors such as are known in the art and commercially available.
  • ThermoFisher/Invitrogen sell a sendai virus reprogramming kit for zero footprint generation of hiPSCs, see catalog number A34546.
  • ThermoFisher also sells EBNA-based systems as well, see catalog number A14703.
  • hiPSC lines there are a number of commercially available hiPSC lines available; see, e.g., the Gibco® Episomal hiPSC line, K18945, which is a zero footprint, viral-integration-free human iPSC cell line (see also Burridge et al, 2011, supra).
  • iPSCs are made from non-pluripotent cells such as CD34+ cord blood cells, fibroblasts, etc., by transiently expressing the reprogramming factors as described herein.
  • successful iPSCs were also generated using only Oct3/4, Sox2 and Klf4, while omitting the C-Myc, although with reduced reprogramming efficiency.
  • iPSCs are characterized by the expression of certain factors that include KLF4, Nanog, OCT4, SOX2, ESRRB, TBX3, c-Myc and TCL1. New or increased expression of these factors for purposes of the invention may be via induction or modulation of an endogenous locus or from expression from a transgene.
  • murine iPSCs can be generated using the methods of Diecke et al, Sci Rep. 2015, Jan. 28 ;5: 8081 (doi: 10.1038/srepO8O81), hereby incorporated by reference in its entirety and specifically for the methods and reagents for the generation of the miPSCs. See also, e.g.. Burridge et al., PLoS One, 2011 6(4): 18293, hereby incorporated by reference in its entirety and specifically for the methods outlined therein.
  • PSCs e.g., iPSCs
  • cardiomyocytes such as to produce a composition highly enriched in cardiomyocytes.
  • the PSCs can be differentiated into cardiomyocytes by any known methods, including but not limited to those described in Murry and Keller, Cell (2008) 132(4):661-80; Burridge et al., Cell Stem Cell (2012) 10: 16-28; Lian et al., Nature Protocols (2013) 8: 162-65; Batalov and Feiberg, Biomark. Insight (2015) 10(Suppl. 1):71-6; Denning et al., Biochim. Biophys. Acta Mol. Cell Res. (2016) 1863:1728-48; Breckwoldt et al., Nature Protocols (2017) 12: 1177-97; Guo et al., Stem Cell Res. And Ther. (2018) 9:44; and Leitolis et al., Front. Cell Dev. Biol. (2019) 8: 164.
  • the cardiomyocytes are allogeneic to a subject receiving a transplant of the cardiomyocytes.
  • the PSCs e.g. iPSCs
  • the cardiomyocytes are derived are engineered to be hypoimmunogenic by any known methods.
  • nucleic acid sequences may be modified within PSCs (e.g., iPSCs) to generate hypoimmunogenic PSCs.
  • PSCs e.g., iPSCs
  • technologies to modify nucleic acid sequences within cells include homologous recombination, knock-in, knock-out, ZFNs (zinc finger nucleases), TALENs (transcription activator-like effector nucleases), CRISPR (clustered regularly interspaced short palindromic repeats )/Cas9, and other site-specific nuclease technologies. These techniques enable double-strand DNA breaks at desired locus sites. These controlled doublestrand breaks promote homologous recombination at the specific locus sites.
  • This process focuses on targeting specific sequences of nucleic acid molecules, such as chromosomes, with endonucleases that recognize and bind to the sequences and induce a double-stranded break in the nucleic acid molecule.
  • the double- strand break is repaired either by an error-prone non- homologous end-joining (NHEJ) or by homologous recombination (HR).
  • NHEJ non- homologous end-joining
  • HR homologous recombination
  • iPSCs iPSCs
  • engineering of the PSCs (iPSCs) to be hypoimmunogenic reduces an immune response of the recipient to the cells, including cardiomyocytes differentiated from the hypoimmunogenic PSCs (e.g., iPSCs).
  • culturing the population of pluripotent stem cells can be performed by seeding pluripotent stem cells at any concentration suitable for forming an aggregate, e.g., an aggregate of between or between about 300 and about 700 pm in diameter by at or about day 2, 3, 4, 5, or 6.
  • the population of pluripotent stem cells has a viable cell concentration of between or between about 1 x 10 5 and 3 x 10 7 cells/mL, 2 x 10 5 and 2 x 10 7 cells/mL, 3 x 10 5 and 1 x 10 7 cells/mL, 4 x 10 5 and 9 x 10 6 cells/mL, 5 x 10 5 and 8 x 10 6 cells/mL, 6 x 10 5 and 7 x 10 6 cells/mL, 7 x 10 5 and 6 x 10 6 cells/mL, 8 x 10 5 and 5 x 10 6 cells/mL, 9 x 10 5 and 4 x 10 6 cells/mL, 9 x 10 5 and 3 x 10 6 cells/mL, 1 x 10 6 and 2 x 10 6 cells/mL, 1.1 x 10 6 and 1.9 x 10 6 cells/mL, 1.2 x 10 6 and 1.8 x 10 6 cells/mL, or 1.25 x 10 6 and 1.75 x 10 6
  • the population of pluripotent stem cells has a viable cell concentration of between or between about 1 x 10 5 and 3 x 10 7 cells/mL, 2 x 10 5 and 2 x 10 7 cells/mL, 3 x 10 5 and 1 x 10 7 cells/mL, 4 x 10 5 and 9 x 10 6 cells/mL, 5 x 10 5 and 8 x 10 6 cells/mL, 6 x 10 5 and 7 x 10 6 cells/mL, 7 x 10 5 and 6 x 10 6 cells/mL, 8 x 10 5 and 5 x 10 6 cells/mL, 9 x 10 5 and 4 x 10 6 cells/mL, 9 x 10 5 and 3 x 10 6 cells/mL, 1 x 10 6 and 2 x 10 6 cells/mL, 1.1 x 10 6 and 1.9 x 10 6 cells/mL, 1.2 x 10 6 and 1.8 x 10 6 cells/mL, or 1.25 x 10 6 and
  • the population of pluripotent stem cells has a viable cell concentration of or of about 1.0 x 10 6 cells/mL, 1.05 x 10 6 cells/mL, 1.1 x 10 6 cells/mL, 1.15 x 10 6 cells/mL, 1.2 x 10 6 cells/mL, 1.25 x 10 6 cells/mL, 1.3 x 10 6 cells/mL, 1.35 x 10 6 cells/mL, 1.4 x 10 6 cells/mL, 1.45 x 10 6 cells/mL, 1.5 x 10 6 cells/mL, 1.55 x 10 6 cells/mL, 1.6 x 10 6 cells/mL, 1.65 x 10 6 cells/mL, 1.7 x 10 6 cells/mL, 1.75 x 10 6 cells/mL, 1.8 x 10 6 cells/mL, 1.85 x 10 6 cells/mL, 1.9 x 10 6 cells/mL, 1.95 x 10 6 cells/mL, 2.0 x 10 7 cells/m
  • the population of pluripotent stem cells has a viable cell concentration of or of about 1.0 x 10 6 cells/mL, 1.05 x 10 6 cells/mL, 1.1 x 10 6 cells/mL, 1.15 x 10 6 cells/mL, 1.2 x 10 6 cells/mL, 1.25 x 10 6 cells/mL, 1.3 x 10 6 cells/mL, 1.35 x 10 6 cells/mL, 1.4 x 10 6 cells/mL, 1.45 x 10 6 cells/mL, 1.5 x 10 6 cells/mL, 1.55 x 10 6 cells/mL, 1.6 x 10 6 cells/mL, 1.65 x 10 6 cells/mL, 1.7 x 10 6 cells/mL, 1.75 x 10 6 cells/mL, 1.8 x 10 6 cells/mL, 1.85 x 10 6 cells/mL, 1.9 x 10 6 cells/mL, 1.95 x 10 6 cells/mL, 2.0 x 10 7 cells/mL, 2.05
  • the population of pluripotent stem cells has a viable cell concentration of or of about 1.4 x 10 6 cells/mL, 1.45 x 10 6 cells/mL, 1.5 x 10 6 cells/mL, 1.55 x 10 6 cells/mL, 1.6 x 10 6 cells/mL, 1.65 x 10 6 cells/mL, 1.7 x 10 6 cells/mL, 1.75 x 10 6 cells/mL, or 1.8 x 10 6 cells/mL.
  • the population of pluripotent stem cells has a viable cell concentration of or of about 1.4 x 10 6 cells/mL, 1.45 x 10 6 cells/mL, 1.5 x 10 6 cells/mL, 1.55 x 10 6 cells/mL, 1.6 x 10 6 cells/mL, 1.65 x 10 6 cells/mL, 1.7 x 10 6 cells/mL, 1.75 x 10 6 cells/mL, or 1.8 x 10 6 cells/mL at or about the time of seeding on day 0 or at or about the time the first incubation is initiated on day 0.
  • one or more of the reagents and conditions for differentiating cardiomyocytes from pluripotent stem cells can include, or be modified from, known methods.
  • Soluble factors important for embryonic cardiac development include Activin A, BMP4, nodal, Wnt agonists and antagonists, bFGF and other molecules (Conlon et al, Development 120(7): 1919 (1994); Lough et al, Dev. Biol. (1996) 178(1): 198; Mima et al, PNAS (1995) 92(2):467; Zaffran and Frasch, Circ. Res. (2002) 91 (6), 457). Any suitable method of inducing cardiomyocyte differentiation may be used, for example, any of those described in Fujiwara et al., PLoS One. (2001) 6(2):el6734; Dambrot et al., Biochem J.
  • PSCs e.g., iPSCs or ESCs
  • W02013013206 and W02013056072 can also be differentiated into cardiomyocytes by any of the methods described in W02013013206 and W02013056072, each incorporated by reference in its entirety.
  • the method of differentiating cardiomyocytes from pluripotent stem cells comprises: a) performing a first incubation comprising culturing a population of pluripotent stem cells under conditions to form a first aggregate, wherein the first incubation is initiated on day 0; b) contacting the first aggregate with a dissociating agent to form a population of dissociated cells; c) performing a second incubation comprising culturing the population of dissociated cells under conditions to aggregate the dissociated cells into a second aggregate; and (d) performing a third incubation comprising culturing the second aggregate under conditions to differentiate the population of cells in the second aggregate into a population of cardiomyocytes.
  • Exemplary reagents and conditions for the first incubation, dissociation via contacting the first aggregate with a dissociating agent, the second incubation, and the third incubation are provided below, which are not intended to be limiting.
  • the first incubation is initiated on day 0 and comprises culturing a population of pluripotent stem cells under conditions to form a first aggregate.
  • the first incubation comprises culturing the population of pluripotent stem cells under any conditions suitable for allowing the pluripotent stem cells to form a first aggregate, such as any medias, reagents, and/or conditions used on one or more of days 0, 1, 2, 3, 4, 5, and 6 in any known method for differentiating cardiomyocytes from pluripotent stem cells, such as, e.g., described in Fujiwara et al., PLoS One. (2001) 6(2):el6734; Dambrot et al., Biochem J. (2011) 434( l):25-35; Foldes et al., J Mol Cell Cardiol.
  • any conditions suitable for allowing the pluripotent stem cells to form a first aggregate such as any medias, reagents, and/or conditions used on one or more of days 0, 1, 2, 3, 4, 5, and 6 in any known method for differentiating cardiomyocytes from pluripotent stem cells, such as, e.g., described in Fujiwara e
  • the first incubation comprises culturing the population of pluripotent stem cells in any one or more medias suitable for allowing the pluripotent stem cells to form a first aggregate, such as any media used on one or more of days 0, 1, 2, 3, 4, 5, and 6, that is known, e.g., as described in Fujiwara et al., PLoS One. (2001) 6(2):el6734; Dambrot et al., Biochem J. (2011) 434(l):25-35; Foldes et al., J Mol Cell Cardiol. (2011) 50(2):367-76; Wang et al., Sci China Life Sci. (2010) 53(5):581-9; Chen et al., J Cell Biochem.
  • the first incubation comprises culturing the population of pluripotent stem cells in a differentiation day (DD) 0 (DD0) media comprising an inhibitor of glycogen synthase kinase 3 (GSK3)/activator of Wnt/p-catenin signaling.
  • the first incubation comprises culturing the population of pluripotent stem cells in a DD0 media comprising an inhibitor of GSK3 /activator of Wnt/p-catenin signaling, and L- alanyl-L-glutamine (a dipeptide substitute for L-glutamine).
  • the DD0 media further comprises a serum-free and insulin-free growth supplement.
  • the serum-free and insulin-free growth supplement is a B27TM minus insulin supplement (Cat#A18956-01; Life Technologies).
  • the DD0 media comprises a base media that is MCDB 131 medium (Cat#10372-019; Life Technologies).
  • the L-alanyl-L-glutamine is GlutaMaxTM, such as CTS GlutamMaxTM (Cat #A 12860-01; Life Technologies) or GlutaMaxTM (Cat#35050-061; Life Technologies).
  • the inhibitor of GSK-3a and/or GSK-3P is an inhibitor of GSK-3a and GSK-3p.
  • the inhibitor of GSK-3a and/or GSK-3P is CHIR9902L
  • the concentration of CHIR99021 is between about 3 pM and about 10 pM. In some embodiments, the concentration of CHIR99021 is or is about 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10, or any value between any of the foregoing. In some embodiments, the concentration of CHIR99021 is between about 4 pM and about 8 pM.
  • the concentration of CHIR99021 is between about 5 pM and about 7 pM. In some embodiments, the concentration of CHIR99021 is about 5, 5.5, 6, 6.5, or 7 pM, or any value in between any of the aforementioned values. In some embodiments, the concentration of CHIR99021 is or is about 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7 pM. In some embodiments, the concentration of CHIR99021 is between about 5 pM and about 6 pM or is between about 4.5 pM and about 6.5 pM.
  • the concentration of CHIR99021 is about 5 pM. In some embodiments, the concentration of CHIR99021 is about 6 pM. In some embodiments, the concentration of CHIR99021 is about 5 pM or about 6 pM. In some embodiments, the concentration of CHIR99021 is about 5
  • the first incubation comprises culturing the population of pluripotent stem cells in the media comprising the inhibitor of GSK-3a and/or GSK-3P, e.g., the DDO media, on day 0. In some embodiments, the first incubation comprises culturing the population of pluripotent stem cells in the media comprising the inhibitor of GSK-3a and/or GSK-3P, e.g., the DDO media, on day 0 and day 1.
  • the first incubation comprises culturing the population of pluripotent stem cells in the media comprising the inhibitor of GSK-3a and/or GSK-3P, e.g., the DDO media, on days 0 and 1, and one or more subsequent days of the first incubation.
  • the first incubation comprises culturing the population of pluripotent stem cells in a media, e.g., a differentiation day 1 (DD1) media, comprising L- alanyl-L-glutamine and a serum-free and insulin-free growth supplement.
  • a media e.g., a differentiation day 1 (DD1) media
  • the media e.g., DD1 media
  • the L-alanyl-L-glutamine is GlutaMaxTM (Cat#35050-061; Life Technologies).
  • the serum-free and insulin-free growth supplement is a B27TM minus insulin supplement (Cat#A18956-01; Life Technologies).
  • the first incubation comprises culturing the population of pluripotent stem cells in a media, e.g., DD1 media, comprising L-alanyl-L-glutamine and a serum-free and insulin-free growth supplement on or about day 1.
  • the first incubation comprises culturing the population of pluripotent stem cells in a media, e.g., DD1 media, comprising L-alanyl-L-glutamine and a serum- free and insulin-free growth supplement on day 1 and day 2.
  • the first incubation comprises culturing the population of pluripotent stem cells in a media, e.g., DD1 media, comprising L-alanyl-L- glutamine and a serum-free and insulin-free growth supplement on day 1 and day 2, and one or more subsequent days of the first incubation.
  • a media e.g., DD1 media, comprising L-alanyl-L- glutamine and a serum-free and insulin-free growth supplement on day 1 and day 2, and one or more subsequent days of the first incubation.
  • the first incubation comprises culturing the population of pluripotent stem cells in a media, e.g., a differentiation day 2 (DD2) media, comprising an inhibitor of Wnt/p-catenin signaling.
  • a media e.g., a differentiation day 2 (DD2) media
  • the media e.g., DD2 media
  • the media, e.g., DD2 media comprises L-alanyl-L-glutamine and a serum-free differentiation supplement.
  • the media, e.g., DD2 media comprises a base media that is MCDB 131 medium.
  • the inhibitor of Wnt/p-catenin signaling is selected from the group consisting of WIKI4, NSC668036, iCRT3, iCRT5, iCRT14, IWP-2, XAV-939, ICG-001, LGK- 974, OMP-18R5, FJ9, IWR-l-endo, KY02111, PFK115-584, Wnt-059, DKK1, FH-535, Box5, and Peptide Pen-N3.
  • the inhibitor of Wnt/p-catenin signaling is WIKI4.
  • the L-alanyl-L-glutamine is GlutaMaxTM (Cat#35050-061; Life Technologies).
  • the serum-free differentiation supplement is a B27TM supplement (Cat#A1486701; Life Technologies).
  • the culturing the population of pluripotent stem cells in the media, e.g., DD2 media, comprising the inhibitor of Wnt/p-catenin signaling begins approximately 36 to 48 hours after the first incubation is initiated on day 0. In some embodiments, the culturing the population of pluripotent stem cells in the media, e.g., DD2 media, comprising the inhibitor of Wnt/p-catenin signaling begins approximately 40 hours after the first incubation is initiated on day 0.
  • the first incubation comprises culturing the population of pluripotent stem cells in a media, e.g., DD2 media, comprising an inhibitor of Wnt/p-catenin signaling on or about day 2.
  • the first incubation comprises culturing the population of pluripotent stem cells in a media, e.g., DD2 media, comprising an inhibitor of Wnt/p-catenin signaling on or about one or more of day 1, day 2, and day 3.
  • the first incubation comprises culturing the population of pluripotent stem cells in a media, e.g., DD2 media, comprising an inhibitor of Wnt/p-catenin signaling on or about day 2 and day 3. In some embodiments, the first incubation comprises culturing the population of pluripotent stem cells in a media, e.g., DD2 media, comprising an inhibitor of Wnt/p-catenin signaling on or about day 2 and day 3, and one or more subsequent days of the first incubation.
  • a media e.g., DD2 media
  • the first incubation comprises culturing the population of pluripotent stem cells in a media, e.g., a differentiation day 3 (DD3) media, comprising L- alanyl-L-glutamine and a serum-free differentiation supplement.
  • a media e.g., a differentiation day 3 (DD3) media
  • the media e.g., DD3 media
  • the L-alanyl-L-glutamine is GlutaMaxTM (Cat#35050-061; Life Technologies).
  • the serum-free differentiation supplement is a B27TM supplement (Cat#A1486701; Life Technologies).
  • the first incubation comprises culturing the population of pluripotent stem cells in a media, e.g., a differentiation day 3 (DD3) media, comprising L- alanyl-L-glutamine and a serum-free differentiation supplement on or about day 3.
  • a media e.g., a differentiation day 3 (DD3) media, comprising L-alanyl-L-glutamine and a serum-free differentiation supplement on or about one or more of about day 3, day 4, day 5, and day 6.
  • the first incubation comprises culturing the population of pluripotent stem cells in a media, e.g., a differentiation day 3 (DD3) media, comprising L- alanyl-L-glutamine and a serum-free differentiation supplement on or about day 3 and day 4.
  • a media e.g., a differentiation day 3 (DD3) media, comprising L-alanyl-L-glutamine and a serum-free differentiation supplement on or about day 3 and day 4, and one or more subsequent days of the first incubation.
  • the first aggregate that is formed during the first incubation is between or between about 300 and about 1,000 pm in diameter. In some embodiments, the first aggregate that is formed during the first incubation is between or between about 300 and about 500, 600, 700, 800, 900, or 1,000 pm in diameter. In some embodiments, the first aggregate that is formed during the first incubation is between or between about 300 and about 1,000 pm in diameter on or about day 2, day 3, day 4, day 5, or day 6. In some embodiments, the first aggregate that is formed during the first incubation is between or between about 300 and about 500, 600, 700, 800, 900, or 1,000 pm in diameter on or about day 2, day 3, day 4, day 5, or day 6.
