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WO2012045096A2 - Compositions et méthodes de production de cellules progénitrices cardiaques - Google Patents

Compositions et méthodes de production de cellules progénitrices cardiaques Download PDF

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WO2012045096A2
WO2012045096A2 PCT/US2011/054662 US2011054662W WO2012045096A2 WO 2012045096 A2 WO2012045096 A2 WO 2012045096A2 US 2011054662 W US2011054662 W US 2011054662W WO 2012045096 A2 WO2012045096 A2 WO 2012045096A2
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progenitor cell
oct
cardiac
cell
cells
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WO2012045096A3 (fr
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Jeremy J. Mao
Kimi Kong
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Columbia University in the City of New York
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Columbia University in the City of New York
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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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/20—Transition metals
    • C12N2500/24—Iron; Fe chelators; Transferrin
    • C12N2500/25—Insulin-transferrin; Insulin-transferrin-selenium
    • 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
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2301—Interleukin-1 (IL-1)
    • 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
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2303—Interleukin-3 (IL-3)
    • 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
    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention generally relates to cardiac progenitor cells.
  • the POU domain transcription factor Oct-4 (also termed Pou5f1) is required for the maintenance of pluripotency of embryonic stem cells (ESCs).
  • the POU domain transcription factor Oct-4 is developmentally regulated in mouse ontogeny. Maternal Oct-4 RNA and protein are present in fertilized oocytes until the two-cell stage. Zygotic Oct-4 gene expression starts at the four- to eight-cell stage. During early cleavage, uniform amounts of Oct-4 RNA are found in all blastomeres, but the level decreases in the outer cells of the morula as they polarize and form the trophectoderm.
  • Oct-4 RNA and protein levels are low in the epithelial cell layer and become undetectable one day later.
  • Oct-4 protein expression is maintained in the inner cell mass (ICM) of the blastocyst.
  • Embryonic stem cells are derived from the ICM of the pre- implantation embryo, and are roughly equivalent to epiblast in vivo.
  • Recent studies have shown that endogenous Oct-4 mRNA and protein expression continues for at least four days in ESCs upon induction of differentiation, which may mirror the expression of Oct-4 in epiblasts during early embryogenesis.
  • it was shown that the presence of Oct-4 during the early stage of differentiation ESCs (day 0 to day 2 post- differentiation) is crucial for successful hematopoiesis.
  • Previous reports have shown that the precise level of Oct-4 tightly regulates the differentiation capacity of ESCs.
  • Oct-4 expression level in ESCs can re-direct the cell fate of ESCs toward either primitive endodermal and early mesodermal differentiation (Oct-4 over-expression) or trophectoderm differentiation (Oct-4 under-expression).
  • Oct-4 is transcriptionally regulated by itself or by Nanog, Sox2 and FoxD3 through various feedback regulatory loops. Other than regulation of transcription, Oct-4 is also regulated at the post-translational level. Oct-4 protein has been shown to be targeted by an E3 Ub ligase, Wwp2, for degradation. [0009] Under convention culture conditions, it has been reported that Oct-4 has no effect on other non-cardiogenic mesodermal development.
  • compositions and methods for generating cardiac progenitor cells via a novel culture medium, manipulation of Oct-4 levels, or a combination thereof is the provision of a compositions and methods for generating cardiac progenitor cells via a novel culture medium, manipulation of Oct-4 levels, or a combination thereof.
  • the culture medium includes at least one of interleukin-3, interleukin-1 , insulin, and transferin. In some configurations, the culture medium includes interleukin-3, interleukin-1 , insulin, and transferin. In some configurations, the culture medium contains interleukin-3 at a concentration of at least about 0.5 ng/ml up to about 50 ng/ml. In one embodiment, the culture medium contains interleukin-3 at a concentration of about 5 ng/ml. In some configurations, the culture medium contains interleukin-1 at a concentration of at least about 0.5 ng/ml up to about 50 ng/ml.
  • the culture medium contains interleukin-1 at a concentration of about 5 ng/ml. In some configurations, the culture medium contains insulin at a concentration of at least about 1 ng/ml up to about 100 ng/ml. In one embodiment, the culture medium contains insulin at a concentration of about 10 ng/ml. In some configurations, the culture medium contains transferin at a concentration of at least about 20 ng/ml up to about 2 g/ml. In one embodiment, the culture medium contains transferin at a concentration of about 200 ng/ml. In some embodiments, the culture medium includes FBS or monothioglycerol. In some configurations the culture medium includes FBS. In some configurations the culture medium includes monothioglycerol. In some
  • the culture medium includes FBS and monothioglycerol.
