Cardiomyocytes and methods of producing and purifying cardiomyocytes

Abstract

The invention provides methods for producing a culture of cardiomyocytes and cultures of cardiomyocytes. Exemplary methods of producing and cultures of cardiomyocytes include a population of cells including cells having spontaneous and periodic electrical activity, and/or including nodal, sino-atrial or pacemaker cells; immature cardiomyocytes (cardiomyoblasts); mature contractile cardiomyocytes; or a mixed population of two or more of such cells.

1 . A method for producing a culture of cardiomyocytes, comprising:
 A) Providing a culture of stem cells that are at least 90% confluent or the cells have overgrown to form multiple layers of cells, or proliferating stem cells until the cells are at least 90% confluent or the cells have overgrown to form multiple layers of cells; and   B) Inducing formation of mesoderm by contacting the overgrown stem cells with a bone morphogenic protein (BMP) receptor ligand and an fibroblast growth factor (FGF) receptor ligand for a period of about 2 to 15 days;   C) promoting cardiomyocyte formation by contacting mesoderm cells with neural cells or endoderm cells, or a neural cell or endoderm cell conditioned culture supernatant, for a period of about 1 to 21 days, thereby producing a culture of cardiomyocytes.   
     
     
         2 . The method of  claim 1 , wherein the cardiomyocytes comprise a population of cells comprising cells having spontaneous and periodic electrical activity. 
     
     
         3 . The method of  claim 2 , wherein the cells having spontaneous and periodic electrical activity comprise nodal, sino-atrial or pacemaker cells; immature cardiomyocytes (cardiomyoblasts); mature contractile cardiomyocytes; or a mixed population thereof. 
     
     
         4 . The method of  claim 1 , wherein the stem cells of step A) comprise a cell monolayer with 100% or more confluency. 
     
     
         5 . The method of  claim 1 , wherein the stem cells of step A) form a multilayer culture. 
     
     
         6 . The method of  claim 1 , wherein the stem cells of step A) have a density of about 150,000 to about 250,000 cells/cm 2 . 
     
     
         7 . The method of  claim 1 , wherein step B) further comprises addition of an acid. 
     
     
         8 . The method of  claim 7 , wherein the acid comprises lactic, citric, fumaric, ascorbic, folic, malic, succinic, oxalacetic, or ketoglutaric acid. 
     
     
         9 . The method of  claim 1 , wherein step B) further comprises subjecting the mesoderm cells to hypoxia. 
     
     
         10 . The method of  claim 1 , wherein the stem cells, mesoderm cells or cardiomyocytes are subjected to an oxygen concentration of about 0.5 to 2.0%. 
     
     
         11 . The method of  claim 1 , wherein step B) further comprises addition of an agent that inhibits cell death or apoptosis. 
     
     
         12 . The method of  claim 11 , wherein the agent comprises selenium, lithium; ascorbic acid or ascorbate or superoxide dismutase (SOD). 
     
     
         13 . The method of  claim 12 , wherein the selenium comprises sodium selenite. 
     
     
         14 . The method of  claim 12 , wherein the selenium is at a concentration of 1 to 20 ng/ml, or 0.5 to 50 ng/ml. 
     
     
         15 . The method of  claim 12 , wherein the lithium comprises lithium carbonate. 
     
     
         16 . The method of  claim 12 , wherein the lithium is at a concentration of 1 to 20 ng/ml, or 0.5-50 ng/ml. 
     
     
         17 . The method of  claim 12 , wherein the ascorbic acid or ascorbate comprises sodium or calcium ascorbate. 
     
     
         18 . The method of  claim 12 , wherein the ascorbic acid or ascorbate is at a concentration of 0.1 to 20 ug/ml, or 1 ug/ml. 
     
     
         19 . The method of  claim 12 , wherein the superoxide dismutase (SOD) is at a concentration of 1 to 100 u/ml, or 1 to 10 u/ml. 
     
     
         20 . The method of  claim 1 , wherein step B) further comprises contacting the mesoderm cells with a wingless-int-1 (Wnt) family member. 
     
     
         21 . The method of  claim 20 , wherein the wingless-int-1 (Wnt) family member is Wnt-5a. 
     
     
         22 . The method of  claim 20 , wherein the Wnt-5a is at a concentration of 1 to 10 ng/ml. 
     
     
         23 . The method of  claim 1 , wherein following step B), a portion of the cells express Nkx2.5/Csx marker. 
     