  • the first aggregate that is formed during the first incubation is between or between about 300 and about 700 pm in diameter. In some embodiments, the first aggregate that is formed during the first incubation is between or between about 300 and about 700 pm in diameter on or about day 2, day 3, day 4, day 5, or day 6. In some embodiments, the first aggregate that is formed during the first incubation is between or between about 350 and 650 pm, 400 and 650 pm, 450 and 650 pm, 450 and 600 pm, 500 and 650 pm, 550 and 650 pm, or 550 and 600 pm in diameter on or about day 2, day 3, day 4, day 5, or day 6. In some embodiments, the first aggregate that is formed during the first incubation is between or between about 500 and about 650 pm in diameter.
  • the first aggregate that is formed during the first incubation is between or between about 550 and about 600 pm in diameter. In some embodiments, the first aggregate that is formed during the first incubation is between or between about 300 and about 700 pm in diameter on or about day 3 or day 4. In some embodiments, the first aggregate that is formed during the first incubation is between or between about 350 and 650 pm, 400 and 650 pm, 450 and 650 pm, 450 and 600 pm, 500 and 650 pm, 550 and 650 pm, or 550 and 600 pm in diameter on or about day 3 or day 4. In some embodiments, the first aggregate that is formed during the first incubation is between or between about 500 and about 650 m in diameter on or about day 3 or day 4. In some embodiments, the first aggregate that is formed during the first incubation is between or between about 550 and about 600 gm in diameter on or about day 3 or day 4.
  • the first incubation is initiated when the population of pluripotent stem cells is first contacted by the DD0 media.
  • the first incubation occurs in suspension. In some embodiments, the first incubation comprises culturing the population of pluripotent stem cells in suspension. In some embodiments, the first incubation comprises culturing the population of pluripotent stem cells in suspension for one or more days, two or more days, three or more days, or four or more days, or five or more days of the first incubation. In some embodiments, the first incubation comprises culturing the population of pluripotent stem cells in suspension for the entirety of the first incubation.
  • the first aggregate that is formed during the first incubation is dissociated using a dissociating agent to form a population of dissociated cells.
  • the method comprises contacting the first aggregate with a dissociating agent to form a population of dissociated cells.
  • the contacting occurs when the first aggregate is between or between about 300 and 1,000 pm in diameter. In some embodiments, the contacting occurs when the first aggregate is between or between about 300 and 500, 600, 700, 800, 900, or 1,000 pm in diameter.
  • the contacting the first aggregate with the dissociating agent occurs when the first aggregate is between or between about 300 and about 1,000 pm in diameter, such as between or between about 300 and 500, 600, 700, 800, 900, or 1,000 pm in diameter, or between or between about 350 and 650 pm, 400 and 650 pm, 450 and 650 pm, 450 and 600 pm, 500 and 650 pm, 550 and 650 pm, or 550 and 600 pm in diameter.
  • the first aggregate is between or between about 500 and about 650 pm in diameter.
  • the first aggregate is between or between about 550 and about 600 pm in diameter.
  • the contacting the first aggregate with the dissociating agent occurs when the first aggregate is between or between about 400 and 1,000, 400 and 950, 400 and 900, 400 and 850, 400 and 800, 400 and 750, 400 and 700, 450 and 1,000, 450 and 950, 450 and 900, 450 and 850, 450 and 800, 450 and 750, or 450 and 700
  • the contacting the first aggregate with the dissociating agent occurs at a time when the first aggregate is between or between about 300 and about 1,000 m in diameter. In some embodiments, the contacting the first aggregate with the dissociating agent occurs at a time when the first aggregate is between or between about 300 and about 500, 600, 700, 800, 900, or 1,000 pm in diameter. In some embodiments, the contacting the first aggregate with the dissociating agent occurs at a time when the first aggregate is between or between about 350 and 650 pun, 400 and 650 pun, 450 and 650 pun, 450 and 600 pun, 500 and 650 pun, 550 and 650 pun, or 550 and 600 pun in diameter.
  • the contacting the first aggregate with the dissociating agent occurs at a time when the first aggregate is between or between about 400 and 1,000, 400 and 950, 400 and 900, 400 and 850, 400 and 800, 400 and 750, 400 and 700, 450 and 1,000, 450 and 950, 450 and 900, 450 and 850, 450 and 800, 450 and 750, or 450 and 700 pun in diameter. In some embodiments, the contacting the first aggregate with the dissociating agent occurs at a time when the first aggregate is between or between about 500 and about 650 pun in diameter. In some embodiments, the contacting the first aggregate with a dissociating agent occurs at a time when the first aggregate is between or between about 550 and about 600 pun in diameter.
  • the time e.g., the time when the first aggregate is contacted with the dissociating agent, is on or about day 2, day 3, day 4, day 5, or day 6. In some of any of such embodiments, the time, e.g., the time when the first aggregate is contacted with the dissociating agent, is on or about day 2 or day 3. In some of any of such embodiments, the time, e.g., the time when the first aggregate is contacted with the dissociating agent, is on or about day 3 or day 4. In some of any of such embodiments, the time, e.g., the time when the first aggregate is contacted with the dissociating agent, is on or about day 4 or day 5.
  • the time e.g., the time when the first aggregate is contacted with the dissociating agent, is on or about day 3, day 4, or day 5. In some of any of such embodiments, the time, e.g., the time when the first aggregate is contacted with the dissociating agent, is on or about day 4.
  • the contacting occurs on or about any one of days 2, 3, 4, 5, and 6. In some embodiments, the contacting occurs on or about day 2 or day 3. In some embodiments, the contacting occurs on or about day 3 or day 4. In some embodiments, the contacting occurs on or about day 4 or day 5. In some embodiments, the contacting occurs on or about day 5 or day 6. In some embodiments, the contacting occurs on or about day 3, day 4, or day 5. In some embodiments, the contacting occurs on or about day 4. In some embodiments, the contacting occurs on or about any one of days 2 to 6, or on or about any one of days 3 to 6, or on any one of days 4 to 6.
  • the contacting occurs on the last day of the first incubation, following the first incubation. In some embodiments, the contacting occurs on the day following the last day of the first incubation.
  • the first aggregate comprises a frequency of CD56+/PDGFRa+ cells that is or is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95%.
  • the first aggregate comprises a frequency of CD56+/PDGFRa+ cells that is or is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95%.
  • the first aggregate comprises a frequency of CD56+/PDGFRa+ cells that is or is about 80%, 81%, 92%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
  • the dissociating agent can, in some embodiments, be or comprise any agent that allows for an aggregate of cells to dissociate from one another, without affecting the ability of the dissociated cells to subsequently proliferate and/or grow and/or differentiate under suitable culture conditions.
  • the dissociating agent is or comprises a cleavage enzyme.
  • the cleavage enzyme is a protease.
  • the protease is a recombinant enzyme that cleaves a peptide bond on the C-terminal side of a lysine or arginine residue.
  • the protease is TrypLETM Select (Cat#A1217702; Life Technologies).
  • the protease is a recombinant enzyme that cleaves a peptide bond on the C-terminal side of a lysine or arginine residue, such as TrypLETM Select.
  • the protease is an endopeptidase.
  • the endopeptidase is trypsin.
  • the protease is selected from the group consisting of trypsin, collagenase, chymotrypsin, elastase, hyaluronidase, papin, and dispase.
  • the collagenase is collagenase type I, collagenase type II, or collagenase type III.
  • the collagenase is collagenase type I, collagenase type II, or collagenase type III.
  • the protease is collagenase.
  • the protease is hyaluronidase.
  • the contacting occurs for a duration that is sufficient to result in a population of dissociated cells. In some embodiments, the contacting occurs for a duration of about 15 minutes to about 2 hours. In some embodiments, the contacting occurs for a duration of about 30 minutes to about 90 minutes, about 30 minutes to about 75 minutes, about 40 minutes to about 60 minutes, about 40 minutes to about 55 minutes, about 40 minutes to about 50 minutes, about 45 minutes to 55 minutes, or about 45 minutes to about 50 minutes. In some embodiments, the contacting occurs for a duration of about 40 to about 55 minutes. In some embodiments, the contacting occurs for a duration of about 45 to about 50 minutes.
  • the method comprises, during the contacting, agitating the first aggregate.
  • the agitating is performed using a shaker, e.g., a platform shaker.
  • the agitating is performed at a revolutions per minute (RPM) of between or between about 20 and 100 RPM.
  • RPM revolutions per minute
  • the agitating is performed at an RPM of between about 20 and 100, 20 and 90, 20 and 80, 20 and 70, 20 and 60, 20 and 50, 30 and 100, 30 and 90, 30 and 80, 30 and 70, 30 and 60, 30 and 50, 40 and 100, 40 and 90, 40 and 80, 40 and 70, 40 and 60, 40 and 50 RPM, 50 and 100, 50 and 90, 50 and 80, 50 and 70, 50 and 60, 60 and 100, 60 and 90, 60 and 80, or 60 and 70 RPM.
  • the agitating is performed at an RPM of between or between about 45 and 55 RPM, such as at or about 50 RPM.
  • the agitating is performed at or about 50 RPM.
  • the agitating is performed at any one or more of the aforementioned RPMs and/or ranges of RPM, such as one, two, three, or four or more different RPMs, each for a duration of time.
  • the method further comprises, during and/or after the contacting, triturating the population of dissociated cells.
  • the triturating is performed by passing the population of dissociated cells through a pipette one or more times. In some embodiments, the triturating is performed by passing the population of dissociated cells through a pipette at or at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 times. In some embodiments, the triturating is performed by passing the population of dissociated cells through a pipette about 15 to about 25 times, such as about 20 times. In some embodiments, the triturating results in a population of dissociated cells that has a higher frequency of single cells than if the method did not comprise triturating the population of dissociated cells.
  • the population of dissociated cells has a viable cell density of between or between about 1 x 10 5 and 3 x 10 7 cells/mL, 2 x 10 5 and 2 x 10 7 cells/mL, 3 x 10 5 and 1 x 10 7 cells/mL, 4 x 10 5 and 9 x 10 6 cells/mL, 5 x 10 5 and 8 x 10 6 cells/mL, 6 x 10 5 and 7 x 10 6 cells/mL, 7 x 10 5 and 6 x 10 6 cells/mL, 8 x 10 5 and 5 x 10 6 cells/mL, 9 x 10 5 and 4 x 10 6 cells/mL, 9 x 10 5 and 3 x 10 6 cells/mL, 1 x 10 6 and 2 x 10 6 cells/mL, 1.1 x 10 6 and 1.9 x 10 6 cells/mL, 1.2 x 10 6 and 1.8 x 10 6 cells/mL, or 1.25 x 10 6 and 1.75 x 10 6 cells/
  • the population of dissociated cells has a viable cell density of between or between about 1 x 10 5 and 3 x 10 7 cells/mL, 2 x 10 5 and 2 x 10 7 cells/mL, 3 x 10 5 and 1 x 10 7 cells/mL, 4 x 10 5 and 9 x 10 6 cells/mL, 5 x 10 5 and 8 x 10 6 cells/mL, 6 x 10 5 and 7 x 10 6 cells/mL, 7 x 10 5 and 6 x 10 6 cells/mL, 8 x 10 5 and 5 x 10 6 cells/mL, 9 x 10 5 and 4 x 10 6 cells/mL, 9 x 10 5 and 3 x 10 6 cells/mL, 1 x 10 6 and 2 x 10 6 cells/mL, 1.1 x 10 6 and 1.9 x 10 6 cells/mL, 1.2 x 10 6 and 1.8 x 10 6 cells/mL, or 1.25 x
  • the population of dissociated cells has a viable cell density of between or between about 1 x 10 5 and 3 x 10 7 cells/mL, 2 x 10 5 and 2 x 10 7 cells/mL, 3 x 10 5 and 1 x 10 7 cells/mL, 4 x 10 5 and 9 x 10 6 cells/mL, 5 x 10 5 and 8 x 10 6 cells/mL, 6 x 10 5 and 7 x 10 6 cells/mL, 7 x 10 5 and 6 x 10 6 cells/mL, 8 x 10 5 and 5 x 10 6 cells/mL, 9 x 10 5 and 4 x 10 6 cells/mL, 9 x 10 5 and 3 x 10 6 cells/mL, 1 x 10 6 and 2 x 10 6 cells/mL, 1.1 x 10 6 and 1.9 x 10 6 cells/mL, 1.2 x 10 6 and 1.8 x 10 6 cells/mL, or 1.
  • the contacting occurs in suspension. In some embodiments, the contacting comprises contacting the first aggregate with the dissociating agent in suspension. In some embodiments, the triturating and/or agitating is performed in suspension.
  • the second incubation is performed following the contacting and comprises culturing the population of dissociated cells under conditions to aggregate the dissociated cells into a second aggregate.
  • the second incubation occurs for a duration of time that is sufficient to allow the population of dissociated cells to aggregate into a second aggregate.
  • the duration of time that is sufficient to allow the population of dissociated cells to aggregate into a second aggregate is or is about one to three days.
  • the duration of time that is sufficient to allow the population of dissociated cells to aggregate into a second aggregate is or is about one day or two days.
  • the duration of time that is sufficient to allow the population of dissociated cells to aggregate into a second aggregate is or is about one day.
  • the second incubation comprises culturing the population of dissociated cells in a media, e.g., a differentiation day 4 (DD4) media, comprising L-alanyl-L- glutamine and a serum-free differentiation supplement.
  • a media e.g., a differentiation day 4 (DD4) media
  • the media e.g., DD4 media
  • the L- alanyl-L-glutamine is GlutaMaxTM, such as CTS GlutamMaxTM (Cat #A 12860-01; Life Technologies) .
  • the serum-free differentiation supplement is a B27TM supplement (Cat#A1486701; Life Technologies).
  • the second incubation comprises culturing the population of dissociated cells, e.g., in DD4 media, on one day or two consecutive days from among days 2-7.
  • the second incubation comprises culturing the population of dissociated cells, e.g., in DD4 media, on or about day 2, 3, 4, 5, 6, or 7; and/or the second incubation comprises culturing the population of dissociated cells, e.g., in DD4 media, on or about days 2 and 3, on or about days 3 and 4, on or about days 4 and 5, on or about days 5 and 6, or on or about days 6 and 7.
  • the second incubation comprises culturing the population of dissociated cells, e.g., in DD4 media, on two consecutive days selected from among days 2 and 3, days 3 and 4, days 4 and 5, days 5 and 6, or days 6 and 7. In some embodiments, the second incubation comprises culturing the population of dissociated cells, e.g., in DD4 media, on or about day 4 and day 5.
  • the second aggregate is smaller in diameter than the first aggregate. In some embodiments, the second aggregate has a diameter that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% smaller than the diameter of first aggregate. In some embodiments, the second aggregate, at or about one day or two days after the contacting, is smaller in diameter than the first aggregate immediately prior to the contacting. In some embodiments, the second aggregate, at or about one day or two days after the contacting, has a diameter that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% smaller than the diameter of first aggregate immediately prior to the contacting.
  • the diameter of aggregates is based on the average diameter of a plurality of such aggregates, e.g., a plurality of first aggregates or a plurality of second aggregates, cultured under the same conditions.
  • the second aggregate that is formed is between or between about 25 and 200 pm in diameter.
  • the second aggregate that is formed is between or between about 25 and 200, 25 and 175, 25 and 150, 25 and 125, 30 and 200, 30 and 175, 30 and 150, 30 and 125, 35 and 200, 35 and 175, 35 and 150, 35 and 125, 40 and 200, 40 and 175, 40 and 150, 40 and 125, 45 and 200, 45 and 175, 45 and 150, 45 and 125, 50 and 200, 50 and 175, 50 and 150, 50 and 125, 55 and 200, 55 and 175, 55 and 150, 55 and 125, 60 and 200, 60 and 175, 60 and 150, 60 and 125, 65 and 200, 65 and 175, 65 and 150, 65 and 125, 70 and 200, 70 and 175, 70 and 150, 70 and 125, 75 and 200, 75 and 175, 75 and 150, or 75 and 125
  • the second aggregate that is formed on the second day of the second incubation and/or at or about 24 hours after contacting, e.g., at or about 24 hours after the contacting is initiated: (a) has a diameter that is less than 50% of the diameter of the first aggregate immediately prior to being contacted with the dissociating agent; and/or (b) has a diameter that is between or between about 5-50% of the diameter of the first aggregate immediately prior to being contacted with the dissociating agent; and/or (c) has a diameter that is at or about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the diameter of the first aggregate immediately prior to being contacted with the dissociating agent; and/or (d) has a diameter that is between or between about 5-25% of the diameter of the first aggregate immediately prior to being contacted with the dissociating agent.
  • the second aggregate has a diameter that is between or between about 5-25% of the diameter of the first aggregate immediately prior to being contacted with the dissociating agent.
  • the second aggregate has a diameter that is between or between about 25 and 200, 25 and 175, 25 and 150, 25 and 125, 30 and 200, 30 and 175, 30 and 150, 30 and 125, 35 and 200, 35 and 175, 35 and 150, 35 and 125, 40 and 200, 40 and 175, 40 and 150, 40 and 125, 45 and 200, 45 and 175, 45 and 150, 45 and 125, 50 and 200, 50 and 175, 50 and 150, 50 and 125, 55 and 200, 55 and 175, 55 and 150, 55 and 125, 60 and 200, 60 and 175, 60 and 150, 60 and 125, 65 and 200, 65 and 175, 65 and 150, 65 and 125, 70 and 200, 70 and 175, 70 and 150, 70 and 125, 75 and 200, 75 and 175, 75 and 150, or 75 and 125 pm.
  • the day following the day of the contacting with the dissociating agent is day 3, 4, 5, 6, or 7. In some embodiments, the day following the day of the contacting with the dissociating agent is day 5. [0222] In some embodiments, on the second day of the second incubation, the second aggregate has a diameter that is between or between about 25 and 200, 25 and 175, 25 and 150, 25 and 125, 30 and 200, 30 and 175, 30 and 150, 30 and 125, 35 and 200, 35 and 175, 35 and 150, 35 and 125, 40 and 200, 40 and 175, 40 and 150, 40 and 125, 45 and 200, 45 and 175, 45 and 150, 45 and 125, 50 and 200, 50 and 175, 50 and 150, 50 and 125, 55 and 200, 55 and 175, 55 and 150, 55 and 125, 60 and 200, 60 and 175, 60 and 150, 60 and 125, 65 and 200, 65 and 175, 65 and 150, 65 and 125, 70 and 200, 70 and 175, 70 and 150, 70 and 125, 75
  • the second aggregate has a diameter that is between or between about 25 and 300, 25 and 275, 25 and 250, 40 and 300, 40 and 275, 40 and 250, 50 and 300, 50 and 275, 50 and 250, 60 and 300, 60 and 275, 60 and 250, 70 and 300, 70 and 275, 70 and 250, 80 and 300, 80 and 275, or 80 and 250 gm.
  • the second day following the day of the contacting with the dissociating agent and/or the second day following the initiation of the second incubation and/or on the first day of the third incubation is or is about day 4, 5, 6, 7, or 8. In some embodiments, the second day following the day of the contacting with the dissociating agent and/or the second day following the initiation of the second incubation and/or on the first day of the third incubation is or is about day 6.
  • the second aggregate has a diameter that is between or between about 25 and 200, 25 and 175, 25 and 150, 25 and 125, 30 and 200, 30 and 175, 30 and 150, 30 and 125, 35 and 200, 35 and 175, 35 and 150, 35 and 125, 40 and 200, 40 and 175, 40 and 150, 40 and 125, 45 and 200, 45 and 175, 45 and 150, 45 and 125, 50 and 200, 50 and 175, 50 and 150, 50 and 125, 55 and 200, 55 and 175, 55 and 150, 55 and 125, 60 and 200, 60 and 175, 60 and 150, 60 and 125, 65 and 200, 65 and 175, 65 and 150, 65 and 125, 70 and 200, 70 and 175, 70 and 150, 70 and 125, 75 and 200, 75 and 175, 75 and 150, or 75 and 125 gm, on the second day of the second incubation and/or on the first day of the third incubation.