  • Another aspect provides a method of forming a cardiac progenitor cell.
  • the method includes contacting a progenitor cell and a culture medium described herein; and culturing the progenitor cell so as to form a cardiac progenitor cell.
  • the method includes increasing a level of Oct-4 in the progenitor cell.
  • the method includes increasing a level of Oct-4 in a progenitor cell; and culturing the progenitor cell so as to form a cardiac progenitor cell.
  • increasing the level of Oct-4 in the progenitor cell includes introducing exogenous Oct-4 to the progenitor cell. In some embodiments, increasing the level of Oct-4 in the progenitor cell includes increasing expression of endogenous Oct-4 in the progenitor cell. In some embodiments, increasing the level of Oct-4 in the progenitor cell includes introducing exogenous Oct-4 to the progenitor cell and increasing expression of endogenous Oct-4 in the progenitor cell.
  • the method includes detecting a level of Oct-4 in the progenitor cell.
  • the level of Oct-4 in the progenitor cell is increased to be about the level of Oct-4 in embryonic stem cell line CGR8 cultured under equivalent conditions.
  • the level of Oct-4 in the progenitor cell is increased so as promote expression of early mesodermal marker Brachyury.
  • the level of Oct-4 in the progenitor cell is increased prior to 1 1 days of culturing the progenitor cell.
  • the number of formed cardiac progenitor cells is at least about 100% greater than the number of cardiac progenitor cells formed under culture conditions not comprising at least one of interleukin-3, interleukin-1 , insulin, and transferin. In some embodiments of the method, the number of formed cardiac progenitor cells is at least about 100% greater than the number of cardiac progenitor cells formed in the absence of increasing the level of Oct-4.
  • the number of formed cardiac progenitor cells is at least about 100% greater than the number of cardiac progenitor cells formed under culture conditions not comprising at least one of interleukin-3, interleukin-1 , insulin, and transferin and the number of cardiac progenitor cells formed in the absence of increasing the level of Oct-4.
  • the cardiac progenitor cell displays one or more cellular markers selected from the group consisting of Nkx2.5, Tnnt2, Myh7, Myh6, and Brachyury.
  • the progenitor cell is an embryonic progenitor cell. In some embodiments of the method, the progenitor cell is an induced pluripotent stem cell. In some embodiments of the method, progenitor cells include embryonic progenitor cells and induced pluripotent stem cells. In some embodiments of the method, the progenitor cell is a human embryonic progenitor cell. In some embodiments of the method, the progenitor cell is a human induced pluripotent stem cell. In some embodiments of the method, progenitor cells include human embryonic progenitor cells and human induced pluripotent stem cells.
  • a first progenitor cell and a second progenitor cell are co-cultured.
  • the first progenitor cell comprising a embryonic progenitor cell and the second progenitor cell comprising an endothelial stem/progenitor cell (HUVEC).
  • embryonic progenitor cells are present at about 90% to 99% and the HUVECs are present at about 10% to about 1 % in the progenitor cell culture.
  • the cardiac progenitor cell comprises a contracting embryoid body.
  • FIG. 1 is a series of images showing Formation of contracting cardiogenic progenitors from day 9 CGR8 and ZHBTc4 EBs when cultured in cardiogenic
  • FIG. 1A and FIG. 1 B shows single frames taken from videos showing that beating cells/focus (white arrow heads) derived from wt CGR8 ESCs when cultured in previously established cardiogenic differentiating liquid medium.
  • FIG. 1A 20X objective
  • FIG. 1 B 10X objective.
  • FIG. 1 C shows RT-PCR analyses of cardiogenic gene expression markers in cells collected from day 9 EBs derived from wt CGR8 or ZHBTc4 ESCs, with or without tet-treatment. 18S was used as an internal control.
  • ANF-atrial natriuretic factor Nkx 2.5- cardiac specific homoebox protein (Csx); Myh6- cardiac a- myosin heavy chain; Myh7- cardiac ⁇ -myosin heavy chain. Tet- tetracycline. Untx- untreated. TDO- tet-treated at day 0, before differentiation induction. TD2- tet-treated at day 2 of differentiation. TD4- tet-treated at day 4 of differentiation.