     
         24 . The method of  claim 1 , wherein the neural cells of step C) are produced by addition of a retinoic acid receptor ligand to the mesoderm cells of step B). 
     
     
         25 . The method of  claim 24 , wherein the retinoic acid receptor ligand comprises retinoic acid. 
     
     
         26 . The method of  claim 1 , wherein the retinoic acid is at a concentration of 1-20 mMol, or 5-10 mMol. 
     
     
         27 . The method of  claim 1 , wherein step C) comprises contact with neural cells or endoderm cells for about 12-48 hours. 
     
     
         28 . The method of  claim 1 , wherein the neural cells of step C) comprise pre-differentiated or primary neural cell cultures or neural progenitor cell cultures. 
     
     
         29 . The method of  claim 1 , wherein the endoderm cells of step C) are produced by addition of a BMP receptor ligand to the mesoderm cells of step B). 
     
     
         30 . The method of  claim 1 , wherein the endoderm cells of step C) comprise a primary culture of endo-epithelial cells. 
     
     
         31 . The method of  claim 1 , wherein step C) further comprises contacting the mesoderm cells with insulin; a thyroid hormone, or an insulin-like growth factor (IGF). 
     
     
         32 . The method of  claim 31 , wherein the insulin is at a concentration of 5 to 50 μg/ml, or 15 μg/ml. 
     
     
         33 . The method of  claim 31 , wherein the thyroid hormone is at a concentration of 1 to 40 ng/ml, or 20 ng/ml. 
     
     
         34 . The method of  claim 31 , wherein the thyroid hormone is T3/4. 
     
     
         35 . The method of  claim 31 , wherein the IGF is IGF-1. 
     
     
         36 . The method of  claim 31 , wherein the IGF is at a concentration of 5 to 50 ng/ml, or 10 ng/ml. 
     
     
         37 . The method of  claim 1 , wherein the cardiomyocytes express a marker selected from a GATA binding family transcription factor (GATA binding protein), MEF2 (Myocyte Enhancer Factor 2), HAND (heart and neural crest derivatives), Irx, Tbx, and HRT families of transcription factors, SRF (serum response factor), Isl1 (Islet1), LIM (named from the Lin-11, Isl-1 and Mec-3 genes) and alpha-actin. 
     
     
         38 . The method of  claim 1 , wherein the FGF receptor ligand comprises FGF basic (FGF2, bFGF), acidic FGF (FGF1, aFGF), or a combination thereof. 
     
     
         39 . The method of  claim 38 , wherein the FGF is between about 2 to 200 ng/ml, or 5 to 20 ng/ml. 
     
     
         40 . The method of  claim 1 , wherein the BMP receptor ligand comprises BMP4, BMP2, BMP7 or any combination thereof. 
     
     
         41 . The method of  claim 40 , wherein the BMP is between about 0.1 to 100 ng/ml, or 0.5 to 10 ng/ml. 
     
     
         42 . The method of  claim 1 , wherein during or following steps A), B), or C) a fresh media is added. 
     
     
         43 . The method of  claim 42 , wherein the fresh media added during step A) comprises a stem cell media. 
     
     
         44 . The method of  claim 42 , wherein the fresh media added during step B) comprises a cardio cell media. 
     
     
         45 . The method of  claim 44 , wherein the cardio cell media comprises a basal media and one or more of the following supplements: human albumin, essential amino acids, non essential amino acids, L-glutamine, a thyroid hormone, insulin, transferrin, ethanolamine, sodium selenite, a hydrosoluble vitamin, a liposoluble vitamin and B27 supplement. 
     
     
         46 . The method of  claim 44 , wherein the basal media comprises DMEM, F12 or DMEM:F12. 
     
     
         47 . The method of  claim 46 , wherein the DMEM:F12 is in a ratio of about 1:1. 
     
     
         48 . The method of  claim 1 , wherein a population of beating contractile cardiomyocytes is produced. 
     
     
         49 . The method of  claim 48 , wherein the frequency of beating is modulated by modulating culture media pH, temperature, or a modulator drug. 
     
     
         50 . The method of  claim 49 , wherein the modulator drug is catecholamine, a calcium channel blocker, or potassium. 
     
     
         51 . The method of  claim 1 , further comprising step D), contacting cardiomyocytes with a cardioplegic solution or treatment. 
     