  • the second aggregate has a diameter that is between or between about 25 and 200, 25 and 175, 25 and 150, 25 and 125, 30 and 200, 30 and 175, 30 and 150, 30 and 125, 35 and 200, 35 and 175, 35 and 150, 35 and 125, 40 and 200, 40 and 175, 40 and 150, 40 and 125, 45 and 200, 45 and 175, 45 and 150, 45 and 125, 50 and 200, 50 and 175, 50 and 150, 50 and 125, 55 and 200, 55 and 175, 55 and 150, 55 and 125, 60 and 200, 60 and 175, 60 and 150, 60 and 125, 65 and 200, 65 and 175, 65 and 150, 65 and 125, 70 and 200, 70 and 175, 70 and 150, 70 and 125, 75 and 200, 75 and 175, 75 and 150, or 75 and 125
  • the second aggregate has a diameter that is between or between about 25 and 300, 25 and 275, 25 and 250, 40 and 300, 40 and 275, 40 and 250, 50 and 300, 50 and 275, 50 and 250, 60 and 300, 60 and 275, 60 and 250, 70 and 300, 70 and 275, 70 and 250, 80 and 300, 80 and 275, or 80 and 250 pun on the second day following the initiation of the second incubation and/or on the first day of the third incubation, the second aggregate has a diameter that is between or between about 25 and 300, 25 and 275, 25 and 250, 40 and 300, 40 and 275, 40 and 250, 50 and 300, 50 and 275, 50 and 250, 60 and 300, 60 and 275, 60 and 250, 70 and 300, 70 and 275, 70 and 250, 80 and 300, 80 and 275, or 80 and 250 m on day 6.
  • the second aggregate has a diameter that is between or between about 25 and 400, 25 and 350, 25 and 300, 25 and 275, 25 and 250, 40 and 400, 40 and 350, 40 and 300, 40 and 275, 40 and 250, 50 and 400, 60 and 350, 50 and 300, 50 and 275, 50 and 250, 60 and 400, 60 and 350, 60 and 300, 60 and 275, 60 and 250, 70 and 400, 70 and 350, 70 and 300, 70 and 275, 70 and 250, 80 and 400, 80 and 350, 80 and 300, 80 and 275, or 80 and 250 pm on the third day following the initiation of the second incubation and/or on the second day of the third incubation.
  • the second aggregate has a diameter that is between or between about 25 and 400, 25 and 350, 25 and 300, 25 and 275, 25 and 250, 40 and 400, 40 and 350, 40 and 300, 40 and 275, 40 and 250, 50 and 400, 60 and 350, 50 and 300, 50 and 275, 50 and 250, 60 and 400, 60 and 350, 60 and 300, 60 and 275, 60 and 250, 70 and 400, 70 and 350, 70 and 300, 70 and 275, 70 and 250, 80 and 400, 80 and 350, 80 and 300, 80 and 275, or 80 and 250 pun on day 7.
  • the second aggregate has a diameter that is between or between about 25 and 400, 25 and 350, 25 and 300, 25 and 275, 25 and 250, 40 and 400, 40 and 350, 40 and 300, 40 and 275, 40 and 250, 50 and 400, 60 and 350, 50 and 300, 50 and 275, 50 and 250, 60 and 400, 60 and 350, 60 and 300, 60 and 275, 60 and 250, 70 and 400, 70 and 350, 70 and 300, 70 and 275, 70 and 250, 80 and 400, 80 and 350, 80 and 300, 80 and 275, or 80 and 250 pun on the fourth day following the initiation of the second incubation and/or on the third day of the third incubation.
  • the second aggregate has a diameter that is between or between about 25 and 400, 25 and 350, 25 and 300, 25 and 275, 25 and 250, 40 and 400, 40 and 350, 40 and 300, 40 and 275, 40 and 250, 50 and 400, 60 and 350, 50 and 300, 50 and 275, 50 and 250, 60 and 400, 60 and 350, 60 and 300, 60 and 275, 60 and 250, 70 and 400, 70 and 350, 70 and 300, 70 and 275, 70 and 250, 80 and 400, 80 and 350, 80 and 300, 80 and 275, or 80 and 250
  • the second incubation occurs in suspension. In some embodiments, the second incubation comprises culturing the population of dissociated cells in suspension. In some embodiments, the second incubation comprises culturing the population of dissociated cells in suspension for one or more days, such as one day, two days, or three days, of the second incubation. In some embodiments, the second incubation comprises culturing the population of dissociated cells in suspension for the entirety of the second incubation.
  • the second incubation comprises culturing the population of dissociated cells in suspension for or for about 12 to 72 hours, 12 to 60 hours, 12 to 48 hours, 12 to 36 hours, 12 to 24 hours, 18 to 72 hours, 18 to 60 hours, 18 to 48 hours, 18 to 36 hours, 18 to 24 hours, 24 to 72 hours, 24 to 60 hours, 24 to 48 hours, or 24 to 36 hours.
  • the third incubation is performed following the formation of the second aggregate, e.g., following the second incubation, and comprises culturing the second aggregate under conditions to differentiate the population of cells in the second aggregate into a population of cardiomyocytes.
  • the culturing of the third incubation comprises culturing the second aggregate under any conditions suitable for differentiating the population of cells in the second aggregate into a population of cardiomyocytes.
  • the culturing of the third incubation can comprise medias, reagents, and/or conditions as described in, e.g., Fujiwara et al., PLoS One. (2001) 6(2):el6734; Dambrot et al., Biochem J. (2011) 434(l):25-35; Foldes et al., J Mol Cell Cardiol. (2011) 50(2):367-76; Wang et al., Sci China Life Sci.
  • the third incubation comprises culturing the second aggregate in a media, e.g., differentiation day 6 (DD6) media, comprising glucose, and in a media, e.g., differentiation day 10 (DD10) media, comprising sodium lactate.
  • a media e.g., differentiation day 6 (DD6) media
  • DD10 differentiation day 10
  • the cells are cultured in media comprising glucose and in media comprising sodium lactate non- concurrently.
  • the third incubation comprises culturing the second aggregate in a media, e.g., differentiation day 6 (DD6) media, comprising glucose further comprises a serum-free differentiation supplement.
  • a media e.g., differentiation day 6 (DD6) media
  • the media e.g., DD6 media
  • the serum-free differentiation supplement is a B27TM supplement (Cat#A1486701; Life Technologies).
  • the third incubation comprises culturing the second aggregate in the media, e.g., DD6 media, comprising glucose and the serum-free differentiation supplement for one or more days selected from among days 4 to 22 or until the population of cardiomyocytes are harvested.
  • the third incubation comprises culturing the second aggregate in the media, e.g., DD6 media, comprising glucose and the serum-free differentiation supplement on 3 or more, 4 or more, 5 or more, or 6 or more consecutive days beginning on or about day 4, day 5, day 6, or day 7.
  • the third incubation comprises culturing the second aggregate in the media, e.g., DD6 media, comprising glucose and the serum- free differentiation supplement on 3, 4, or 5 consecutive days beginning on or about day 5, 6, or 7. In some embodiments, the third incubation comprises culturing the second aggregate in the media, e.g., DD6 media, comprising glucose and the serum-free differentiation supplement on 5 consecutive days beginning on or about day 6.
  • the media comprising sodium lactate lacks glucose.
  • the sodium lactate is sodium-S -lactate (Cat#106522; Millipore-Sigma).
  • the third incubation comprises culturing the second aggregate in a media, e.g., DD6 media, comprising glucose beginning at the initiation of the third incubation and continuing until harvest, e.g., on or about day 22, except for a period of one, two, three, or four consecutive days beginning on or about day 7, 8, 9, 10, 11, 12, or 13, where the media, e.g., DD6 media, comprising glucose is replaced with a media, e.g., DD10 media, comprising sodium lactate.
  • a media e.g., DD6 media
  • the third incubation comprises culturing the second aggregate in a media, e.g., DD6 media, comprising glucose and a serum-free differentiation supplement beginning at the initiation of the third incubation and continuing until harvest, e.g., on or about day 22, except for a period of one, two, three, or four consecutive days beginning on or about day 7, 8, 9, 10, 11, 12, or 13, where the media, e.g., DD6 media, comprising glucose and the serum-free differentiation supplement is replaced with a media, e.g., DD10 media, comprising sodium lactate.
  • a media e.g., DD6 media
  • a serum-free differentiation supplement e.g., sodium lactate
  • the third incubation comprises culturing the second aggregate in a media, e.g., DD6 media, comprising glucose beginning at the initiation of the third incubation and continuing until harvest, e.g., on or about day 22, except for a period of three consecutive days beginning on or about day 10, e.g., on days 10, 11, and 12, where the media, e.g., DD6 media, comprising glucose is replaced with a media, e.g., DD10 media, comprising sodium lactate.
  • a media e.g., DD6 media
  • the third incubation comprises culturing the second aggregate in a media, e.g., DD6 media, comprising glucose and a serum-free differentiation supplement beginning at the initiation of the third incubation and continuing until harvest, e.g., on or about day 22, except for a period of three consecutive days beginning on or about day 10, e.g., on days 10, 11, and 12, where the media, e.g., DD6 media, comprising glucose and the serum-free differentiation supplement is replaced with a media, e.g., DD10 media, comprising sodium lactate.
  • a media e.g., DD6 media
  • a serum-free differentiation supplement e.g., sodium lactate
  • the third incubation comprises culturing the second aggregate in a media, e.g., DD10 media, comprising glucose and a serum-free differentiation supplement on one or more days selected from among days 7 to 15.
  • the third incubation comprises culturing the second aggregate in a media, e.g., DD10 media, comprising glucose and a serum- free differentiation supplement on 1, 2 or more, 3 or more, or 4 or more consecutive days selected from among days 7 to 15.
  • the third incubation comprises culturing the second aggregate in a media, e.g., DD10 media, comprising glucose and a serum- free differentiation supplement on 1, 2, 3, or 4 consecutive days beginning on or about day 7, 8, 9, 10, 11, 12, or 13.
  • the third incubation comprises culturing the second aggregate in a media, e.g., DD10 media, comprising glucose and a serum-free differentiation supplement on 3 consecutive days beginning on or about day 9, 10, or 11.
  • the third incubation comprises culturing the second aggregate in a media, e.g., DD10 media, comprising glucose and a serum-free differentiation supplement on days 10, 11, and 12.
  • the culturing the second aggregate in the media comprising glucose comprises culturing the second aggregate in the media comprising glucose on or about days 6-10 and 12-22; and the culturing the second aggregate in the media comprising sodium lactate comprises culturing the second aggregate in the media comprising sodium lactate on or about days 10-12; or (b) the culturing the second aggregate in the media comprising glucose comprises culturing the second aggregate in the media comprising glucose on or about days 4-8 and 12-22; and the culturing the second aggregate in the media comprising sodium lactate comprises culturing the second aggregate in the media comprising sodium lactate on or about days 8-12; or (c) the culturing the second aggregate in the media comprising glucose comprises culturing the second aggregate in the media comprising glucose on or about days 5-9 and 13-22; and the culturing the second aggregate in the media comprising sodium lactate comprises culturing the second aggregate in the media comprising sodium lactate on or about days 9-13; or
  • the culturing the second aggregate in the media comprising glucose comprises culturing the second aggregate in the media comprising glucose on or about days 6-10 and 12-22; and the culturing the second aggregate in the media comprising sodium lactate comprises culturing the second aggregate in the media comprising sodium lactate on or about days 10-12.
  • the culturing the second aggregate in the media comprising glucose comprises culturing the second aggregate in the media comprising glucose on or about days 4-8 and 12-22; and the culturing the second aggregate in the media comprising sodium lactate comprises culturing the second aggregate in the media comprising sodium lactate on or about days 8-12.
  • the culturing the second aggregate in the media comprising glucose comprises culturing the second aggregate in the media comprising glucose on or about days 5-9 and 13-22; and the culturing the second aggregate in the media comprising sodium lactate comprises culturing the second aggregate in the media comprising sodium lactate on or about days 9-13.
  • the culturing the second aggregate in the media comprising glucose comprises culturing the second aggregate in the media comprising glucose on or about days 7-11 and 13-22; and the culturing the second aggregate in the media comprising sodium lactate comprises culturing the second aggregate in the media comprising sodium lactate on or about days 11-13.
  • the culturing the second aggregate in the media comprising glucose comprises culturing the second aggregate in the media comprising glucose on or about days 8-12 and 14-22; and the culturing the second aggregate in the media comprising sodium lactate comprises culturing the second aggregate in the media comprising sodium lactate on or about days 12-14.
  • culturing the second aggregate in the media comprising sodium lactate comprises culturing the second aggregate in the media comprising sodium lactate on one or more of any of about days 9-13. In some embodiments, culturing the second aggregate in the media comprising sodium lactate comprises culturing the second aggregate in the media comprising sodium lactate on one or more of days 10-12. In some embodiments, culturing the second aggregate in the media comprising sodium lactate comprises culturing the second aggregate in the media comprising sodium lactate on or about days 10-12.
  • the third incubation continues until the population of cardiomyocytes are harvested. In some embodiments, the third incubation begins on or about any one of days 4 to 8, e.g., day 4, 5, 6, 7, or 8, and continues until the population of cardiomyocytes are harvested. In some embodiments, the third incubation begins on or about day 4 or day 5. In some embodiments, the third incubation begins on or about any one of days 6 to 8, e.g., day 6, 7, or 8, and continues until the population of cardiomyocytes are harvested. In some embodiments, the third incubation begins on or about day 6 or day 7. In some embodiments, the third incubation begins on or about day 6.
  • the population of cardiomyocytes comprises a frequency of one or more cardiomyocyte markers that is or is at least 75%, 76%, 77%, 78%, 79% 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on one or more of any of days 8, 9, 10, 11, and 12 and/or on the sixth day following the day of the contacting with the dissociating agent.
  • the population of cardiomyocytes comprises a frequency of one or more cardiomyocyte markers that is or is at least 85% on one or more of any of days 8, 9, 10, 11, and 12 and/or on the sixth day following the day of the contacting with the dissociating agent. In some embodiments, the population of cardiomyocytes comprises a frequency of one or more cardiomyocyte markers that is or is at least 90% on one or more of any of days 8, 9, 10, 11, and 12 and/or on the sixth day following the day of the contacting with the dissociating agent. In some embodiments, the population of cardiomyocytes comprises a frequency of one or more cardiomyocyte markers that is or is at least 90% on day 10 and/or on the sixth day following the day of the contacting with the dissociating agent.
  • cardiomyocyte markers include, e.g., the proteins NKX2.5, cTNT, ACTN2, TNNI1, TNNI3, MYH6, MYH7, MYL2, and MYL7.
  • NKX2.5 is NK2 homeobox 5 (also known as NKX2-5) and is encoded by the NKX2-5 gene (NM_001166175; NCBI Gene ID 1482).
  • cTNT is troponin T2 cardiac type (also known as TNNT2) and is encoded by the TNNT2 gene (NG_007556; NCBI Gene ID 7139).
  • ACTN2 is actinin alpha 2 and is encoded by the ACTN2 gene (NCBI Accession: NG_009081; NCBI Gene ID 88).
  • TNNI1 is troponin II slow skeletal type (also known as SSTNII) and is encoded by the TNNI1 gene (NG_016649; NCBI Gene ID 7135).
  • TNNI3 is troponin 13 cardiac type and is encoded by the TNNI3 gene (NM_000363; NCBI Gene ID 7137).
  • MYH6 is myosin heavy chain 6 and is encoded by the MYH6 gene (NM_002471; NCBI Gene ID 4624).
  • MYH7 is myosin heavy chain 7 and is encoded by the MYH7 gene (NM_000257; NCBI Gene ID 4625).
  • MYL2 is myosin light chain 2 and is encoded by the MYL2 gene (NM_000432; NCBI Gene ID 4633).
  • MYL7 is myosin light chain 7 and is encoded by the MYL2 gene (NM_021223; NCBI Gene ID 58498).
  • the one or more cardiomyocyte markers are selected from the group consisting of NKX2.5, cTNT, ACTN2, TNNI1, TNNI3, MYH6, MYH7, MYL2, and MYL7.
  • the one or more cardiomyocyte markers comprise NKX2.5 and cTNT.
  • the one or more cardiomyocyte markers comprise MYH6 and MYL7.
  • the one or more cardiomyocyte markers comprise NKX2.5, cTNT, MYH6, and MYL7.
  • the population of cardiomyocytes comprises a frequency of NKX2.5+/cTNT+ cells that is or is at least 75%, 76%, 77%, 78%, 79% 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on one or more of any of days 8-12, i.e., any one or more of days 8, 9, 10, 11, and 12, and/or on the sixth day following the day of the contacting with the dissociating agent.
  • the frequency of NKX2.5+/cTNT+ cells is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on one or more of any of days 8-12, i.e., any one or more of days 8, 9, 10, 11, and 12, and/or on the sixth day following the day of the contacting with the dissociating agent.
  • the population of cardiomyocytes comprises a frequency of NKX2.5+/cTNT+ cells that is or is at least 75%, 76%, 77%, 78%, 79% 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on one or more of any of days 9-11, i.e., any one or more of days 9, 10, and 11, and/or on the sixth day following the day of the contacting with the dissociating agent.
  • the frequency of NKX2.5+/cTNT+ cells is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on one or more of any of days 9-11, i.e., any one or more of days 9, 10, and 11, and/or on the sixth day following the day of the contacting with the dissociating agent.
  • the population of cardiomyocytes comprises a frequency of NKX2.5+/cTNT+ cells that is or is at least 75%, 76%, 77%, 78%, 79% 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 10 and/or on the sixth day following the day of the contacting with the dissociating agent.
  • the frequency of NKX2.5+/cTNT+ cells is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 10 and/or on the sixth day following the day of the contacting with the dissociating agent.
  • the frequency of NKX2.5+/cTNT+ cells is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 10.
  • the frequency of NKX2.5+/cTNT+ cells is or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on one or more of any of days 8-12, i.e., any one or more of days 8, 9, 10, 11, and 12, and/or on the sixth day following the day of the contacting with the dissociating agent.
  • the frequency of NKX2.5+/cTNT+ cells is or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on one or more of any of days 9-11, i.e., on one or more of any of days 9, 10, and 11, and/or on the sixth day following the day of the contacting with the dissociating agent.
  • the frequency of NKX2.5+/cTNT+ cells is or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 10 and/or on the sixth day following the day of the contacting with the dissociating agent.
  • the frequency of NKX2.5+/cTNT+ cells is or is at least 90% on or about day 10 and/or on the sixth day following the day of the contacting with the dissociating agent.
  • the third incubation occurs in suspension. In some embodiments, the third incubation comprises culturing the second aggregate in suspension. In some embodiments, the third incubation comprises culturing the second aggregate in suspension beginning on or about day 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the third incubation comprises culturing the second aggregate in suspension beginning on or about day 6. In some embodiments, the third incubation comprises culturing the second aggregate in suspension beginning at or about 1 day, 2 days, 3 days, or 4 days after the contacting with the dissociating agent. In some embodiments, the third incubation comprises culturing the second aggregate in suspension beginning at or about 2 days after the contacting with the dissociating agent. 5. Population of Cardiomyocytes
  • the population of cardiomyocytes comprises a frequency of one or more cardiomyocyte markers that is or is at least 75%, 76%, 77%, 78%, 79% 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%.
  • the one or more cardiomyocyte markers are selected from the group consisting of NKX2.5, cTNT, ACTN2, TNNI1, TNNI3, MYH6, MYH7, MYL2, and MYL7.
  • the one or more cardiomyocyte markers comprise NKX2.5 and cTNT.
  • the one or more cardiomyocyte markers comprise MYH6 and MYL7.
  • the one or more cardiomyocyte markers comprise NKX2.5, cTNT, MYH6, and MYL7.
  • the population of cardiomyocytes comprises a frequency of one or more cardiomyocyte markers selected from the group consisting of NKX2.5, cTNT, ACTN2, TNNI1, TNNI3, MYH6, MYH7, MYL2, and MYL7 that is or is at least 75%, 76%, 77%, 78%, 79% 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%.
  • the population of cardiomyocytes comprises a frequency of NKX2.5+/cTNT+ that is or is at least 75%, 76%, 77%, 78%, 79% 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%.
  • the population of cardiomyocytes comprises a frequency of NKX2.5+/cTNT+ that is or is at least 85%.
  • the population of cardiomyocytes comprises a frequency of NKX2.5+/cTNT+ that is or is at least 90%.
  • the population of cardiomyocytes comprises a frequency of MYH6+/MYL7+ that is or is at least 75%, 76%, 77%, 78%, 79% 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%.
  • the population of cardiomyocytes comprises a frequency of NKX2.5+/cTNT+/MYH6+/MYL7+ that is or is at least 75%, 76%, 77%, 78%, 79% 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%.
  • the population of cardiomyocytes comprises a frequency of NKX2.5+/cTNT+ that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%.
  • the population of cardiomyocytes comprises a frequency of MYH6+/MYL7+ that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%.
  • the population of cardiomyocytes comprises a frequency of NKX2.5+/cTNT+/MYH6+/MYL7+ that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%.
  • the population of cardiomyocytes in the second aggregate comprises a frequency of mature cardiomyocytes that is higher as compared to a reference population of cardiomyocytes in an aggregate comprising cells that were not previously dissociated.