  • FIG. 2 is a pair of images and a bar graph showing formation of contracting cardiogenic progenitors from day 1 1 CGR8 EBs when cultured in semi-solid
  • FIG. 3 is a series of images and a bar graph showing Oct-4 protein expression levels.
  • FIG. 3A shows immunoblot analysis of Oct-4 expression in CGR8, tet-treated and untreated ZHTc6 ESCs before and after induction for differentiation, ⁇ - actin protein expression is shown to demonstrate equal loading of lanes.
  • FIG. 3B shows quantification of Oct-4 protein expression levels in undifferentiated (DO, with LIF) or differentiating (D1 , 24 hrs without LIF) CGR8 and ZHT6 ESCs. Levels of Oct-4 protein in wt CGR8 cells were set at 100% for comparison against Oct-4 protein levels in ZHTc6 cells. Oct-4 expression levels were normalized by the expression of ⁇ -actin in the same sample.
  • ZHTc6 Tet- ZHTc6 cultured with tet before and after induction for differentiation ZHTc6 24h- ZHTc6 ESCs removed from tet 24 hr before induction of differentiation and remained tet-free.
  • FIG. 4 is a pair of bar graphs showing formation of contracting cardiogenic EBs from day 1 1 CGR8 and ZHTc6 EBs when cultured in the modified cardiogenic differentiating condition B.
  • FIG. 4A shows ZHTc6 when cultured in cardiogenic differentiating condition B, tet-treated, differentiation induced.
  • FIG. 4B shows contracting EBs formed from wt CGR8 and ZHTc6 ESCs, with or without tet-treatment, when cultured in cardiogenic differentiation culture condition B.
  • Percentages of beating EBs were calculated to compare untreated (CGR8, ZHTc6 24h and ZHTc6 48h) and tet- treated (CGR8 TD0, ZHTc6 Tet) ESCs. Actual EB number count from each culture condition is shown (number of beating EBs/total number of EBs). Data are expressed as the average +/- SEM of 4 experiments.
  • ZHTc6 24h- ZHTc6 Tet ESCs removed from tet- treatment 24 hr before induction of differentiation and remained tet-free.
  • ZHTc6 48h- ZHTc6 ESCs removed from tet-treatment 48 hr before induction for differentiation and remained tet-free.
  • TDO- tet-treatment began at day of differentiation.
  • TD2- tet-treatment began at day 2 post-differentiation.
  • FIG. 5 is a series of bar graphs showing qRT-PCR analyses of cardiogenic marker expression from CGR8 and ZHTc6 ESCs.
  • FIG. 5A-D show a summary of the relative expression of cardiac markers Nkx2.5, Tnnt2, Myh7 and Myh6 from day 0 ESCs and day 18 EBs;
  • FIG. 5E-G show day 10 EBs. Data are expressed as the average +/- SEM of at least 3 experiments.
  • TDO- tet-treatment began at day of differentiation.
  • TD2- tet-treatment at day 2 post- differentiation.
  • FIG. 6 is a cartoon depicting culturing methods applied to generate contracting cells or EBs.
  • Conventional protocol for cardiac differentiation of ESCs in liquid medium condition A, left panel
  • modified protocol for cardiac differentiation of ESCs in 3-dimensional methylcellulose condition B, right panel
  • FIG. 7 is a series of scatter plots showing flow cytometry analyses of formation of hematopoietic progenitors from day 12 EBs derived from CRG8 and ZHTc6 ESCs.
  • FIG. 7A shows representative flow cytometric analyses on wt untreated CGR8, TDO CGR8, tet-treat ZHTc6 (ZHTc6 Tet) and untreated ZHTc6 (ZHTc6 24h and ZHTc6 48h).
  • CD45 marker was analyzed on the FL4 channel (y-axis) and CD1 1 b marker was analyzed by the FL2 channel (x-axis).
  • FIG. 7B shows calculation of the CD45/CD1 1 b double positive cell populations from each clone.
  • FIG. 8 is a bar graph showing CD45+CD1 1 b+ cell population (%) for wt untreated CGR8, TDO CGR8, tet-treat ZHTc6 (ZHTc6 Tet) and untreated ZHTc6
  • FIG. 9 shows percentages of beating EBs when wt CGR8 ESCs were cultured in condition A (CGR8A) or condition B (CGR8B). Data are expressed as the average +/- SEM of 4 experiments (beating EBs/total number of EBs).
  • FIG. 10A is a western blot analysis on Oct-4 protein expression in CGR8 or Oct-4 knock-in ZHTc6 ESCs before (DO) and after induction for differentiation (D1 ).