     
         52 . The method of  claim 51 , wherein the cardioplegic solution or treatment induces diastolic arrest of beating cardiomyocytes. 
     
     
         53 . The method of  claim 51 , wherein the cardioplegic solution or treatment induces reduces energy requirement of beating cardiomyocytes. 
     
     
         54 . The method of  claim 51 , wherein the cardioplegic solution or treatment inhibits hypoxia induced damage of beating cardiomyocytes. 
     
     
         55 . The method of  claim 51 , wherein the cardioplegic solution comprises a mixture of one or more of: KCl (10-20 mM); MgCl 2  (10 mM). 
     
     
         56 . The method of  claim 51 , wherein the cardioplegic solution further comprises one or more of CaCl2 (1.2 mM); puerarin (0.5 mM), or Nifedipine (1-10 uM). 
     
     
         57 . The method of  claim 51 , wherein the cardioplegic solution comprises a tris(hydroxymethyl)aminomethane or Hanks balanced salt solution adjusted to a pH of approximately 8.6. 
     
     
         58 . The method of  claim 51 , wherein the cardioplegic solution further comprises a mixture of L-Monosodium Glutamate Monohydrate and L-Monosodium Aspartate Monohydrate. 
     
     
         59 . The method of  claim 58 , wherein the mixture comprises about 4.277% of L-Monosodium Glutamate Monohydrate and about 3.923% of L-Monosodium Aspartate Monohydrate. 
     
     
         60 . The method of  claim 1  or  51 , further comprising steps D) or E), isolating immature cardiomyocytes (cardioblasts) prior to beating. 
     
     
         61 . The method of  claim 1  or  51 , further comprising steps D) or E), isolating mature contractile cardiomyocytes. 
     
     
         62 . The method of  claim 1  or  51 , further comprising steps D) or E), preserving, freezing or storing the cardiomyocytes. 
     
     
         63 . The method of  claim 1  or  51 , further comprising steps D) or E), enriching or selecting for cardiomyocytes. 
     
     
         64 . The method of  claim 63 , wherein enriching or selecting for cardiomyocytes comprises subjecting the cells to a treatment that requires anaerobic metabolism so that cells unable to survive by anaerobic metabolism senesce or die. 
     
     
         65 . The method of  claim 63 , wherein enriching or selecting for cardiomyocytes comprises exposure to hypoxia, contact with lactic acid or contact with a cardioplegic solution or treatment. 
     
     
         66 . The method of  claim 63 , wherein enriching or selecting for cardiomyocytes comprises identifying cardiomyocytes and removing the identified cardiomyocytes. 
     
     
         67 . The method of  claims 60  to  63 , further comprising steps E) or F), recovering the enriched or selected cardiomyocytes. 
     
     
         68 . The method of  claims 60  to  63 , further comprising steps E) or F), recovering the enriched or selected cardiomyocytes and distributing the enriched or selected cardiomyocytes in a cell culture dish, plate, vial, tube, flask or bottle. 
     
     
         69 . The method of  claims 60  to  63 , further comprising steps E) or F), recovering the enriched or selected cardiomyocytes and freezing the enriched or selected cardiomyocytes in a solution. 
     
     
         70 . A cardiomyocyte population produced by the method of any of  claims 1  to  69 . 
     
     
         71 . A cardiomyocyte population comprising nodal, sino-atrial or pacemaker cells, mature contractile cardiomyocytes, immature cardiomyocytes (cardioblasts), or a mixed population thereof produced by the method of any of  claims 1  to  69 . 
     
     
         72 . A kit comprising a cardiomyocyte population produced by the method of any of  claims 1  to  69 . 
     
     
         73 . A method for identifying a cardioactive agent, comprising:
 A) contacting a cardiomyocyte population produced by the method of any of  claims 1  to  69  with a test agent; and   B) determining if the test agent modulates an activity or function of cardiomyocytes within the population, wherein modulating an activity or function of cardiomyocytes within the population identifies the test agent as a cardioactive agent.   
     
     
         74 . The method of  claim 73 , wherein the activity or function comprises contraction or beating. 
     
     
         75 . The method of  claim 73 , wherein the activity or function comprises production of a metabolic product or intracellular enzyme. 
     
     
         76 . The method of  claim 75 , wherein the metabolic product is one or more of urea, creatine or CO2. 
     
     
         77 . The method of  claim 75 , wherein the intracellular enzyme is one or more of lactate dehydrogenase, creatine phosphokinase (CPK), creatine kinase (CK) or troponin. 
     