  • the population of cardiomyocytes in the second aggregate comprises a frequency of mature cardiomyocytes that is higher as compared to a reference population of cardiomyocytes in an aggregate comprising cells that were not previously dissociated.
  • the population of cardiomyocytes in the second aggregate comprises a frequency of mature cardiomyocytes that is higher as compared to a reference population of cardiomyocytes in an aggregate comprising cells that were not previously dissociated.
  • the reference population of cardiomyocytes is differentiated under the same or substantially the same conditions as the population of cardiomyocytes in the second aggregate except for b) and c). In some embodiments, the reference population of cardiomyocytes is differentiated under the same or substantially the same conditions as the population of cardiomyocytes in the second aggregate except for the step b) comprising contacting the first aggregate with a dissociating agent to form a population of dissociated cells, and the step c) comprising performing a second incubation comprising culturing the population of dissociated cells under conditions to aggregate the dissociated cells into a second aggregate.
  • the reference population of cardiomyocytes is differentiated under conditions that do not comprise contacting the aggregate with a dissociating agent to form a population of dissociated cells. In some embodiments, the reference population of cardiomyocytes is differentiated under the same or substantially the same conditions as the population of cardiomyocytes in the second aggregate except that it does not comprise contacting the aggregate with a dissociating agent. In some embodiments, the reference population of cardiomyocytes is differentiated under the same or substantially the same conditions as the population of cardiomyocytes in the second aggregate except that it does not comprise contacting the aggregate with a dissociating agent. In some embodiments, the reference population of cardiomyocytes had not been previous dissociated using a dissociating agent.
  • the frequency of mature cardiomyocytes is based on a frequency of the presence of one or more mature cardiomyocyte markers in the population of cardiomyocytes in the second aggregate.
  • the cardiomyocyte markers can be any marker, e.g., any cell surface marker, used to identify cardiomyocytes.
  • the one or more cardiomyocyte markers comprises one or more markers selected from the group consisting of NKX2.5, cTNT, ACTN2, TNNI1, TNNI3, MYH6, MYH7, MYL2, and MYL7.
  • the one or more cardiomyocyte markers comprise MYH6 and MYL7.
  • the one or more cardiomyocyte markers comprise NKX2.5, cTNT, MYH6, and MYL7.
  • the one or more cardiomyocyte markers comprise NKX2.5 and cTNT.
  • the population of cardiomyocytes comprise one or more modifications that reduce or prevent engraftment arrhythmia (EA) when grafted into a subject.
  • the population of dissociated cells comprise one or more modifications that reduce or prevent EA when grafted into a subject.
  • the population of cells in the second aggregate comprise one or more modifications that reduce or prevent EA when grafted into a subject.
  • the one or more modifications comprise one or more modifications that (i) increase expression of one or more tolerogenic factors; and/or (ii) reduce expression of one or more major histocompatibility complex (MHC) class I molecules and/or MHC class II molecules, relative to a population of cardiomyocytes that do not comprise the one or more modifications.
  • MHC major histocompatibility complex
  • the one or more modifications comprise one or more modifications that (a) reduce expression of one or more of CACNA1G, HCN4, and SLC8A1 and/or (b) increase expression of one or more of KCNJ2, TRDN, SRL, HRC, and CASQ2.
  • the one or more modifications comprise one or more modifications that (a) reduce expression of one or more of CACNA1H, HCN4, and SLC8A1 and/or (b) increase expression of one or more of KCNJ2, TRDN, SRL, HRC, and CASQ2.
  • the one or more modifications comprise one or more modifications that (a) reduce expression of CACNA1H, HCN4, and SLC8AL, and (b) increase expression of KCNJ2.
  • Exemplary methods to introduce modifications into a cell to alter expression are known and are also described herein.
  • any of a variety of methods for overexpressing or increasing expression of a gene or protein may be used, such as by introduction or delivery of an exogenous polynucleotide encoding a protein (i.e. a transgene) or introduction of delivery of a fusion protein of a DNA-targeting domain and a transcriptional activator targeting a gene.
  • any of a variety of methods for reducing or eliminating expression of a gene or protein may be used, including non-gene editing methods such as by introduction or delivery of inhibitory nucleic acids (e.g.
  • RNAi RNAi
  • CRISPR/Cas a targeted nuclease system
  • the method for reducing or eliminating expression is via a nuclease-based gene editing technique.
  • genome editing technologies utilizing rare-cutting endonucleases are used to reduce or eliminate expression of genes, including immune genes (e.g., by deleting genomic DNA of critical immune genes) in human cells.
  • the genome editing technology comprises use of nickases, base editing, prime editing, and gene writing.
  • genome editing technologies or other gene modulation technologies are used to: insert one or more of the KCNJ2, TRDN, SRL, HRC, and CASQ2 genes; reduce or eliminate expression of one or more of the CACNA1G, CACNA1H, HCN4, and SLC8A1 genes; or any combination thereof, thus producing engineered cells, e.g., an engineered population of cardiomyocytes, that can result in reduced or eliminated EA following engraftment in a subject.
  • the cells provided herein e.g., population of cardiomyocytes, or population of dissociated cells, or populations of cells in the second aggregate, can, in some embodiments, exhibit modulated expression (e.g., reduced or eliminated expression) of one or more of CACNA1G, CACNA1H, HCN4, and SLC8A1, and/or modulated expression (e.g., increased expression or overexpression) of one or more of KCNJ2, TRDN, SRL, HRC, and CASQ2.
  • modulated expression e.g., reduced or eliminated expression
  • KCNJ2 e.g., TRDN, SRL, HRC, and CASQ2
  • the cells provided herein e.g., population of cardiomyocytes, or population of dissociated cells, or populations of cells in the second aggregate, exhibit modulated expression (e.g., reduced or eliminated expression) of CACNA1G, HCN4, and SLC8A1, and modulated expression (e.g., increased expression or overexpression) of KCNJ2.
  • the cells provided herein e.g., population of cardio myocytes, or population of dissociated cells, or populations of cells in the second aggregate, do not cause engraftment arrhythmia following engraftment in a subject.
  • Methods for reducing expression of a target gene of interest are known. Any method for reducing expression of a target polynucleotide may be used.
  • the modifications e.g., genetic modifications
  • the target polynucleotide or gene is disrupted by introducing a DNA break in the target polynucleotide, such as by using a targeting endonuclease.
  • the modifications e.g., genetic modifications
  • gene repression is achieved using an inhibitory nucleic acid that is complementary to the target polynucleotide to selectively suppress or repress expression of the gene, for instance using antisense techniques, such as by RNA interference (RNAi), short interfering RNA (siRNA), short hairpin (shRNA), and/or ribozymes.
  • RNAi RNA interference
  • siRNA short interfering RNA
  • shRNA short hairpin
  • the target polynucleotide sequence is a genomic sequence. In some embodiments, the target polynucleotide sequence is a human genomic sequence. In some embodiments, the target polynucleotide sequence is a mammalian genomic sequence. In some embodiments, the target polynucleotide sequence is a vertebrate genomic sequence. In some embodiments, gene disruption is carried out by induction of one or more double-stranded breaks and/or one or more single- stranded breaks in the gene, typically in a targeted manner.
  • the double-stranded or single-stranded breaks are made by a nuclease, e.g., an endonuclease, such as a gene-targeted nuclease.
  • the targeted nuclease is selected from zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALENs), and RNA-guided nucleases such as a CRISPR-associated nuclease (Cas), specifically designed to be targeted to the sequence of a gene or a portion thereof.
  • ZFN zinc finger nucleases
  • TALENs transcription activator-like effector nucleases
  • Cas CRISPR-associated nuclease
  • the targeted nuclease generates double-stranded or single- stranded breaks that then undergo repair through error prone non-homologous end joining (NHEJ) or, in some cases, precise homology directed repair (HDR) in which a template is used.
  • NHEJ non-homologous end joining
  • HDR precise homology directed repair
  • the targeted nuclease generates DNA double strand breaks (DSBs).
  • the process of producing and repairing the breaks is typically error prone and results in insertions and deletions (indels) of DNA bases from NHEJ repair.
  • the genetic modification may induce a deletion, insertion or mutation of the nucleotide sequence of the target gene.
  • the genetic modification may result in a frameshift mutation, which can result in a premature stop codon.
  • the targeted edits occur on both alleles of the gene resulting in a biallelic disruption or edit of the gene.
  • all alleles of the gene are targeted by the gene editing.
  • genetic modification with a targeted nuclease such as using a CRISPR/Cas system, leads to complete knockout of the gene.
  • the method further comprises harvesting the population of cardiomyocytes. In some embodiments, the method further comprises harvesting the population of dissociated cells. In some embodiments, the method further comprises harvesting the second aggregate.
  • the population of cardiomyocytes is harvested at any suitable time, e.g., at any suitable during the third incubation or beyond. In some embodiments, the population of cardiomyocytes is harvested based on a frequency of one or more cardiomyocyte markers. In some embodiments, the population of cardiomyocytes is harvested based on criteria comprising a frequency of one or more cardiomyocyte markers being above a threshold. In some embodiments, the threshold is at or above 85%, 96%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
  • the population of cardiomyocytes is harvested on or about day 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24. In some embodiments, the population of cardiomyocytes is harvested on or about day 20, 21, 22, 23, or 24. In some embodiments, the population of cardiomyocytes is harvested on or about day 21, 22, or 23. In some embodiments, the population of cardiomyocytes is harvested on or about day 22.
  • the population of dissociated cells is harvested on or about day 2, 3, 4, 5, or 6. In some embodiments, the population of dissociated cells is harvested on or about day 3, 4, or 5. In some embodiments, the population of dissociated cells is harvested on or about day 4 or day 5. In some embodiments, the population of dissociated cells is harvested on or about day 4.
  • the second aggregate is harvested on or about any one of days 3 to 22. In some embodiments, the second aggregate is harvested on or about any one of days 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the second aggregate is harvested on or about any one of days 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments, the second aggregate is harvested on or about day 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the second aggregate is harvested on or about day 5, 6, or 7.
  • the harvesting e.g., the harvesting of the population of cardiomyocytes, the population of the population of dissociated cells, and/or the second aggregate, can be performed using any suitable method, including any known method, such as any method as described or used in, e.g., Fujiwara et al., PLoS One. (2001) 6(2):el6734; Dambrot et al., Biochem J. (2011) 434(l):25-35; Foldes et al., J Mol Cell Cardiol. (2011) 50(2):367-76; Wang et al., Sci China Life Sci. (2010) 53(5):581-9; Chen et al., J Cell Biochem.
  • the harvesting of the population of cardiomyocytes comprises centrifuging the population of cardiomyocytes into a pellet. In some embodiments, the harvesting of the population of cardiomyocytes further comprises one or more washing steps. In some embodiments, the method further comprises cryopreserving the harvested population of cardiomyocytes.
  • the harvesting of the population of dissociated cells comprises centrifuging the population of dissociated cells into a pellet. In some embodiments, the harvesting of the population of dissociated cells further comprises one or more washing steps. In some embodiments, the method further comprises cryopreserving the harvested population of dissociated cells.
  • the harvesting of the second aggregate comprises centrifuging the second aggregate into a pellet. In some embodiments, the harvesting of the second aggregate further comprises one or more washing steps. In some embodiments, the method further comprises cryopreserving the harvested second aggregate.
  • the cryopreserving can be performed using any suitable method, including any known method.
  • a population of cardiomyocytes produced by any of the methods described herein, such as any of the methods described in Section I.
  • Also provided herein is a population of cardiomyocytes differentiating from pluripotent stem cells, wherein the population has a frequency of the presence of one or more cardiomyocyte markers that is or is at least 75%, 76%, 77%, 78%, 79% 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about any one of days 8 to 22, and wherein the differentiating is initiated on day 0.
  • Also provided herein is a population of cardiomyocytes differentiated from pluripotent stem cells, wherein the population has a frequency of the presence of one or more cardiomyocyte markers that is or is at least 75%, 76%, 77%, 78%, 79% 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about any one of days 8 to 22, and wherein the differentiation is initiated on day 0.
  • the population of cardiomyocytes are comprised within a second aggregate. In some embodiments, the population of cardiomyocytes are comprised within a first aggregate. In some embodiments, the population of cardiomyocytes are dissociated from a first aggregate. In some embodiments, the population of cardiomyocytes are dissociated from a second aggregate.
  • the differentiating is initiated on the first day that the pluripotent stem cells are cultured in a media comprising an inhibitor of glycogen synthase kinase 3 (GSK3)/activator of Wnt/p-catenin signaling.
  • GSK3 glycogen synthase kinase 3
  • the one or more cardiomyocyte markers comprises one or more markers selected from the group consisting of NKX2.5, cTNT, ACTN2, TNNI1, TNNI3, MYH6, MYH7, MYL2, and MYL7. In some embodiments, the one or more cardiomyocyte markers comprise MYH6 and MYL7. In some embodiments, the one or more cardiomyocyte markers comprise NKX2.5, cTNT, MYH6, and MYL7. In some embodiments, the one or more cardiomyocyte markers comprise NKX2.5 and cTNT.
  • the population has a frequency of NKX2.5+/cTNT+ cells that is or is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 8, 9, 10, 11, or 12.
  • the population has a frequency of NKX2.5+/cTNT+ cells that is or is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 9, 10, or 11.
  • the population has a frequency of NKX2.5+/cTNT+ cells that is or is at least 85% on or about day 10.
  • the population has a frequency of NKX2.5+/cTNT+ cells that is or is at least 90% on or about day 10.
  • the population has a frequency of MYH6+/MYL7+ cells that is or is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 8, 9, 10, 11, or 12.
  • the population has a frequency of NKX2.5+/cTNT+/MYH6+/MYL7+ cells that is or is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 8, 9, 10, 11, or 12.
  • the population has a frequency of NKX2.5+/cTNT+ cells that is or is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 20, 21, or 22.
  • the population has a frequency of NKX2.5+/cTNT+ cells that is or is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 22.
  • the population has a frequency of MYH6+/MYL7+ cells that is or is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 20, 21, or 22.
  • the population has a frequency of NKX2.5+/cTNT+/MYH6+/MYL7+ cells that is or is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 20, 21, or 22.
  • the population has a frequency of NKX2.5+/cTNT+ cells, or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 9, 10, or 11.
  • the population has a frequency of NKX2.5+/cTNT+ cells, or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 10.
  • the population has a frequency of NKX2.5+/cTNT+ cells, or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 8, 9, 10, 11, or 12.
  • the population has a frequency of NKX2.5+/cTNT+ cells, or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 9, 10, or 11.
  • the population has a frequency of NKX2.5+/cTNT+ cells, or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 10.
  • the population has a frequency of NKX2.5+/cTNT+ cells, or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 8, 9, 10, 11, or 12.
  • the population has a frequency of NKX2.5+/cTNT+ cells, or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 9, 10, or 11.
  • the population has a frequency of NKX2.5+/cTNT+ cells, or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day
  • the pluripotent stem cells are selected from the group consisting of induced pluripotent stem cells, embryonic stem cells, bone marrow-mesenchymal stem cells, cardiac tissue stem cells, and adipose tissue stem cells. In some embodiments, the pluripotent stem cells are induced pluripotent stem cells.
  • the population of cardiomyocytes was produced using any one or more of the methods, incubations, e.g., first incubation, second incubation, and/or third incubation, and dissociation by contacting, e.g., contacting with a dissociating agent, as described herein, such as in Section I.
  • the population of cardiomyocytes was contacted with a dissociating agent on or about day 2, 3, 4, 5, or 6. In some embodiments, the population of cardiomyocytes was contacted with a dissociating agent on or about day 2, 3, or 4. In some embodiments, the population of cardiomyocytes was contacted with a dissociating agent on or about day 3. In some embodiments, the population of cardiomyocytes was contacted with a dissociating agent on or about day 4. In some embodiments, the population of cardiomyocytes was contacted with a dissociating agent on or about day 5.
  • the dissociating agent is or comprises a cleavage enzyme.
  • the cleavage enzyme is a protease.
  • the protease is a recombinant enzyme that cleaves a peptide bond on the C-terminal side of a lysine or arginine residue.
  • the protease is an endopeptidase.
  • the endopeptidase is trypsin.
  • the protease is selected from the group consisting of trypsin, collagenase, chymotrypsin, elastase, hyaluronidase, papin, and dispase.
  • the collagenase is collagenase type I, collagenase type II, or collagenase type III. In some embodiments, the collagenase is collagenase type I, collagenase type II, or collagenase type III. In some embodiments, the protease is collagenase. In some embodiments, the protease is hyaluronidase.
  • the population of cardiomyocytes is at or about any one of days 8-22. In some embodiments, the population of cardiomyocytes is at or about day 8, 9, 10,
  • the population of cardiomyocytes is at or about day 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the population of cardiomyocytes is at or about day 8, 9, 10, 11, or 12. In some embodiments, the population of cardiomyocytes is at or about day 9, 10, or 11. In some embodiments, the population of cardiomyocytes is at or about day 10.
  • the population of cardiomyocytes was harvested at or about any one of days 8-22. In some embodiments, the population of cardiomyocytes was harvested at or about day 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22. In some embodiments, the population of cardiomyocytes was harvested at or about day 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the population of cardiomyocytes was harvested at or about day 8, 9, 10, 11, or 12. In some embodiments, the population of cardiomyocytes was harvested at or about day 9, 10, or 11. In some embodiments, the population of cardiomyocytes was harvested at or about day 10.
  • the population of cardiomyocytes has a frequency of NKX2.5+/cTNT+ cells, or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%.
  • the population of cardiomyocytes has a frequency of NKX2.5+/cTNT+ cells, or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%.
  • the population of cardiomyocytes has a frequency of NKX2.5+/cTNT+ cells, or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%.
  • the population of cardiomyocytes has a frequency of NKX2.5+/cTNT+ cells, or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%.
  • the population of cardiomyocytes has a frequency of NKX2.5+/cTNT+ cells, or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%.
  • the population of differentiating cardiomyocytes has a frequency of NKX2.5+/cTNT+ cells, or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%.
  • the population of differentiating cardiomyocytes has a frequency of NKX2.5+/cTNT+ cells, or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%.
  • the population of differentiating cardiomyocytes has a frequency of NKX2.5+/cTNT+ cells, or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 95%, 96%, 97%, or 98%.
  • mixed populations of cells comprising one or more populations of cells selected from among a population of cells of any of the first aggregates, a population of dissociated cells, e.g., a population of cells dissociated from a first aggregate, a population of cells of any of the second aggregates, and/or a populations of cardiomyocytes.
  • harvesting cells at a certain time point may include harvesting a mixed populations of cells, such as a second aggregate comprising cardiomyocytes and a population of dissociated cells that did not form a second aggregate.
  • a mixed population of cells comprising one or more of (a) first aggregate, such as any of the first aggregates described herein, e.g., in Section I or II; (b) a population of dissociated cells, such as any of the populations of dissociated cells described herein, e.g., in Section I; (c) a second aggregate, such as any of the second aggregates described herein, e.g., in Section I or II; and/or (d) a population of cardiomyocytes, e.g., a population of cardiomyocytes of the second aggregate and/or a population of cardiomyocytes differentiated from and/or dissociated from the second aggregate.
  • the population of dissociated cells was cultured in accordance with the second incubation and/or the third incubation but did not form a second aggregate.
  • a mixed population of cells comprising a first aggregate and a second aggregate, such as any of the first aggregates and/or second aggregates described herein, e.g., in Section I or II.
  • a mixed population of cells comprising a first aggregate, a population of dissociated cells, and a second aggregate, such as any of the first aggregates and/or populations of dissociated cells and/or second aggregates described herein, e.g., in Section I or II.
  • a mixed population of cells comprising a first aggregate and population of dissociated cells, such as any of the first aggregates and/or populations of dissociated cells described herein, e.g., in Section I or II.
  • a mixed population of cells comprising a population of dissociated cells and a second aggregate, such as any of the populations of dissociated cells and/or second aggregates described herein, e.g., in Section I or II.
  • a population of cells differentiated from a first aggregate such as any of the first aggregates described herein, e.g., in Section I.
  • the population of cells differentiated from the first aggregate are differentiated in accordance with any of the reagents, conditions, and/or incubations described herein.
  • a population of cells differentiated from a second aggregate such as any of the second aggregates described herein, e.g., in Section I or II.
  • the population of cells differentiated from the second aggregate are differentiated in accordance with any of the reagents, conditions, and/or incubations described herein, including any of the second and/or third incubations described herein.