  • FIG. 10B is a bar graph of Oct-4 protein expression level before (DO) and after induction for differentiation (D1 ).
  • FIG. 10C is a table describing Oct-4 protein level expression in CGR8 or Oct-4 knock-in ZHTc6 ESCs before (DO) and after induction for differentiation (D1 ).
  • FIG. 1 1A-G are bar graphs showing relative expression of cardiac-specific markers, Nkx2.5, Tnnt2, Myh7, and Myh6 in CGR8- and ZHTc6-derived EBs measured by Real Time PCR.
  • FIG. 12A-L are fluorescence microscopy images of immunohistochemically- stained samples of NBEBs and BEBs.
  • the cardiac-specific marker targeted for staining was Myh6/7.
  • FIG. 13 shows a pair of bar graphs showing a percentage of Myh6/7 positive cells in BEBs and NBEBs.
  • beating EBs BEBs
  • NBEBs non-beating EBs
  • FIG. 13B areas of myosin heavy chain staining (Myh6/7) and DAPI staining were measured from each EB.
  • FIG. 14A-C shows immunofluorescence microscopy images of cardiac- specific marker Myh6/7 in BEBs from CGR8 24h ESCs.
  • FIG. 14C is the zoomed-in area of FIG. 14A.
  • FIG. 15A-D shows immunofluorescence microscopy images of cardiac- specific marker Myh6/7 in BEBs from ZHTc6 24h ESCs.
  • FIG. 15C-D are the zoomed-in areas of FIG. 15A-B.
  • FIG. 16 shows immunofluorescence microscopy images of cardiac-specific marker, Myh6/7, and vascular-specific marker, CD31 , in CGR8-derived BEBs (FIG. 16A-F) and NBEBs (FIG. 16G-L).
  • FIG. 17A-B shows a pair of bar graphs showing cardiac potential in samples treated with Endothelial stem/progenitor cells (HUVECs).
  • the present disclosure is based, at least in part, on the observation of a 5- fold increase in the number of contracting embryoid bodies when cultured in a novel cardiogenic culture medium containing interleukin-3, interleukin-1 , insulin, transferring, and monothioglycerol as compared to conventional medium.
  • the present disclosure is also based, at least in part, on the observation that an about 20% to 50% decrease of Oct-4 expression in undifferentiated and early differentiating ESCs can severely cripple cardiogenesis and formation of functional cardiogenic progenitors, but this developmental defect can be rescued if Oct-4 levels are increased so as to approximate wild-type levels.
  • an up- regulation of brachyury is seen in Oct-4 over-expressing ESCs (about 50% higher than wild-type), this over-expression does not promote later stage cardiogenesis (or hematopoiesis) as much as the wild-type level expression of Oct-4 (wildtype biallelic expression).
  • Oct-4 a higher expression of Oct- 4 (-150% of wildtype) in undifferentiated or early differentiating ESCs does not promote cardiac differentiation (see Example 5), although it does promote the expression of the early mesodermal marker brachyury (see Example 5), as shown previously.
  • Oct-4 levels must be maintained that a specific level of Oct-4 must be maintained at particular levels for progression to post-brachyury+ differentiation of general mesodermal lineages, for example, when inducing pluripotent stem cells from adult tissues to differentiation into mature tissues for organ replacement. This is in contrast to the role of Oct-4 in sustaining ESC pluripotentiality, where there is more tolerance to Oct-4 levels.
  • Oct-4 protein levels in ESCs does not necessarily target cardiac differentiation specifically, as has been previously reported.
  • the requirement for Oct-4 appears to be earlier in differentiation, perhaps in the specification of lateral plate mesoderm, which is the precursor for cardiac,
  • Oct-4 hematopoietic and vascular cells. Therefore, not only is the presence of Oct-4 required for both cardiogenesis and hematopoiesis, but a specific concentration of Oct-4 is required for both pre- and post-brachyury positive mesodormal development.
  • the present disclosure shows that specific Oct-4 levels are key regulators for mesodermal lineage specification.
  • ESCs are more tolerant of varying Oct-4 levels to maintain pluripotentiality than they are for mesodermal linage differentiation.
  • ZHTc6 ESCs are able to remain undifferentiated despite decreased level of Oct-4.
  • even a small decrease in Oct-4 markedly reduced cardiogenic differentiation see e.g.,
  • Example 2 Such finding implicates using Oct-4 to induce pluripotent stem cells from adult tissues.