     
         78 . The method of  claim 73 , wherein the activity or function comprises cellular apoptosis, necrosis, death; or dedifferentiation, maturation, division. 
     
     
         79 . A method for treating a subject in need of increased numbers or function of cardiomyocytes, comprising transplanting into the subject a cardiomyocyte population produced by the method of any of  claims 1  to  69 . 
     
     
         80 . A method for treating a subject in need of increased numbers or function of cardiomyocytes, comprising grafting a cardiomyocyte population produced by the method of any of  claims 1  to  69  into the heart of a subject. 
     
     
         81 . A cell culture of immature cardiomyocytes (cardiomyblasts), wherein 50% or more of said culture comprises immature cardiomyocyte (cardiomyblast) cells, and wherein no more than about 30% of said cells beat or contract. 
     
     
         82 . The cell culture of  claim 81 , wherein 60% or more of said culture comprises immature cardiomyocyte (cardiomyblast) cells. 
     
     
         83 . The cell culture of  claim 81 , wherein 70% or more of said culture comprises immature cardiomyocyte (cardiomyblast) cells. 
     
     
         84 . The cell culture of  claim 81 , wherein 80% or more of said culture comprises immature cardiomyocyte (cardiomyblast) cells. 
     
     
         85 . The cell culture of  claim 81 , wherein 90% or more of said culture comprises immature cardiomyocyte (cardiomyblast) cells. 
     
     
         86 . The cell culture of  claim 81 , wherein no more than about 25% of said cells beat or contract. 
     
     
         87 . The cell culture of  claim 81 , wherein no more than about 20% of said cells beat or contract. 
     
     
         88 . The cell culture of  claim 81 , wherein no more than about 15% of said cells beat or contract. 
     
     
         89 . The cell culture of  claim 81 , wherein no more than about 10% of said cells beat or contract. 
     
     
         90 . The cell culture of  claim 81 , wherein no more than about 5% of said cells beat or contract. 
     
     
         91 . A cell culture of immature cardiomyocytes (cardiomyblasts), wherein 50% or more of said cell culture comprises immature cardiomyocyte (cardiomyblast) cells, and wherein 75% or more of said immature cardiomyocyte (cardiomyblast) cells survive anaerobic conditions for 30, 60 or more minutes. 
     
     
         92 . A cell culture of immature cardiomyocytes (cardiomyblasts), wherein 50% or more of said culture comprises immature cardiomyocyte (cardiomyblast) cells, and wherein no more than about 25% of said immature cardiomyocyte (cardiomyblast) cells die when subjected to anaerobic conditions for 30, 60 or more minutes. 
     
     
         93 . The cell culture of  claim 92 , wherein no more than about 20% of said immature cardiomyocyte (cardiomyblast) cells die when subjected to anaerobic conditions for 30, 60 or more minutes. 
     
     
         94 . The cell culture of  claim 92 , wherein no more than about 15% of said immature cardiomyocyte (cardiomyblast) cells die when subjected to anaerobic conditions for 30, 60 or more minutes. 
     
     
         95 . The cell culture of  claim 92 , wherein no more than about 10% of said immature cardiomyocyte (cardiomyblast) cells die when subjected to anaerobic conditions for 30, 60 or more minutes. 
     
     
         96 . The cell culture of  claim 92 , wherein no more than about 5% of said immature cardiomyocyte (cardiomyblast) cells die when subjected to anaerobic conditions for 30, 60 or more minutes. 
     
     
         97 . The cell culture of  claim 92 , wherein said anaerobic condition comprises an atmosphere of 0%-2% oxygen. 
     
     
         98 . The cell culture of  claim 92 , wherein the immature cardiomyocyte (cardiomyblast) cells proliferate. 
     
     
         99 . The cell culture of  claim 92 , wherein no more than about 30% of said immature cardiomyocyte (cardiomyblast) cells exhibit a function associated with contractile cardiomyocytes. 
     
     
         100 . The cell culture of any of  claim 81 ,  91  or  92 , further comprising a substrate to which the cells are attached. 
     
     
         101 . The cell culture of  claim 100 , wherein the substrate comprises a multiwell plate or dish having disposed thereon the cardiomyocyte (cardiomyblast) cells in one or more of said wells. 
     
     
         102 . A kit comprising the immature cardiomyocyte (cardiomyblast) cells of any of  claim 81 ,  91  or  92 .