  • a population of cells dissociated from a first aggregate such as any of the first aggregates described herein, e.g., in Section I.
  • the dissociation of the population of cells from the first aggregate can occur by, or result from, any means and/or reagents and/or conditions, e.g., those described in Section I.B.2.
  • the population of dissociated cells was cultured in accordance with the second incubation and/or the third incubation but did not form a second aggregate.
  • the first aggregate was cultured in accordance with the contacting with a dissociating agent, and/or the second incubation and/or the third incubation, but did not result in a population of dissociated cells.
  • a mixed population of cells comprising one or more populations of cells selected from the group consisting of (a) a first aggregate, such as any of the first aggregates described herein, (b) a population of dissociated cells, such as any of the populations of dissociated cells described herein, (c) a second aggregate, such as any of the second aggregates described herein, (d) a population of cardiomyocytes, such as any of the populations of cardiomyocytes described herein, (e) a population of cells dissociated from a first aggregate, such as a population of cells dissociated from any of the first aggregates described herein, (f) a population of cells dissociated from a second aggregate, such as a population of cells dissociated from any of the second aggregates described herein, (g) a first aggregate, such as any of the first aggregates described herein, cultured in accordance with the conditions of the second incubation and/or third incubation, without being dissociated into a population of dissociated
  • any of such populations of cells e.g., a population of pluripotent stem cells, a population of dissociated cells, a population of cariomyocytes, and/or aggregates, e.g., a first aggregate and/or second aggregate, including combinations thereof, can be harvested, cryopreserved, and/or administered to a subject, such as for treatment of a disease or condition.
  • compositions e.g., a pharmaceutical composition, comprising any of the populations of cells, including populations of cardiomyocytes or precursors thereof, e.g., pluripotent stem cells and/or cardiomyocyte progenitors.
  • composition e.g., a pharmaceutical composition, comprising any of the populations of cardiomyocytes described herein, such as any of the populations of cardiomyocytes as described in Section II.
  • composition e.g., a pharmaceutical composition, comprising a population of cardiomyocytes produced by any of the methods described herein, such as any of the methods as described in Section I.
  • composition e.g., a pharmaceutical composition, comprising any of the populations of dissociated cells described herein, such as any of the populations of dissociated cells as described in Sections I or II.
  • composition e.g., a pharmaceutical composition, comprising a population of cells in any of the second aggregates described herein, such as the population of cells in any of the second aggregates as described in Sections I or II.
  • composition e.g., a pharmaceutical composition, comprising cells dissociated from any of the first aggregates described herein.
  • a composition e.g., a pharmaceutical composition, comprising a population of dissociated cells, such as any of the populations of dissociated cells described herein.
  • composition e.g., a pharmaceutical composition, comprising cells dissociated from any of the first aggregates described herein.
  • composition e.g., a pharmaceutical composition, comprising cells dissociated from any of the second aggregates described herein.
  • composition e.g., a pharmaceutical composition, comprising cells comprised within any of the second aggregates described herein.
  • composition comprising one or more populations of cells selected from the group consisting of (a) a first aggregate, such as any of the first aggregates described herein, (b) a population of dissociated cells, such as any of the populations of dissociated cells described herein, (c) a second aggregate, such as any of the second aggregates described herein, (d) a population of cardiomyocytes, such as any of the populations of cardiomyocytes described herein, (e) a population of cells dissociated from a first aggregate, such as a population of cells dissociated from any of the first aggregates described herein, (f) a population of cells dissociated from a second aggregate, such as a population of cells dissociated from any of the second aggregates described herein, (g) a first aggregate, such as any of the first aggregates described herein, cultured in accordance with the conditions of the second incubation and/or third incubation, without being dissoci
  • one or more of the first aggregate, the population of dissociated cells, the second aggregate, the population of cardiomyocytes, the population of cells dissociated from a first aggregate, the population of cells dissociated from a second aggregate, the first aggregate cultured in accordance with the conditions of the second incubation and/or third incubation without being dissociated into a population of dissociated cells, and the population of dissociated cells cultured in accordance with the conditions of the second incubation and/or third incubation without forming a second aggregate were harvested on or about any one of days 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24.
  • the composition e.g., a pharmaceutical composition
  • the composition e.g., a pharmaceutical composition
  • the composition e.g., a pharmaceutical composition, comprises a population of cardiomyocytes, such as any of the populations of cardiomyocytes described herein, that was harvested on or about day 21.
  • the composition e.g., a pharmaceutical composition
  • the composition e.g., a pharmaceutical composition
  • the composition e.g., a pharmaceutical composition
  • compositions comprising one or more populations of cells selected from the group consisting of (a) a first aggregate, such as any of the first aggregates described herein, (b) a population of dissociated cells, such as any of the populations of dissociated cells described herein, (c) a second aggregate, such as any of the second aggregates described herein, and (d) a population of cardiomyocytes, such as any of the populations of cardiomyocytes described herein.
  • the first aggregate, the population of dissociated cells, the second aggregate, and the population of cardiomyocytes were harvested on or about any one of days 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24.
  • compositions comprising one or more populations of cells selected from the group consisting of (a) a first aggregate, such as any of the first aggregates described herein, (b) a population of dissociated cells, such as any of the populations of dissociated cells described herein, (c) a second aggregate, such as any of the second aggregates described herein, (d) a population of cells dissociated from a first aggregate, such as a population of cells dissociated from any of the first aggregates described herein, (e) a population of cells dissociated from a second aggregate, such as a population of cells dissociated from any of the second aggregates described herein, (f) a first aggregate, such as any of the first aggregates described herein, cultured in accordance with the conditions of the second incubation and/or third incubation, without being dissociated into a population of dissociated cells, and (g) a population of dissociated cells, such as any
  • one or more of the first aggregate, the population of dissociated cells, the second aggregate, the population of cardiomyocytes, the population of cells dissociated from a first aggregate, the population of cells dissociated from a second aggregate, the first aggregate cultured in accordance with the conditions of the second incubation and/or third incubation without being dissociated into a population of dissociated cells, and the population of dissociated cells cultured in accordance with the conditions of the second incubation and/or third incubation without forming a second aggregate were harvested on or about any one of days 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24.
  • compositions e.g., a pharmaceutical composition, comprising (a) a first aggregate, such as any of the first aggregates described herein, (b) a population of dissociated cells, such as any of the populations of dissociated cells described herein, (c) a second aggregate, such as any of the second aggregates described herein, and (d) a population of cardiomyocytes, such as any of the populations of cardiomyocytes described herein.
  • first aggregate, the population of dissociated cells, the second aggregate, and the population of cardiomyocytes were harvested on or about any one of days 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24.
  • compositions e.g., a pharmaceutical composition, comprising one or more populations of cells selected from the group consisting of (a) a first aggregate, such as any of the first aggregates described herein, and (b) a population of dissociated cells, such as any of the populations of dissociated cells described herein.
  • the first aggregate and the population of dissociated cells were harvested on or about any one of days 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24.
  • compositions comprising a first aggregate, such as any of the first aggregates described herein, and one or more populations of cells selected from the group consisting of (a) a population of dissociated cells, such as any of the populations of dissociated cells described herein, (b) a second aggregate, such as any of the second aggregates described herein, (c) a population of cardiomyocytes, such as any of the populations of cardiomyocytes described herein, (d) a population of cells dissociated from a first aggregate, such as a population of cells dissociated from any of the first aggregates described herein, (e) a population of cells dissociated from a second aggregate, such as a population of cells dissociated from any of the second aggregates described herein, (f) a first aggregate, such as any of the first aggregates described herein, cultured in accordance with the conditions of the second incubation and/or third incubation, without being dissociated into
  • compositions comprising a second aggregate, such as any of the second aggregates described herein, and one or more populations of cells selected from the group consisting of (a) a population of dissociated cells, such as any of the populations of dissociated cells described herein, (b) a first aggregate, such as any of the first aggregates described herein, (c) a population of cardiomyocytes, such as any of the populations of cardiomyocytes described herein, (d) a population of cells dissociated from a first aggregate, such as a population of cells dissociated from any of the first aggregates described herein, (e) a population of cells dissociated from a second aggregate, such as a population of cells dissociated from any of the second aggregates described herein, (f) a first aggregate, such as any of the first aggregates described herein, cultured in accordance with the conditions of the second incubation and/or third incubation, without being dissociated into
  • compositions comprising a population of cardiomyocytes, such as any of the populations of cardiomyocytes described herein, and one or more populations of cells selected from the group consisting of (a) a population of dissociated cells, such as any of the populations of dissociated cells described herein, (b) a first aggregate, such as any of the first aggregates described herein, (c) a second aggregate, such as any of the second aggregates described herein, (d) a population of cells dissociated from a first aggregate, such as a population of cells dissociated from any of the first aggregates described herein, (e) a population of cells dissociated from a second aggregate, such as a population of cells dissociated from any of the second aggregates described herein, (f) a first aggregate, such as any of the first aggregates described herein, cultured in accordance with the conditions of the second incubation and/or third incubation, without being dissociated into
  • composition e.g., a pharmaceutical composition, comprising (a) a first aggregate, such as any of the first aggregates described herein, and (b) a second aggregate, such as any of the second aggregates described herein.
  • a composition comprising a mixture of any two or more populations of cells described herein.
  • the two or more populations of cells described herein include, e.g., two or more of any of the following: (a) a first aggregate, such as any of the first aggregates described herein, (b) a population of dissociated cells, such as any of the populations of dissociated cells described herein, (c) a second aggregate, such as any of the second aggregates described herein, (d) a population of cardiomyocytes, such as any of the populations of cardiomyocytes described herein, (e) a population of cells dissociated from a first aggregate, such as a population of cells dissociated from any of the first aggregates described herein, (f) a population of cells dissociated from a second aggregate, such as a population of cells dissociated from any of the second aggregates described herein, (g) a first aggregate, such as any of
  • a composition comprising a mixture of any three or more populations of cells described herein.
  • the three or more populations of cells described herein include, e.g., three or more of any of the following: (a) a first aggregate, such as any of the first aggregates described herein, (b) a population of dissociated cells, such as any of the populations of dissociated cells described herein, (c) a second aggregate, such as any of the second aggregates described herein, (d) a population of cardiomyocytes, such as any of the populations of cardiomyocytes described herein, (e) a population of cells dissociated from a first aggregate, such as a population of cells dissociated from any of the first aggregates described herein, (f) a population of cells dissociated from a second aggregate, such as a population of cells dissociated from any of the second aggregates described herein, (g) a first aggregate, such as any of
  • composition e.g., a pharmaceutical composition, comprising a second aggregate, wherein the second aggregate is formed by a method comprising: a) performing a first incubation comprising culturing a population of pluripotent stem cells under conditions to form a first aggregate, wherein the first incubation is initiated on day 0; b) contacting the first aggregate with a dissociating agent to form a population of dissociated cells; and c) performing a second incubation comprising culturing the population of dissociated cells under conditions to aggregate the dissociated cells into a second aggregate.
  • the composition comprising the second aggregate is used as a cardiac cell therapy, e.g., for injection of cardiomyocytes as an aggregate instead of as single cells.
  • compositions e.g., a pharmaceutical composition, comprising cells dissociated from a second aggregate, wherein the second aggregate is formed by a method comprising: a) performing a first incubation comprising culturing a population of pluripotent stem cells under conditions to form a first aggregate, wherein the first incubation is initiated on day 0; b) contacting the first aggregate with a dissociating agent to form a population of dissociated cells; and c) performing a second incubation comprising culturing the population of dissociated cells under conditions to aggregate the dissociated cells into a second aggregate.
  • the cells dissociated from the second aggregate can be dissociated by any agent and/or means.
  • the composition comprising the second aggregate is used as a cardiac cell therapy, e.g., for injection of cardiomyocytes as an aggregate instead of as single cells.
  • the method is performed in suspension. [0330] In some embodiments, the method comprises the first incubation and/or the second incubation and/or the contacting, as described herein, such as in Section I.
  • the contacting occurs on or about day, 2, 3, 4, 5, or 6. In some embodiments, the contacting occurs on or about day 4.
  • the dissociating agent is or comprises a cleavage enzyme.
  • the cleavage enzyme is a protease.
  • the protease is a recombinant enzyme that cleaves a peptide bond on the C-terminal side of a lysine or arginine residue.
  • the protease is an endopeptidase.
  • the endopeptidase is trypsin.
  • the protease is selected from the group consisting of trypsin, collagenase, chymotrypsin, elastase, hyaluronidase, papin, and dispase.
  • the collagenase is collagenase type I, collagenase type II, or collagenase type III. In some embodiments, the collagenase is collagenase type I, collagenase type II, or collagenase type III. In some embodiments, the protease is collagenase. In some embodiments, the protease is hyaluronidase.
  • the contacting occurs for a duration of time that is sufficient to dissociate the cells in the first aggregate. In some embodiments, the contacting occurs for a duration of about 15 minutes to about 2 hours. In some embodiments, the contacting occurs for a duration of about 30 minutes to about 90 minutes, about 30 minutes to about 75 minutes, about 40 minutes to about 60 minutes, about 40 minutes to about 55 minutes, about 40 minutes to about 50 minutes, about 45 minutes to 55 minutes, or about 45 minutes to about 50 minutes. In some embodiments, the contacting occurs for a duration of about 40 to about 55 minutes.
  • the contacting occurs for a duration that is sufficient to result in a population of dissociated cells.
  • the method further comprises, during and/or after the contacting, triturating the population of dissociated cells.
  • the population of dissociated cells has a viable cell density of between or between about 1 x 10 5 and 3 x 10 7 cells/mL, 2 x 10 5 and 2 x 10 7 cells/mL, 3 x 10 5 and 1 x 10 7 cells/mL, 4 x 10 5 and 9 x 10 6 cells/mL, 5 x 10 5 and 8 x 10 6 cells/mL, 6 x 10 5 and 7 x 10 6 cells/mL, 7 x 10 5 and 6 x 10 6 cells/mL, 8 x 10 5 and 5 x 10 6 cells/mL, 9 x 10 5 and 4 x 10 6 cells/mL, 9 x 10 5 and 3 x 10 6 cells/mL, 1 x 10 6 and 2 x 10 6 cells/mL, 1.1 x 10 6 and 1.9 x 10 6 cells/mL, 1.2 x 10 6 and 1.8 x 10 6 cells/mL, or 1.25 x 10 6 and 1.75 x 10 6 cells/
  • the population of dissociated cells has a viable cell density of between or between about 1 x 10 5 and 3 x 10 7 cells/mL, 2 x 10 5 and 2 x 10 7 cells/mL, 3 x 10 5 and 1 x 10 7 cells/mL, 4 x 10 5 and 9 x 10 6 cells/mL, 5 x 10 5 and 8 x 10 6 cells/mL, 6 x 10 5 and 7 x 10 6 cells/mL, 7 x 10 5 and 6 x 10 6 cells/mL, 8 x 10 5 and 5 x 10 6 cells/mL, 9 x 10 5 and 4 x 10 6 cells/mL, 9 x 10 5 and 3 x 10 6 cells/mL, 1 x 10 6 and 2 x 10 6 cells/mL, 1.1 x 10 6 and 1.9 x 10 6 cells/mL, 1.2 x 10 6 and 1.8 x 10 6 cells/mL, or 1.25 x 10 6 and 1.75 x 10 6 cells/mL at or
  • the method further comprises harvesting the second aggregate.
  • the second aggregate was harvested on or about day 2, 3, 4, 5, 6, or 7.
  • the second aggregate was harvested on or about day 4 or day 5.
  • the second aggregate is cryopreserved.
  • the method further comprises: c) performing a third incubation comprising culturing the second aggregate under conditions to differentiate the population of cells in the second aggregate into a population of cardiomyocytes.
  • the method comprises any third incubation as described herein, such as in Section I.
  • the population has a frequency of NKX2.5+/cTNT+ cells, or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 9, 10, or 11.
  • the population has a frequency of NKX2.5+/cTNT+ cells, or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 10.
  • the population has a frequency of NKX2.5+/cTNT+ cells, or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 8, 9, 10, 11, or 12.
  • the population has a frequency of NKX2.5+/cTNT+ cells, or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 10.
  • the population has a frequency of NKX2.5+/cTNT+ cells, or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 85% on or about day 10.
  • the population has a frequency of NKX2.5+/cTNT+ cells that is or is at least 85% on or about day 10. In some embodiments, the population has a frequency of NKX2.5+/cTNT+ cells that is or is at least 90% on or about day 10.
  • the second aggregate is cryopreserved.
  • the second aggregate was harvested on or about day 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22. In some embodiments, the second aggregate was cryopreserved on or about day 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22. In some embodiments, the second aggregate is cryopreserved on or about day 6, 7, 8, 9, 10, 11, or 12.
  • the composition e.g., pharmaceutical composition
  • the composition comprises between about 5 x 10 8 and about 1 x 10 10 cardiomyocytes, inclusive of each.
  • the composition comprises between about 1 x 10 9 and about 5 x 10 9 cardiomyocytes, inclusive of each.
  • the composition is a pharmaceutical composition.
  • the composition comprises a pharmaceutically acceptable carrier.
  • the composition e.g., a pharmaceutical composition
  • suitable pharmaceutical carriers are described in “Remington’s Pharmaceutical Sciences” by E. W. Martin.
  • Such compositions will contain a therapeutically effective amount of tacrolimus, generally in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil.
  • the pharmaceutical compositions can contain any one or more of a diluents(s), adjuvant(s), antiadherent(s), binder(s), coating(s), filler(s), flavor(s), color(s), lubricant(s), glidant(s), preservative(s), detergent(s), sorbent(s), emulsifying agent(s), pharmaceutical excipient(s), pH buffering agent(s), or sweetener(s) and a combination thereof.
  • the pharmaceutical composition can be liquid, solid, a lyophilized powder, in gel form, and/or combination thereof.
  • the choice of carrier is determined in part by the particular inhibitor and/or by the method of administration.
  • Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine
  • the pharmaceutical composition can be formulated for administration by any route known to those of skill in the art including intramuscular, intravenous, intradermal, intralesional, intraperitoneal injection, subcutaneous, intratumoral, epidural, nasal, oral, vaginal, rectal, topical, local, otic, inhalational, buccal (e.g., sublingual), and transdermal administration or any route.
  • routes known to those of skill in the art including intramuscular, intravenous, intradermal, intralesional, intraperitoneal injection, subcutaneous, intratumoral, epidural, nasal, oral, vaginal, rectal, topical, local, otic, inhalational, buccal (e.g., sublingual), and transdermal administration or any route.
  • other modes of administration also are contemplated.
  • the administration is by bolus infusion, by injection, e.g., intravenous or subcutaneous injections, intraocular injection, periocular injection, subretinal injection, intravitreal injection, trans-septal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjectval injection, subconjuntival injection, sub-Tenon’s injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery.
  • administration is by parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.
  • compositions also can be administered with other biologically active agents, either sequentially, intermittently or in the same composition.
  • administration also can include controlled release systems including controlled release formulations and device-controlled release, such as by means of a pump.
  • the administration is oral.
  • the administration is intravenous.
  • a cardiac cell therapy comprising any of the compositions described herein, e.g., in Section III, or any of the populations of cardiomyocytes described herein, e.g., as described in Sections I and II.
  • a cardiac cell therapy comprising any of the populations of cardiomyocytes described herein.
  • a cardiac cell therapy comprising any of the compositions comprising a second aggregate described herein.
  • a cardiac cell therapy comprising a composition, e.g., a pharmaceutical composition, comprising one or more populations of cells, such as any one or more populations of cells, first aggregates, or second aggregates, or cells dissociated therefrom, as described herein.
  • a composition e.g., a pharmaceutical composition
  • populations of cells such as any one or more populations of cells, first aggregates, or second aggregates, or cells dissociated therefrom, as described herein.
  • a cardiac cell therapy comprising a composition, e.g., a pharmaceutical composition, comprising one or more populations of cells selected from the group consisting of (a) a first aggregate, such as any of the first aggregates described herein, (b) a population of dissociated cells, such as any of the populations of dissociated cells described herein, (c) a second aggregate, such as any of the second aggregates described herein, (d) a population of cardiomyocytes, such as any of the populations of cardiomyocytes described herein, (e) a population of cells dissociated from a first aggregate, such as a population of cells dissociated from any of the first aggregates described herein, (f) a population of cells dissociated from a second aggregate, such as a population of cells dissociated from any of the second aggregates described herein, (g) a first aggregate, such as any of the first aggregates described herein, cultured in accordance with the conditions of the second incubation and/or third in
  • the cardiac cell therapy is a pharmaceutical composition comprising cardiomyocytes and a pharmaceutically acceptable carrier.
  • the cardiac cell therapy comprises a suspension of cardiomyocytes.
  • the cardiac cell therapy is a suspension of cardiomyocytes, such as a suspension of dissociated cardiomyocytes.
  • the cardiac cell therapy comprises is an engineered tissue graft containing cardiomyocytes and a matrix. In some embodiments, the cardiac cell therapy does not comprise or is not an organ.
  • the cardiac cell therapy comprises a suspension of aggregates comprising a population of cardiomyocytes, such as any of the second aggregates described herein.
  • a method of treatment comprising administering any of the cardiac cell therapies described herein, such as any of the cardiac cell therapies comprising any of the compositions described herein.
  • compositions described herein such as any of those described in Section III.
  • Also provided herein are methods of treatment comprising administering any of the populations of cardiomyocytes described herein, or precursors thereof, such as any of those described in Section II.
  • provided herein is a method of treatment comprising administering (a) any of the cardiac cell therapies described herein; or (b) any of the compositions, e.g., pharmaceutical compositions, described herein; or (c) any of the populations of cardiomyocytes described herein.
  • a method of treatment comprising administering a cardiac cell therapy comprising any of the populations of cardiomyocytes described herein.
  • a method of treatment comprising administering a cardiac cell therapy, wherein the cardiac cell therapy comprises a composition, e.g., a pharmaceutical composition, comprising one or more populations of cells, such as any one or more populations of cells, first aggregates, or second aggregates, or cells dissociated therefrom, as described herein.
  • a composition e.g., a pharmaceutical composition, comprising one or more populations of cells, such as any one or more populations of cells, first aggregates, or second aggregates, or cells dissociated therefrom, as described herein.
  • a method of treatment comprising administering a cardiac cell therapy, wherein the cardiac cell therapy comprises a composition, e.g., a pharmaceutical composition, comprising one or more populations of cells selected from the group consisting of (a) a first aggregate, such as any of the first aggregates described herein, (b) a population of dissociated cells, such as any of the populations of dissociated cells described herein, (c) a second aggregate, such as any of the second aggregates described herein, (d) a population of cardiomyocytes, such as any of the populations of cardiomyocytes described herein, (e) a population of cells dissociated from a first aggregate, such as a population of cells dissociated from any of the first aggregates described herein, (f) a population of cells dissociated from a second aggregate, such as a population of cells dissociated from any of the second aggregates described herein, (g) a first aggregate, such as any of the first aggregates described herein, cultured in
  • a method of treatment comprising administering a cardiac cell therapy to a subject, wherein the cardiac cell therapy comprises a population of cardiomyocytes, wherein the population of cardio myocytes is produced by a differentiation method comprising: a) performing a first incubation comprising culturing a population of pluripotent stem cells under conditions to form a first aggregate, wherein the first incubation is initiated on day 0; b) contacting the first aggregate with a dissociating agent to form a population of dissociated cells; c) performing a second incubation comprising culturing the population of dissociated cells under conditions to aggregate the dissociated cells into a second aggregate; and (d) performing a third incubation comprising culturing the second aggregate under conditions to differentiate the population of cells in the second aggregate into a population of cardiomyocytes.
  • any one or more of the first incubation, the second incubation, the third incubation, and the contacting is performed according to any of the embodiments as described herein, such as described in Section I.
  • the differentiation method is performed in suspension. In some embodiments, the contacting occurs on or about day, 2, 3, 4, 5, or 6. In some embodiments, the contacting occurs on or about day 4.
  • the dissociating agent is or comprises a cleavage enzyme.
  • the cleavage enzyme is a protease.
  • the protease is a recombinant enzyme that cleaves a peptide bond on the C-terminal side of a lysine or arginine residue.
  • the protease is an endopeptidase.
  • the endopeptidase is trypsin.
  • the protease is selected from the group consisting of trypsin, collagenase, chymotrypsin, elastase, hyaluronidase, papin, and dispase.
  • the collagenase is collagenase type I, collagenase type II, or collagenase type III. In some embodiments, the collagenase is collagenase type I, collagenase type II, or collagenase type III. In some embodiments, the protease is collagenase. In some embodiments, the protease is hyaluronidase. [0365] In some embodiments, the contacting occurs for a duration of about 15 minutes to about 2 hours.
  • the contacting occurs for a duration of about 30 minutes to about 90 minutes, about 30 minutes to about 75 minutes, about 40 minutes to about 60 minutes, about 40 minutes to about 55 minutes, about 40 minutes to about 50 minutes, about 45 minutes to 55 minutes, or about 45 minutes to about 50 minutes. In some embodiments, the contacting occurs for a duration of about 40 to about 55 minutes.
  • the contacting occurs for a duration that is sufficient to result in a population of dissociated cells.
  • the differentiation method further comprises, during and/or after the contacting, triturating the population of dissociated cells.
  • the population of dissociated cells has a viable cell density of between or between about 1 x 10 5 and 3 x 10 7 cells/mL, 2 x 10 5 and 2 x 10 7 cells/mL, 3 x 10 5 and 1 x 10 7 cells/mL, 4 x 10 5 and 9 x 10 6 cells/mL, 5 x 10 5 and 8 x 10 6 cells/mL, 6 x 10 5 and 7 x 10 6 cells/mL, 7 x 10 5 and 6 x 10 6 cells/mL, 8 x 10 5 and 5 x 10 6 cells/mL, 9 x 10 5 and 4 x 10 6 cells/mL, 9 x 10 5 and 3 x 10 6 cells/mL, 1 x 10 6 and 2 x 10 6 cells/mL, 1.1 x 10 6 and 1.9 x 10 6 cells/mL, 1.2 x 10 6 and 1.8 x 10 6 cells/mL, or 1.25 x 10 6 and 1.75 x 10 6 cells/
  • the population of dissociated cells has a viable cell density of between or between about 1 x 10 5 and 3 x 10 7 cells/mL, 2 x 10 5 and 2 x 10 7 cells/mL, 3 x 10 5 and 1 x 10 7 cells/mL, 4 x 10 5 and 9 x 10 6 cells/mL, 5 x 10 5 and 8 x 10 6 cells/mL, 6 x 10 5 and 7 x 10 6 cells/mL, 7 x 10 5 and 6 x 10 6 cells/mL, 8 x 10 5 and 5 x 10 6 cells/mL, 9 x 10 5 and 4 x 10 6 cells/mL, 9 x 10 5 and 3 x 10 6 cells/mL, 1 x 10 6 and 2 x 10 6 cells/mL, 1.1 x 10 6 and 1.9 x 10 6 cells/mL, 1.2 x 10 6 and 1.8 x 10 6 cells/mL, or 1.25 x 10 6 and 1.75 x 10 6 cells/mL on or
  • the population of cardiomyocytes differentiated from pluripotent stem cells wherein the population has a frequency of the presence of one or more cardiomyocyte markers that is or is at least 75%, 76%, 77%, 78%, 79% 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about any one of days 8 to 22, and wherein the differentiation is initiated on day 0.
  • the one or more cardiomyocyte markers comprises one or more markers selected from the group consisting of NKX2.5, cTNT, ACTN2, TNNI1, TNNI3, MYH6, MYH7, MYL2, and MYL7. In some embodiments, the one or more cardiomyocyte markers comprise MYH6 and MYL7. In some embodiments, the one or more cardiomyocyte markers comprise NKX2.5, cTNT, MYH6, and MYL7. In some embodiments, the one or more cardiomyocyte markers comprise NKX2.5 and cTNT.
  • the population has a frequency of NKX2.5+/cTNT+ cells, or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 9, 10, or 11.
  • the population has a frequency of NKX2.5+/cTNT+ cells, or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 10.
  • the population has a frequency of NKX2.5+/cTNT+ cells, or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 8, 9, 10, 11, or 12.
  • the population has a frequency of NKX2.5+/cTNT+ cells, or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 10.
  • the population has a frequency of NKX2.5+/cTNT+ cells that is or is at least 85% on or about day 10.
  • the population has a frequency of NKX2.5+/cTNT+ cells that is or is at least 90% on or about day 10.
  • the second aggregate was harvested on or about day 20, 21, or 22.
  • the cardiac cell therapy is administered as a suspension of cardiomyocytes or as an engineered tissue graft comprising cardiomyocytes and a matrix.
  • administration of the cardiac cell therapy comprises delivery into a subject’s heart tissue, optionally by intravenous injection, intraarterial injection, intracoronary injection, intramuscular injection, intraperitoneal injection, intramyocardial injection, trans-endocardial injection, trans-epicardial injection, and/or infusion.
  • the cardiac cell therapy comprises between about 5 x 10 8 and 1 x 10 10 engineered cardiomyocytes, inclusive of each. In some embodiments, the cardiac cell therapy comprises between about 1 x 10 9 and about 5 x 10 9 engineered cardiomyocytes, inclusive of each.
  • the cardiac cell therapy comprises a pharmaceutically acceptable carrier.
  • the subject is a non-human primate (NHP).
  • the subject is a human.
  • the cardiomyocytes are human or are human-derived.
  • the provided methods include administration of a cardiac cell therapy comprising human cardiomyocytes to a human subject.
  • the subject has a heart disease or condition.
  • Exemplary heart diseases and conditions can include diseases and conditions associated with the loss (e.g., death) of cardiomyocytes, such as myocardial infarction (MI).
  • MI myocardial infarction
  • the heart disease or condition is pediatric cardiomyopathy, age-related cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, chronic ischemic cardiomyopathy, peripartum cardiomyopathy, inflammatory cardiomyopathy, other cardiomyopathy, myocarditis, myocardial infarction, myocardial ischemic reperfusion injury, ventricular dysfunction, heart failure, congestive heart failure, coronary artery disease, end stage heart disease, atherosclerosis, ischemia, hypertension, restenosis, angina pectoris, rheumatic heart, arterial inflammation, or cardiovascular disease.
  • the heart disease or condition is myocardial infarction.
  • a subject’s endogenous cardiomyocytes have reduced physiological function, relative to normal cardiomyocyte physiological function (e.g. prior to the heart disease or condition). In some embodiments, a subject’s endogenous cardiomyocytes have reduced physiological function as a result of a MI. In some embodiments, the physiological function of a subject’s endogenous cardiomyocytes is reduced by between about 0.1% and about 50%, between about 1% and 25%, or between about 5% and 10%, inclusive of each, relative to normal cardiomyocyte physiological function. In some embodiments, the physiological function of a subject’s endogenous cardiomyocytes is reduced by between about 0.1% and about 1%, inclusive of each, relative to normal cardiomyocyte physiological function.
  • the physiological function of a subject’s endogenous cardiomyocytes is reduced by between about 0.5% and 5%, inclusive of each, relative to normal cardio myocyte physiological function. In some embodiments, the physiological function of a subject’s endogenous cardiomyocytes is reduced by between about 1% and 10%, inclusive of each, relative to normal cardiomyocyte physiological function. In some embodiments, the physiological function of a subject’s endogenous cardiomyocytes is reduced by between about 2% and 20%, inclusive of each, relative to normal cardiomyocyte physiological function. In some embodiments, the physiological function of a subject’s endogenous cardiomyocytes is reduced by between about 3% and 30%, inclusive of each, relative to normal cardiomyocyte physiological function.
  • the physiological function of a subject’s endogenous cardiomyocytes is reduced by between about 4% and 40%, inclusive of each, relative to normal cardiomyocyte physiological function. In some embodiments, the physiological function of a subject’s endogenous cardiomyocytes is reduced by between about 5% and 50%, inclusive of each, relative to normal cardiomyocyte physiological function.
  • the heart disease or condition is pediatric cardiomyopathy, age- related cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, chronic ischemic cardiomyopathy, peripartum cardiomyopathy, inflammatory cardiomyopathy, other cardiomyopathy, myocarditis, myocardial infarction, myocardial ischemic reperfusion injury, ventricular dysfunction, heart failure, congestive heart failure, coronary artery disease, end stage heart disease, atherosclerosis, ischemia, hypertension, restenosis, angina pectoris, rheumatic heart, arterial inflammation, or cardiovascular disease.
  • the heart disease or condition is myocardial infarction (MI).
  • Also provided herein are used of any of the cell therapies, any of the compositions, or any of the populations of cardiomyocytes or any of the second aggregates as described herein, in the manufacture of a medicament for treating a subject in need of a cardiac cell therapy.
  • the subject in need of a cardiac cell therapy has a heart disease or condition.
  • the heart disease or condition is pediatric cardiomyopathy, age-related cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, chronic ischemic cardiomyopathy, peripartum cardiomyopathy, inflammatory cardiomyopathy, other cardiomyopathy, myocarditis, myocardial infarction, myocardial ischemic reperfusion injury, ventricular dysfunction, heart failure, congestive heart failure, coronary artery disease, end stage heart disease, atherosclerosis, ischemia, hypertension, restenosis, angina pectoris, rheumatic heart, arterial inflammation, or cardiovascular disease.
  • the heart disease or condition is myocardial infarction.
  • the subject is a human.
  • the subject in need of a cardiac cell therapy has a heart disease or condition.
  • the heart disease or condition is pediatric cardiomyopathy, age-related cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, chronic ischemic cardiomyopathy, peripartum cardiomyopathy, inflammatory cardiomyopathy, other cardiomyopathy, myocarditis, myocardial infarction, myocardial ischemic reperfusion injury, ventricular dysfunction, heart failure, congestive heart failure, coronary artery disease, end stage heart disease, atherosclerosis, ischemia, hypertension, restenosis, angina pectoris, rheumatic heart, arterial inflammation, or cardiovascular disease.
  • the heart disease or condition is myocardial infarction.
  • the subject is a human.
  • cardiomyocyte compositions e.g., compositions comprising a population of cardiomyocytes, or compositions comprising an aggregate of cardiomyocyte cells, or a cardiac cell therapy comprising cardiomyocytes, are known and may be used in connection with the provided methods and compositions.
  • administration of the cardiac cell therapy comprises delivery into a subject’s heart tissue.
  • delivery into a subject’s heart tissue comprises intravenous injection, intraarterial injection, intracoronary injection, intramuscular injection, intraperitoneal injection, intramyocardial injection, trans-endocardial injection, transepicardial injection, and/or infusion.
  • delivery into a subject’s heart tissue comprises intramyocardial injection.
  • delivery into a subject’s heart tissue comprises trans-epicardial injection.
  • delivery into a subject’s heart tissue comprises trans-endocardial injection.
  • delivery into a subject’s heart tissue comprises delivery at the site of a myocardial infarct (MI). In some embodiments, delivery into a subject’s heart tissue comprises delivery near the site of a myocardial infarct (MI). In some embodiments, delivery into a subject’s heart tissue comprises delivery within about 1 mm, 5 mm, 10 mm, 20 mm, 30 mm, 40 mm, 50 mm, 60 mm, 70 mm, 80 mm, or 90 mm of the site of an injury, such as a MI.
  • delivery into a subject’s heart tissue comprises delivery within about 0.1 cm, 0.2 cm, 0.3 cm, 0.4 cm, 0.5 cm, 0.6 cm, 0.7 cm, 0.8 cm, 0.9 cm, 1.0 cm, 1.2 cm, 1.4 cm, 1.6 cm, 1.8 cm, 2.0 cm, 2.5 cm, 3.0 cm, 3.5 cm, 4.0 cm, 4.5 cm, or 5.0 of the site of an injury such as a MI.
  • the cardiac cell therapy is a pharmaceutical composition comprising cardiomyocytes and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier comprises cell culture medium.
  • the pharmaceutical acceptable carrier is cell culture medium.
  • the pharmaceutically acceptable carrier comprises between about 1 % and 20% of the total volume of the cardiac cell therapy composition.
  • the pharmaceutically acceptable carrier comprises between about 5% and 10% of the total volume of the cardiac cell therapy composition.
  • the pharmaceutically acceptable carrier comprises about 5% of the total volume of the cardiac cell therapy composition.
  • the pharmaceutically acceptable carrier comprises about 10% of the total volume of the cardiac cell therapy composition.
  • the pharmaceutically acceptable carrier comprises about 15% of the total volume of the cardiac cell therapy composition.
  • the cardiac cell therapy is a suspension of cardiomyocytes. In some embodiments, the cardiac cell therapy is an engineered tissue graft comprising cardiomyocytes.
  • the subject administered the cardiac cell therapy has a condition or disease, such as a heart condition or disease.
  • the heart condition or disease is selected from the group consisting of pediatric cardiomyopathy, age- related cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, chronic ischemic cardiomyopathy, peripartum cardiomyopathy, inflammatory cardiomyopathy, other cardiomyopathy, myocarditis, myocardial infarction (MI), myocardial ischemic reperfusion injury, ventricular dysfunction, heart failure, congestive heart failure, coronary artery disease, end stage heart disease, atherosclerosis, ischemia, hypertension, restenosis, angina pectoris, rheumatic heart, arterial inflammation, or cardiovascular disease.
  • the heart condition or disease is myocardial infarction (MI).
  • the cardiac cell therapy is administered to a subject to treat a MI (e.g. as a composition comprising cardio
  • the subject is a candidate for a left ventricular assist device (LVAD).
  • the subject is a candidate for a LVAD at the time of administration of the cardiomyocyte cell therapy.
  • the subject has a left ventricular assist device (LVAD).
  • the subject has a left ventricular assist device (LVAD) at the time of administration of the cardiac cell therapy.
  • the subject has a LVAD at the time of administration of the cardiac cell therapy. In some embodiments, the subject receives a LVAD at a time subsequent to administration of the cardiac cell therapy. In some embodiments, the subject receives a LVAD at a time subsequent to administration of the cardiac cell therapy.
  • the subject is a human. In some embodiments, the subject is a non-human primate (NHP). VI. ARTICLES OF MANUFACTURE AND KITS
  • compositions described herein such as any of the compositions as described in Section III.
  • the articles of manufacture may include a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container in some embodiments holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing a disease or condition.
  • the container has a sterile access port.
  • Exemplary containers include an intravenous solution bags, vials, including those with stoppers pierceable by a needle for injection, or bottles or vials for orally administered agents.
  • the label or package insert may indicate that the composition is used for treating a disease or condition.
  • the article of manufacture may include a first container with a composition contained therein, wherein the composition is a cardiac cell therapy (e.g. a pharmaceutical composition comprising a population of cardiomyocytes and a pharmaceutically acceptable carrier); (b) a second container comprising one or more additional agents.
  • a cardiac cell therapy e.g. a pharmaceutical composition comprising a population of cardiomyocytes and a pharmaceutically acceptable carrier
  • the article of manufacture may further include a package insert indicating that the compositions can be used to treat a particular condition, e.g., a heart disease or condition.
  • the article of manufacture may further include another or the same container comprising a pharmaceutically-acceptable buffer or excipient. It may further include other materials such as other buffers, diluents, filters, needles, and/or syringes.
  • treatment refers to complete or partial amelioration or reduction of a sign or symptom associated with a disease or condition being treated, e.g., a heart disease or condition.
  • the terms do not imply complete prevention or cure of any sign or symptom of the disease or condition, or of the disease or condition itself.
  • an “effective amount” of an agent e.g., a pharmaceutical formulation, cells, or composition, in the context of administration, refers to an amount effective, at dosages/amounts and for periods of time necessary, to achieve a desired result, such as a therapeutic or prophylactic result.
  • a “therapeutically effective amount” of an agent refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result, such as for treatment of a disease, condition, or disorder (e.g. rejection of a cardiomyocyte cell therapy), and/or pharmacokinetic or pharmacodynamics effect of the treatment.
  • the therapeutically effective amount may vary according to factors such as the disease or condition state, age, sex, and weight of the subject, and the populations of cells administered.
  • the provided methods involve administering the molecules, cells, and/or compositions at effective amounts, e.g., therapeutically effective amounts.
  • a “subject” or an “individual” is a mammal.
  • a “mammal” includes humans, non-human primates, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, rabbits, cattle, pigs, hamsters, gerbils, mice, ferrets, rats, cats, monkeys, etc.
  • the subject is human.
  • pluripotent cells refers to cells that can self-renew and proliferate while remaining in an undifferentiated state and that can, under the proper conditions, be induced to differentiate into specialized cell types.
  • Exemplary human stem cell lines include the H9 human embryonic stem cell line. Additional exemplary stem cell lines include those made available through the National Institutes of Health Human Embryonic Stem Cell Registry and the Howard Hughes Medical Institute HUES collection (as described in Cowan, C. A. et. al, New England J. Med. 350: 13. (2004), incorporated by reference herein in its entirety.)
  • pluripotent stem cells as used herein have the potential to differentiate into any of the three germ layers: endoderm, mesoderm, or ectoderm.
  • pluripotent stem cells also encompasses “induced pluripotent stem cells”, or “iPSCs”, a type of pluripotent stem cell derived from a non-pluripotent cell. Examples of parent cells include somatic cells that have been reprogrammed to induce a pluripotent, undifferentiated phenotype by various means.
  • Such "iPS” or “iPSC” cells can be created by inducing the expression of certain regulatory genes or by the exogenous application of certain proteins.
  • iPSCs are human induced pluripotent stem cells
  • riPSCs are rhesus induced pluripotent stem cells
  • Pluripotent stem cell characteristics refer to characteristics of a cell that distinguish pluripotent stem cells from other cells. The ability to give rise to progeny that can undergo differentiation, under the appropriate conditions, into cell types that collectively demonstrate characteristics associated with cell lineages from all of the three germinal layers (endoderm, mesoderm, and ectoderm) is a pluripotent stem cell characteristic. Expression or non-expression of certain combinations of molecular markers are also pluripotent stem cell characteristics.
  • human pluripotent stem cells express at least several, and in some embodiments, all of the markers from the following non-limiting list: S SEA-3, S SEA-4, TRA-1-60, TRA-1-81, TRA-2-49/6E, ALP, Sox2, E-cadherin, UTF-1, Oct4, Rexl, and Nanog.
  • Cell morphologies associated with pluripotent stem cells are also pluripotent stem cell characteristics.
  • multipotent or “multipotent cell” refers to a cell type that can give rise to a limited number of other particular cell types. For example, induced multipotent cells are capable of forming endodermal cells. Additionally, multipotent blood stem cells can differentiate itself into several types of blood cells, including lymphocytes, monocytes, neutrophils, etc.
  • oligopotent refers to the ability of an adult stem cell to differentiate into only a few different cell types.
  • lymphoid or myeloid stem cells are capable of forming cells of either the lymphoid or myeloid lineages, respectively.
  • spermatogonial stem cells are only capable of forming sperm cells.
  • totipotent means the ability of a cell to form an entire organism. For example, in mammals, only the zygote and the first cleavage stage blastomeres are totipotent.
  • non-pluripotent cells refer to mammalian cells that are not pluripotent cells. Examples of such cells include differentiated cells as well as progenitor cells. Examples of differentiated cells include, but are not limited to, cells from a tissue selected from bone marrow, skin, skeletal muscle, fat tissue and peripheral blood.
  • Exemplary cell types include, but are not limited to, fibroblasts, hepatocytes, myoblasts, neurons, osteoblasts, osteoclasts, and T-cells.
  • the starting cells employed for generating the induced multipotent cells can be non-pluripotent cells.
  • Differentiated cells include, but are not limited to, multipotent cells, oligopotent cells, unipotent cells, progenitor cells, and terminally differentiated cells.
  • a less potent cell is considered “differentiated” in reference to a more potent cell.
  • a "somatic cell” is a cell forming the body of an organism. Somatic cells include cells making up organs, skin, blood, bones and connective tissue in an organism, but not germ cells.
  • Cells can be from, for example, human or non-human mammals.
  • exemplary nonhuman mammals include, but are not limited to, mice, rats, cats, dogs, rabbits, guinea pigs, hamsters, sheep, pigs, horses, bovines, and non-human primates.
  • a cell is from an adult human or non-human mammal.
  • a cell is from a neonatal human, an adult human, or non-human mammal.
  • HLA human leukocyte antigen
  • MHC major histocompatibility complex
  • HLA-I major histocompatibility complex
  • HLA-I includes three proteins, HLA- A, HLA-B and HLA-C, which present peptides from the inside of the cell, and antigens presented by the HLA-I complex attract killer T-cells (also known as CD8+ T-cells or cytotoxic T cells).
  • P 2M P-2 microglobulin
  • HLA-II includes five proteins, HLA-DP, HLA-DM, HLA-DOB, HLA-DQ and HLA-DR, which present antigens from outside the cell to T lymphocytes. This stimulates CD4+ cells (also known as T-helper cells).
  • MHC human hemangiomaline
  • HLA-DOB human hemangiomaline
  • HLA-DQ human hemangiomaline
  • HLA-DR CD4+ cells
  • hypoimmunogenic cell herein is meant a cell that gives rise to a reduced immunological rejection response when transferred into an allogeneic host. In preferred embodiments, hypoimmunogenic cells do not give rise to an immune response.
  • hypo- immunogenic or “hypoimmune” refers to a significantly reduced or eliminated immune response when compared to the immune response of a parental (i.e., wild-type” or “wt”) cell prior to immunoengineering as outlined herein.
  • polypeptide and “protein” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length.
  • Polypeptides, including the antibodies and antibody chains and other peptides may include amino acid residues including natural and/or non-natural amino acid residues.
  • the terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like.
  • the polypeptides may contain modifications with respect to a native or natural sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes the vector as a selfreplicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced.
  • Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”
  • package insert is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.
  • a “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject.
  • a pharmaceutically acceptable carrier includes, but is not limited to, a cell culture medium, a buffer, excipient, stabilizer, or preservative.
  • composition refers to any mixture of two or more products, substances, or compounds, including cells. It may be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or any combination thereof.
  • a statement that a cell or population of cells is “positive” for a particular marker refers to the detectable presence on or in the cell of a particular marker, typically a surface marker.
  • a surface marker refers to the presence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is detectable by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control under otherwise identical conditions and/or at a level substantially similar to that for cell known to be positive for the marker, and/or at a level substantially higher than that for a cell known to be negative for the marker.
  • a statement that a cell or population of cells is “negative” for a particular marker refers to the absence of substantial detectable presence on or in the cell of a particular marker, typically a surface marker.
  • a surface marker refers to the absence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is not detected by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control under otherwise identical conditions, and/or at a level substantially lower than that for cell known to be positive for the marker, and/or at a level substantially similar as compared to that for a cell known to be negative for the marker.
  • a statement that a media is used “on day #” or that cells are cultured in a media “on day #” or that an incubation, a first, second, or third incubation, or a contacting, occurs “on day #” is not to be construed to require that the cells are cultured in that media for the entirety of that particular day, but instead allows for the media to be used for only a portion of the day.
  • both medias could be said to have been used “on” that particular day, even though the cells were cultured in only one of the two medias at a time, with the first media being used in a first part of the day and the second media being used for the remainder of the day.
  • population of dissociated cells refers to a population of cells comprising cells that dissociated from the first aggregate.
  • the term includes single cells in addition to one or more smaller aggregates or clusters of cells that were dissociated from the first aggregate.
  • a method of differentiating cardiomyocytes from pluripotent stem cells comprising: a) performing a first incubation comprising culturing a population of pluripotent stem cells under conditions to form a first aggregate, wherein the first incubation is initiated on day 0; b) contacting the first aggregate with a dissociating agent to form a population of dissociated cells; c) performing a second incubation comprising culturing the population of dissociated cells under conditions to aggregate the dissociated cells into a second aggregate; and
  • pluripotent stem cells are selected from the group consisting of induced pluripotent stem cells, embryonic stem cells, bone marrow- mesenchymal stem cells, cardiac tissue stem cells, and adipose tissue stem cells.
  • the population of pluripotent stem cells has a viable cell concentration of between or between about 1 x 10 5 and 3 x 10 7 cells/mL, 2 x 10 5 and 2 x 10 7 cells/mL, 3 x 10 5 and 1 x 10 7 cells/mL, 4 x 10 5 and 9 x 10 6 cells/mL, 5 x 10 5 and 8 x 10 6 cells/mL, 6 x 10 5 and 7 x 10 6 cells/mL, 7 x 10 5 and 6 x 10 6 cells/mL, 8 x 10 5 and 5 x 10 6 cells/mL, 9 x 10 5 and 4 x 10 6 cells/mL, 9 x 10 5 and 3 x 10 6 cells/mL, 1 x 10 6 and 2 x 10 6 cells/mL, 1.1 x 10 6 and 1.9 x 10 6 cells/mL, 1.2 x 10 6 and 1.8 x 10 6 cells/mL, or 1.25 x 10 6 and 1.
  • the population of pluripotent stem cells has a viable cell concentration of or of about 1.0 x 10 6 cells/mL, 1.05 x 10 6 cells/mL, 1.1 x 10 6 cells/mL, 1.15 x 10 6 cells/mL, 1.2 x 10 6 cells/mL, 1.25 x 10 6 cells/mL, 1.3 x 10 6 cells/mL, 1.35 x 10 6 cells/mL, 1.4 x 10 6 cells/mL, 1.45 x 10 6 cells/mL, 1.5 x 10 6 cells/mL, 1.55 x 10 6 cells/mL, 1.6 x 10 6 cells/mL, 1.65 x 10 6 cells/mL, 1.7 x 10 6 cells/mL, 1.75 x 10 6 cells/mL, 1.8 x 10 6 cells/mL, 1.85 x 10 6 cells/mL, 1.9 x 10 6 cells/mL, 1.95 x 10 6 cells/mL, 2.0 x
  • the population of pluripotent stem cells has a viable cell concentration of or of about 1.4 x 10 6 cells/mL, 1.45 x 10 6 cells/mL, 1.5 x 10 6 cells/mL, 1.55 x 10 6 cells/mL, 1.6 x 10 6 cells/mL, 1.65 x 10 6 cells/mL, 1.7 x 10 6 cells/mL, 1.75 x 10 6 cells/mL, or 1.8 x 10 6 cells/mL. 7.
  • the first incubation comprises culturing the population of pluripotent stem cells in a media comprising an inhibitor of glycogen synthase kinase 3 (GSK3)/activator of Wnt/p-catenin signaling.
  • GSK3 glycogen synthase kinase 3
  • the first incubation comprises culturing the population of pluripotent stem cells in a media comprising an inhibitor of Wnt/p-catenin signaling.
  • protease is a recombinant enzyme that cleaves a peptide bond on the C-terminal side of a lysine or arginine residue.
  • any one of embodiments 1-56 wherein the population of dissociated cells has a viable cell density of between or between about 1 x 10 5 and 3 x 10 7 cells/mL, 2 x 10 5 and 2 x 10 7 cells/mL, 3 x 10 5 and 1 x 10 7 cells/mL, 4 x 10 5 and 9 x 10 6 cells/mL, 5 x 10 5 and 8 x 10 6 cells/mL, 6 x 10 5 and 7 x 10 6 cells/mL, 7 x 10 5 and 6 x 10 6 cells/mL, 8 x 10 5 and 5 x 10 6 cells/mL, 9 x 10 5 and 4 x 10 6 cells/mL, 9 x 10 5 and 3 x 10 6 cells/mL, 1 x 10 6 and 2 x 10 6 cells/mL, 1.1 x 10 6 and 1.9 x 10 6 cells/mL, 1.2 x 10 6 and 1.8 x 10 6 cells/mL, or 1.25 x 10 6 and 1.
  • the second incubation comprises culturing the population of dissociated cells on or about day 2, 3, 4, 5, 6, or 7; and/or the second incubation comprises culturing the population of dissociated cells on or about days 2 and 3, on or about days 3 and 4, on or about days 4 and 5, on or about days 5 and 6, or on or about days 6 and 7.
  • the second aggregate that is formed is between or between about 25 and 200, 25 and 175, 25 and 150, 25 and 125, 30 and 200, 30 and 175, 30 and 150, 30 and 125, 35 and 200, 35 and 175, 35 and 150, 35 and 125, 40 and 200,
  • (b) has a diameter that is between or between about 5-50% of the diameter of the first aggregate immediately prior to being contacted with the dissociating agent;
  • (c) has a diameter that is at or about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the diameter of the first aggregate immediately prior to being contacted with the dissociating agent;
  • (d) has a diameter that is between or between about 5-25% of the diameter of the first aggregate immediately prior to being contacted with the dissociating agent.
  • (d) has a diameter that is between or between about 5-25% of the diameter of the first aggregate immediately prior to being contacted with the dissociating agent.
  • 67. The method of any one of embodiments 1-66, wherein, on the second day of the second incubation and/or at or about 24 hours after the contacting, the second aggregate has a diameter that is between or between about 5-25% of the diameter of the first aggregate immediately prior to being contacted with the dissociating agent.
  • the second aggregate has a diameter that is between or between about 25 and 200, 25 and 175, 25 and 150, 25 and 125, 30 and 200, 30 and 175, 30 and 150, 30 and 125, 35 and 200, 35 and 175, 35 and 150, 35 and 125, 40 and 200, 40 and 175, 40 and 150, 40 and 125, 45 and 200, 45 and 175, 45 and 150, 45 and 125, 50 and 200, 50 and 175, 50 and 150, 50 and 125, 55 and 200, 55 and 175, 55 and 150, 55 and 125, 60 and 200, 60 and 175, 60 and 150, 60 and 125, 65 and 200, 65 and 175, 65 and 150, 65 and 125, 70 and 200, 70 and 175, 70 and 150, 70 and 125, 75 and 200, 75 and 175, 75 and 150, or 75 and 125 pm.
  • the second aggregate has a diameter that is between or between about 25 and 200, 25 and 175, 25 and 150, 25 and 125, 30 and 200, 30 and 175, 30 and 150, 30 and 125, 35 and 200, 35 and 175, 35 and 150, 35 and 125, 40 and 200, 40 and 175, 40 and 150, 40 and 125, 45 and 200, 45 and 175, 45 and 150, 45 and 125, 50 and 200, 50 and 175, 50 and 150, 50 and 125, 55 and 200, 55 and 175, 55 and 150, 55 and 125, 60 and 200, 60 and 175, 60 and 150, 60 and 125, 65 and 200, 65 and 175, 65 and 150, 65 and 125, 70 and 200, 70 and 175, 70 and 150, 70 and 125, 75 and 200, 75 and 175, 75 and 150, or 75 and 125 pm.
  • the second aggregate has a diameter that is between or between about 25 and 300, 25 and 275, 25 and 250, 40 and 300, 40 and 275, 40 and 250, 50 and 300, 50 and 275, 50 and 250, 60 and 300, 60 and 275, 60 and 250, 70 and 300, 70 and 275, 70 and 250, 80 and 300, 80 and 275, or 80 and 250
  • the second aggregate has a diameter that is between or between about 25 and 300, 25 and 275, 25 and 250, 40 and 300, 40 and 275, 40 and 250, 50 and 300, 50 and 275, 50 and 250, 60 and 300, 60 and 275, 60 and 250, 70 and 300, 70 and 275, 70 and 250, 80 and 300, 80 and 275, or 80 and 250
  • the second aggregate has a diameter that is between or between about 25 and 400, 25 and 350, 25 and 300, 25 and 275, 25 and 250, 40 and 400, 40 and 350, 40 and 300, 40 and 275, 40 and 250, 50 and 400, 60 and 350, 50 and 300, 50 and 275, 50 and 250, 60 and 400, 60 and 350, 60 and 300, 60 and 275, 60 and 250, 70 and 400, 70 and 350, 70 and 300, 70 and 275, 70 and 250, 80 and 400, 80 and 350, 80 and 300, 80 and 275, or 80 and 250 m on the third day following the initiation of the second incubation and/or on the second day of the third incubation.
  • the second aggregate has a diameter that is between or between about 25 and 400, 25 and 350, 25 and 300, 25 and 275, 25 and 250, 40 and 400, 40 and 350, 40 and 300, 40 and 275, 40 and 250, 50 and 400, 60 and 350, 50 and 300, 50 and 275, 50 and 250, 60 and 400, 60 and 350, 60 and 300, 60 and 275, 60 and 250, 70 and 400, 70 and 350, 70 and 300, 70 and 275, 70 and 250, 80 and 400, 80 and 350, 80 and 300, 80 and 275, or 80 and 250 pm on day 7.
  • the second aggregate has a diameter that is between or between about 25 and 400, 25 and 350, 25 and 300, 25 and 275, 25 and 250, 40 and 400, 40 and 350, 40 and 300, 40 and 275, 40 and 250, 50 and 400, 60 and 350, 50 and 300, 50 and 275, 50 and 250, 60 and 400, 60 and 350, 60 and 300, 60 and 275, 60 and 250, 70 and 400, 70 and 350, 70 and 300, 70 and 275, 70 and 250, 80 and 400, 80 and 350, 80 and 300, 80 and 275, or 80 and 250 pun on day 8.
  • the first aggregate comprises a frequency of CD56+/PDGFRa+ cells that is or is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95%.
  • the first aggregate comprises a frequency of CD56+/PDGFRa+ cells that is or is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95%.
  • the first aggregate comprises a frequency of CD56+/PDGFRa+ cells that is or is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95%.
  • the first aggregate comprises a frequency of CD56+/PDGFRa+ cells that is or is about 80%, 81%, 92%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
  • culturing the second aggregate in the media comprising glucose comprises culturing the second aggregate in the media comprising glucose on one or more of any of about days 4-22.
  • the culturing the second aggregate in the media comprising glucose comprises culturing the second aggregate in the media comprising glucose on or about days 6-10 and 12- 22; and the culturing the second aggregate in the media comprising sodium lactate comprises culturing the second aggregate in the media comprising sodium lactate on or about days 10-12; or
  • the culturing the second aggregate in the media comprising glucose comprises culturing the second aggregate in the media comprising glucose on or about days 4-8 and 12-22; and the culturing the second aggregate in the media comprising sodium lactate comprises culturing the second aggregate in the media comprising sodium lactate on or about days 8-12; or
  • the culturing the second aggregate in the media comprising glucose comprises culturing the second aggregate in the media comprising glucose on or about days 5-9 and 13-22; and the culturing the second aggregate in the media comprising sodium lactate comprises culturing the second aggregate in the media comprising sodium lactate on or about days 9-13; or
  • the culturing the second aggregate in the media comprising glucose comprises culturing the second aggregate in the media comprising glucose on or about days 7-11 and 13- 22; and the culturing the second aggregate in the media comprising sodium lactate comprises culturing the second aggregate in the media comprising sodium lactate on or about days 11-13; or
  • the culturing the second aggregate in the media comprising glucose comprises culturing the second aggregate in the media comprising glucose on or about days 8-12 and 14- 22; and the culturing the second aggregate in the media comprising sodium lactate comprises culturing the second aggregate in the media comprising sodium lactate on or about days 12-14.
  • culturing the second aggregate in the media comprising sodium lactate comprises culturing the second aggregate in the media comprising sodium lactate on one or more of any of about days 9-13.
  • culturing the second aggregate in the media comprising sodium lactate comprises culturing the second aggregate in the media comprising sodium lactate on one or more of days 10-12.
  • culturing the second aggregate in the media comprising sodium lactate comprises culturing the second aggregate in the media comprising sodium lactate on or about days 10-12.
  • the population of cardiomyocytes comprises a frequency of NKX2.5+/cTNT+ cells that is or is at least 75%, 76%, 77%, 78%, 79% 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on one or more of any of days 8-12 and/or on the sixth day following the day of the contacting with the dissociating agent.
  • the population of cardiomyocytes comprises a frequency of NKX2.5+/cTNT+ cells that is or is at least 75%, 76%, 77%, 78%, 79% 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on one or more of any of days 9-11 and/or on the sixth day following the day of the contacting with the dissociating agent.
  • the population of cardiomyocytes comprises a frequency of NKX2.5+/cTNT+ cells that is or is at least 75%, 76%, 77%, 78%, 79% 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 10 and/or on the sixth day following the day of the contacting with the dissociating agent.
  • 114 The method of any one of embodiments 108-113, wherein the frequency of NKX2.5+/cTNT+ cells is or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on one or more of any of days 8-12 and/or on the sixth day following the day of the contacting with the dissociating agent.
  • the population of cardiomyocytes in the second aggregate comprises a frequency of mature cardiomyocytes that is higher as compared to a reference population of cardiomyocytes in an aggregate comprising cells that were not previously dissociated.
  • 121 The method of any one of embodiments 117-120, wherein the frequency of mature cardiomyocytes is based on a frequency of the presence of one or more mature cardiomyocyte markers in the population of cardiomyocytes in the second aggregate.
  • the one or more cardiomyocyte markers comprises one or more markers selected from the group consisting of NKX2.5, cTNT, ACTN2,
  • TNNI1, TNNI3, MYH6, MYH7, MYL2, and MYL7 are examples of MYH6, MYH7, MYL2, and MYL7.
  • cardiomyocyte markers comprise NKX2.5, cTNT, MYH6, and MYL7.
  • cardiomyocyte markers comprise NKX2.5 and cTNT.
  • the population of cardiomyocytes comprise one or more modifications that (a) reduce expression of one or more of CACNA1G, HCN4, and SLC8A C, and/or (b) increase expression of one or more of KCNJ2, TRDN, SRL, HRC, and CASQ2.
  • the population of cardiomyocytes comprise one or more modifications that: (i) increase expression of one or more tolerogenic factors; and/or (ii) reduce expression of one or more major histocompatibility complex (MHC) class I molecules and/or MHC class II molecules, relative to a population of cardiomyocytes that do not comprise the one or more modifications.
  • MHC major histocompatibility complex
  • the population of cardiomyocytes comprise one or more modifications that (a) reduce expression of one or more of CACNA1G, HCN4, and SLC8A1 and/or (b) increase expression of one or more of KCNJ2, TRDN, SRL, HRC, and CASQ2.
  • the population of cardiomyocytes comprise one or more modifications that (a) reduce expression of CACNA1G, HCN4, and SLC8A1 and/or
  • the population of cardiomyocytes comprise one or more modifications that (a) reduce expression of one or more of CACNA1H, HCN4, and SLC8A1 and/or (b) increase expression of one or more of KCNJ2, TRDN, SRL, HRC, and CASQ2.
  • a population of cardiomyocytes differentiating from pluripotent stem cells wherein the population has a frequency of the presence of one or more cardiomyocyte markers that is or is at least 75%, 76%, 77%, 78%, 79% 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about any one of days 8 to 22, and wherein the differentiating is initiated on day 0.
  • a population of cardiomyocytes differentiated from pluripotent stem cells wherein the population has a frequency of the presence of one or more cardiomyocyte markers that is or is at least 75%, 76%, 77%, 78%, 79% 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about any one of days 8 to 22, and wherein the differentiation is initiated on day 0.
  • cardiomyocyte markers comprises one or more markers selected from the group consisting of NKX2.5, cTNT, ACTN2, TNNI1, TNNI3, MYH6, MYH7, MYL2, and MYL7.
  • cardiomyocyte markers comprise NKX2.5, cTNT, MYH6, and MYL7.
  • 143 The population of any one of embodiments 136-138, wherein the population has a frequency of NKX2.5+/cTNT+ cells that is or is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 9, 10, or 11.
  • 145 The population of any one of embodiments 136-138, wherein the population has a frequency of NKX2.5+/cTNT+/MYH6+/MYL7+ cells that is or is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 8, 9, 10, 11, or 12.
  • 146 The population of any one of embodiments 136-138, wherein the population has a frequency of NKX2.5+/cTNT+ cells that is or is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 20, 21, or 22.
  • 147 The population of any one of embodiments 136-138, wherein the population has a frequency of NKX2.5+/cTNT+ cells that is or is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 22.
  • 149 The population of any one of embodiments 136-138, wherein the population has a frequency of NKX2.5+/cTNT+/MYH6+/MYL7+ cells that is or is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 20, 21, or 22.
  • NKX2.5+/cTNT+ cells or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 9, 10, or 11.
  • NKX2.5+/cTNT+ cells or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 10.
  • NKX2.5+/cTNT+ cells or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 8, 9, 10, 11, or 12.
  • NKX2.5+/cTNT+ cells or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 9, 10, or 11.
  • 155 The population of any one of embodiments 136-138 and 150, wherein the population has a frequency of NKX2.5+/cTNT+ cells, or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 10.
  • 156 The population of any one of embodiments 136-138 and 150, wherein the population has a frequency of NKX2.5+/cTNT+ cells, or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 8, 9, 10, 11, or 12.
  • 157 The population of any one of embodiments 136-138 and 150, wherein the population has a frequency of NKX2.5+/cTNT+ cells, or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 9, 10, or 11.
  • NKX2.5+/cTNT+ cells or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 10.
  • pluripotent stem cells are selected from the group consisting of induced pluripotent stem cells, embryonic stem cells, bone marrow-mesenchymal stem cells, cardiac tissue stem cells, and adipose tissue stem cells.
  • pluripotent stem cells are induced pluripotent stem cells.
  • protease is a recombinant enzyme that cleaves a peptide bond on the C-terminal side of a lysine or arginine residue.
  • the population of any one of embodiments 136-169, wherein the population of cardiomyocytes is at or about day 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22.
  • NKX2.5+/cTNT+ cells or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%.
  • NKX2.5+/cTNT+ cells or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%.
  • NKX2.5+/cTNT+ cells or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%.
  • a mixed population of cells comprising a first aggregate, a population of dissociated cells, and a second aggregate, wherein the first aggregate, the population of dissociated cells, and the second aggregate are formed by a method comprising: a) performing a first incubation comprising culturing a population of pluripotent stem cells under conditions to form a first aggregate, wherein the first incubation is initiated on day 0; b) contacting the first aggregate with a dissociating agent to form a population of dissociated cells; and c) performing a second incubation comprising culturing the population of dissociated cells under conditions to aggregate the dissociated cells into a second aggregate.
  • a mixed population of cells comprising a population of dissociated cells, a second aggregate, and a population of cardiomyocytes, wherein the first aggregate, the population of dissociated cells, and the second aggregate are formed by a method comprising: a) performing a first incubation comprising culturing a population of pluripotent stem cells under conditions to form a first aggregate, wherein the first incubation is initiated on day 0; b) contacting the first aggregate with a dissociating agent to form a population of dissociated cells; c) performing a second incubation comprising culturing the population of dissociated cells under conditions to aggregate the dissociated cells into a second aggregate; and d) performing a third incubation comprising culturing the second aggregate under conditions to differentiate the population of cells in the second aggregate into a population of cardiomyocytes.
  • a mixed population of cells comprising a population of dissociated cells and a second aggregate, wherein the population of dissociated cells and the second aggregate are formed by a method comprising: a) performing a first incubation comprising culturing a population of pluripotent stem cells under conditions to form a first aggregate, wherein the first incubation is initiated on day 0; b) contacting the first aggregate with a dissociating agent to form a population of dissociated cells; c) performing a second incubation comprising culturing the population of dissociated cells under conditions to aggregate the dissociated cells into a second aggregate; and d) performing a third incubation comprising culturing the second aggregate under conditions to differentiate the population of cells in the second aggregate into a population of cardiomyocytes.
  • a mixed population of cells comprising a population of cells dissociated from a second aggregate, wherein the population of cells dissociated from the second aggregate is formed by a method comprising: a) performing a first incubation comprising culturing a population of pluripotent stem cells under conditions to form a first aggregate, wherein the first incubation is initiated on day 0; b) contacting the first aggregate with a dissociating agent to form a population of dissociated cells; c) performing a second incubation comprising culturing the population of dissociated cells under conditions to aggregate the dissociated cells into a second aggregate; and d) performing a third incubation comprising culturing the second aggregate under conditions to differentiate the population of cells in the second aggregate into a population of cardiomyocytes, wherein the third incubation results in a population of cells dissociated from the second aggregate.
  • a mixed population of cells comprising a first aggregate and a population of dissociated cells, wherein the first aggregate and the population of dissociated cells are formed by a method comprising: a) performing a first incubation comprising culturing a population of pluripotent stem cells under conditions to form a first aggregate, wherein the first incubation is initiated on day 0; and b) contacting the first aggregate with a dissociating agent to form a population of dissociated cells.
  • a mixed population of cells comprising a second aggregate and a population of cells dissociated from the second aggregate, wherein the second aggregate and the population of cells dissociated from the second aggregate are formed by a method comprising: a) performing a first incubation comprising culturing a population of pluripotent stem cells under conditions to form a first aggregate, wherein the first incubation is initiated on day 0; b) contacting the first aggregate with a dissociating agent to form a population of dissociated cells; c) performing a second incubation comprising culturing the population of dissociated cells under conditions to aggregate the dissociated cells into a second aggregate; and d) performing a third incubation comprising culturing the second aggregate under conditions to differentiate the population of cells in the second aggregate into a population of cardiomyocytes, wherein the third incubation results in a population of cells dissociated from the second aggregate.
  • composition comprising the population of cardiomyocytes of any one of embodiments 135-178.
  • composition comprising the mixed population of cells of any one of embodiments 179-185.
  • a composition comprising a second aggregate, wherein the second aggregate is formed by a method comprising: a) performing a first incubation comprising culturing a population of pluripotent stem cells under conditions to form a first aggregate, wherein the first incubation is initiated on day 0; b) contacting the first aggregate with a dissociating agent to form a population of dissociated cells; and c) performing a second incubation comprising culturing the population of dissociated cells under conditions to aggregate the dissociated cells into a second aggregate.
  • composition of embodiment 192, wherein the cleavage enzyme is a protease.
  • composition of embodiment 193, wherein the protease is a recombinant enzyme that cleaves a peptide bond on the C-terminal side of a lysine or arginine residue.
  • composition of embodiment 203, wherein the second aggregate was harvested on or about day 4 or day 5.
  • composition of embodiment 206, wherein the population has a frequency of NKX2.5+/cTNT+ cells, or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 9, 10, or 11.
  • composition of any one of embodiments 206-210, wherein the second aggregate is cryopreserved is cryopreserved.
  • composition of embodiment 211, wherein the second aggregate was harvested on or about day 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22.
  • composition 213. The composition of embodiment 211 or embodiment 212, wherein the second aggregate is cryopreserved on or about day 6, 7, 8, 9, 10, 11, or 12. 214.
  • composition of any one of embodiments 186-213, wherein the composition comprises between about 5 x 10 8 and about 1 x IO 10 cardiomyocytes, inclusive of each.
  • a container comprising the composition of any one of embodiments 186-216.
  • a cardiac cell therapy comprising the population of cardiomyocytes of any one of embodiments 135-178.
  • a cardiac cell therapy comprising the mixed population of cardiomyocytes of any one of embodiments 179-185.
  • a cardiac cell therapy comprising the composition of any one of embodiments 186-216.
  • a method of treatment comprising administering the cardiac cell therapy of any one of embodiments 218-220 to a subject.
  • a method of treatment comprising administering the population of any one of embodiments 135-178 or the mixed population of any one of embodiments 179-185 to a subject.
  • a method of treatment comprising administering a cardiac cell therapy comprising the population of cardiomyocytes of any one of embodiments 135-178 to a subject.
  • a method of treatment comprising administering a cardiac cell therapy comprising the mixed population of any one of embodiments 179-185 to a subject.
  • a method of treatment comprising administering a cardiac cell therapy to a subject, wherein the cardiac cell therapy comprises a population of cardiomyocytes, wherein the population of cardiomyocytes is produced by a differentiation method comprising: a) performing a first incubation comprising culturing a population of pluripotent stem cells under conditions to form a first aggregate, wherein the first incubation is initiated on day 0; b) contacting the first aggregate with a dissociating agent to form a population of dissociated cells; c) performing a second incubation comprising culturing the population of dissociated cells under conditions to aggregate the dissociated cells into a second aggregate; and
  • protease is a recombinant enzyme that cleaves a peptide bond on the C-terminal side of a lysine or arginine residue.
  • the population of dissociated cells has a viable cell density of between or between about 1 x 10 5 and 3 x 10 7 cells/mL, 2 x 10 5 and 2 x 10 7 cells/mL, 3 x 10 5 and 1 x 10 7 cells/mL, 4 x 10 5 and 9 x 10 6 cells/mL, 5 x 10 5 and 8 x 10 6 cells/mL, 6 x 10 5 and 7 x 10 6 cells/mL, 7 x 10 5 and 6 x 10 6 cells/mL, 8 x 10 5 and 5 x 10 6 cells/mL, 9 x 10 5 and 4 x 10 6 cells/mL, 9 x 10 5 and 3 x 10 6 cells/mL, 1 x 10 6 and 2 x 10 6 cells/mL, 1.1 x 10 6 and 1.9 x 10 6 cells/mL, 1.2 x 10 6 and 1.8 x 10 6 cells/mL, or 1.25 x 10 6 and 1.
  • NKX2.5+/cTNT+ cells or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 9, 10, or 11.
  • NKX2.5+/cTNT+ cells or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 10.
  • NKX2.5+/cTNT+ cells or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 8, 9, 10, 11, or 12.
  • NKX2.5+/cTNT+ cells or MYH6+/MYL7+ cells, or NKX2.5+/cTNT+/MYH6+/MYL7+ cells, that is or is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% on or about day 10.
  • administration of the cardiac cell therapy comprises delivery into a subject’s heart tissue, optionally by intravenous injection, intraarterial injection, intracoronary injection, intramuscular injection, intraperitoneal injection, intramyocardial injection, trans-endocardial injection, trans-epicardial injection, and/or infusion.
  • the heart disease or condition is pediatric cardiomyopathy, age-related cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, chronic ischemic cardiomyopathy, peripartum cardiomyopathy, inflammatory cardiomyopathy, other cardiomyopathy, myocarditis, myocardial infarction, myocardial ischemic reperfusion injury, ventricular dysfunction, heart failure, congestive heart failure, coronary artery disease, end stage heart disease, atherosclerosis, ischemia, hypertension, restenosis, angina pectoris, rheumatic heart, arterial inflammation, or cardiovascular disease.
  • the heart disease or condition is pediatric cardiomyopathy, age-related cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, chronic ischemic cardiomyopathy, peripartum cardiomyopathy, inflammatory cardiomyopathy, other cardiomyopathy, myocarditis, myocardial infarction, myocardial ischemic reperfusion injury, ventricular dysfunction, heart failure, congestive
  • compositions of any one of embodiments 186-216 in the manufacture of a medicament for treating a subject in need of a cardiac cell therapy.
  • embodiment 255 wherein the heart disease or condition is pediatric cardiomyopathy, age-related cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, chronic ischemic cardiomyopathy, peripartum cardiomyopathy, inflammatory cardiomyopathy, other cardiomyopathy, myocarditis, myocardial infarction, myocardial ischemic reperfusion injury, ventricular dysfunction, heart failure, congestive heart failure, coronary artery disease, end stage heart disease, atherosclerosis, ischemia, hypertension, restenosis, angina pectoris, rheumatic heart, arterial inflammation, or cardiovascular disease.
  • embodiment 256 wherein the heart disease or condition is myocardial infarction.
  • a composition of any one of embodiments 186-216 for use in treating a subject in need of a cardiac cell therapy 260.
  • the composition for use of embodiment 259, wherein the subject in need of a cardiac cell therapy has a heart disease or condition.
  • composition for use of embodiment 260 wherein the heart disease or condition is pediatric cardiomyopathy, age-related cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, chronic ischemic cardiomyopathy, peripartum cardiomyopathy, inflammatory cardiomyopathy, other cardiomyopathy, myocarditis, myocardial infarction, myocardial ischemic reperfusion injury, ventricular dysfunction, heart failure, congestive heart failure, coronary artery disease, end stage heart disease, atherosclerosis, ischemia, hypertension, restenosis, angina pectoris, rheumatic heart, arterial inflammation, or cardiovascular disease.
  • the heart disease or condition is pediatric cardiomyopathy, age-related cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, chronic ischemic cardiomyopathy, peripartum cardiomyopathy, inflammatory cardiomyopathy, other cardiomyopathy, myocarditis, myocardial infarction, myocardial ischemic reperfusion injury,
  • composition for use of embodiment 260 or embodiment 261, wherein the heart disease or condition is myocardial infarction.
  • Example 1 Dissociation and re-aggregation of aggregated cells during differentiation of pluripotent stem cells into cardiomyocytes
  • the study design included (a) differentiating pluripotent stem cells into cardiomyocytes using six (6) vessels of cells having one of two CHIR09921 (CHIR) concentrations (5 pM for vessels 1-3 and 6 pM for vessels 4-6) and, (b) dissociation and reaggregation of aggregates at Differentiation Day (DD) 4 (DD4) or not.
  • CHIR09921 CHIR09921
  • DD4 Differentiation Day
  • the cell aggregates in vessels 2 and 5 underwent dissociation at DD4 and were allowed to re-aggregate, whereas the cells in vessels 1, 3, 4, and 6 did not undergo such dissociation and re-aggregation at DD4 and, instead, continued through the differentiation protocol See Table 1 below.
  • DDO Differentiation Day 0
  • PSCs were cultured in each of vessels 1-6 as described above and in Table 61
  • Cells in all vessels were cultured in DDO MCDB 131 media comprising other supplements, such as L-alanyl-L-glutamine (GlutaMax), B-27 Minus Insulin (Gibco), and CHIR09921 at 5 pM or 6 pM.
  • GlutaMax L-alanyl-L-glutamine
  • Gabco B-27 Minus Insulin
  • CHIR09921 L-alanyl-L-glutamine
  • a total cell counts in each vessel were calculated from duplicate 2mL samples (vessels A and B), see Table 2, following CHIR-Induction . Post- CHIR cell counts are shown in Table 3, displaying viable cell concentrations and viability percentages (97.65% and 97.85%).
  • DD1 DDO media was replaced with DD1 MCDB 131 media comprising L-alanyl-L-glutamine (GlutaMax) and B-27 Minus Insulin.
  • DD2 DD1 media was replaced with DD2 MCDB 131 media comprising 2.5uM of a WIKI4 inhibitor (a small molecule inhibitor of Wnt/B-catenin signaling), CTSTM B-27.
  • WIKI4 inhibitor a small molecule inhibitor of Wnt/B-catenin signaling
  • DD4 MCDB 131 media comprising L-alanyl-L-glutamine (GlutaMax) and CTSTM B-27.
  • Vessels 1, 3, 4, and 6 were dissociated on DD4.
  • the cells in vessels 2 and 5 were dissociated on DD4 by removing media, washing cells, dissociating using trypsin LE (TrypLE), and allowed to re-aggregate in MCDB 131 comprising with L-alanyl-L-glutamine (GlutaMax) and CTSTM B-27. Cell counts are shown in Table 4.
  • Cell counts, cell viability, and expression of CD56, PDGFRa, cTNT, and NKX2.5 were performed on days including DD4, DD10, and DD22.
  • the average cluster size of cell aggregates was determined on DDO, DD2, DD4, DD6, and DD8.
  • Results show a range of about 85-95% CD56/PDGFRa ++ cells from vessels 1-6 on DD4 following treatment with varying concentrations of CHIR at 5pM and 6 pM.
  • the cells in non-dissociated vessels were between about 57-75% cTNT/NKX2.5 ++ on DD10, while the cells in the dissociated vessels (vessels 2 and 5) that were dissociated and re-aggregated on DD4 were between about 91-98% cTNT/NKX2.5 ++ on DD10, see FIG. 1.
  • FIG. 2 A and FIG. 2B The effect of using a CHIR concentration of 5pM (FIG.2A) and 6 pM (FIG. 2B) with or without dissociation on DD4 on number viable cells is shown in FIG. 2 A and FIG. 2B.
  • Cells that were dissociated on DD4 exhibited a higher number of viable cells per mL on DD10 and DD20 with CHIR concentrations of either 5pM or 6 pM (FIGs. 2A and 2B).
  • the average cluster size of the cells on DDO, DD2, DD4, DD6, DD5, and DD7 (all cells), and DD8 (cells differentiated and re-aggregated on DD4) of cells that were (dotted line) or were not (solid line) dissociated on DD4 is shown in FIG. 4A, with CHIR at a concentration of 5 pM or 6 pM.
  • the average cluster size at the time of dissociation was approximately 500-600 pm.
  • Re-aggregated clusters grew larger in vessels with 6 pM CHIR (-300 pm by DD8) as compared to the re-aggregated clusters in vessels with 5 pM CHIR (-80 pm by DD8), as shown in FIG.
  • FIG. 5B shows viable cell counts and estimated cell size, and average cluster size of cells on DDO, DD2, DD3, DD4, DD7, and DD10, for cells that were (dashed line) or were not (solid line) dissociated and re-aggregated on DD4, which also demonstrates a smaller average cluster size for the cells that underwent dissociation and re-aggregation.
  • FIG. 5C also shows the average cluster size of cells on DD5 in addition to the other days noted above.

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Abstract

L'invention concerne des procédés de différenciation de cardiomyocytes à partir de cellules souches pluripotentes, par exemple, des cellules souches pluripotentes induites, qui impliquent la dissociation et la réagrégation d'agrégats cellulaires, ainsi que des populations de cardiomyocytes, des agrégats de cellules, et des compositions de ceux-ci, et des procédés de traitement et des utilisations impliquant de telles populations, agrégats ou compositions, par exemple pour traiter un sujet atteint d'une affection ou d'une maladie cardiaque.
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