  • using induced pluripotent stem cells to engineer organ replacement from mesodermal lineages requires precise levels of Oct-4 for proper mesodermal differentiation.
  • Oct-4 does not promote formation of later stage hematopoietic progenitors.
  • Oct-4 While being under no obligation to supply a mechanistic description, and in no way limited the scope of the invention, the following discussion addresses how Oct-4 maintains pluripotentiality, and yet is necessary for mesodermal lineage differentiation. Partners of Oct-4 may switch as differentiation is induced, thus altering the genes transcriptionally regulated by Oct-4.
  • the epigenetic structure of Oct-4 target promoters may be altered during the early differentiation process, and this alters Oct-4's ability to interact with those promoters. But experiments reported herein show that normal levels of Oct-4 are required not only for hematopoietic differentiation, but also for cardiogenic differentiation.
  • Oct-4 is required for all types of mesodermal differentiation, since there is evidence that hematopoietic, vascular and cardiac tissue may derive from a common precursor. It does, however, imply that Oct-4- induced pluripotent stem cells require appropriate maintenance of Oct-4 expression to form certain mesodermal tissues.
  • One aspect provides a cardiogenic culture medium that increases cardiogenesis or increases differentiation of cardiac progenitor cells from progenitor cells, such as stem cells or embryonic stem cells.
  • Various embodiments of the cardiogenic culture medium promote both cardiogenesis and hematopoiesis
  • cardiogenic culture medium employed in studies reported herein generated cardiogenic EBs at a significantly higher rate than
  • the cardiogenic culture medium can affect cardiogenesis and hematopoiesis. Such dual effect has not been previously reported in a culture medium.
  • both Tall and Nkx 2.5 can be activated in the differentiating ESCs. It has be reported that in Tall over-expressing ESCs, hematopoietic lineage specification became more favorable when cultured with cytokine supplementation, whereas cardiac genes were down- regulated. In contrast, the inventors have discovered that both cardiogenic and hematopoietic genes can be co-expressed in cytokine-supplemented culture medium such as the cardiogenic culture medium described herein.
  • the cardiogenic culture medium can include one or more of interleukin-3, interleukin-1 , insulin, and transferin.
  • cardiogenic culture medium can include two or more, three or more, or all of interleukin-3, interleukin-1 , insulin, and transferin.
  • the cardiogenic culture medium can be a conventional culture medium, such as DMEM or MethoCult methylcellulose, supplemented with the above
  • the cardiogenic culture medium can include further include components such as FBS and monothioglycerol.
  • the cardiogenic culture medium can include a DMEM based medium supplemented with interleukin-3, interleukin-1 , insulin, or transferin.
  • the cardiogenic culture medium can include a DMEM based medium supplemented with FBS, interleukin-3, interleukin-1 , insulin, transferin, or monothioglycerol.
  • the cardiogenic culture medium can be a DMEM medium supplemented with 15% FBS, interleukin-3 (5 ng/ml), interleukin-1 (5 ng/ml) insulin (10 ng/ml), transferrin (200 ng/ml) or monothioglycerol (100 ⁇ ).
  • the cardiogenic culture medium can include a methylcellulose medium supplemented with interleukin-3, interleukin-1 , insulin, or transferin.
  • An exemplary methylcellulose medium is MethoCult medium.
  • the cardiogenic culture medium can include a methylcellulose based medium
  • the cardiogenic culture medium includes interleukin- 3.
  • the cardiogenic culture medium can include interleukin-3 at a concentration of at least about 0.5 ng/ml.
  • the cardiogenic culture medium can include interleukin-3 at a concentration of up to about 50 ng/ml.
  • the cardiogenic culture medium can include interleukin-3 at a concentration of at least about 0.5 ng/ml up to about 50 ng/ml.
  • the cardiogenic culture medium can include interleukin-3 at a concentration of about 0.5 ng/ml, about 0.6 ng/ml, about 0.7 ng/ml, about 0.8 ng/ml, about 0.9 ng/ml, about 1 ng/ml, about 2 ng/ml, about 3 ng/ml, about 4 ng/ml, about 5 ng/ml, about 6 ng/ml, about 7 ng/ml, about 8 ng/ml, about 9 ng/ml, about 10 ng/ml, about 20 ng/ml, about 30 ng/ml, about 40 ng/ml, or about 50 ng/ml.
  • the cardiogenic culture medium can include interleukin-3 at a concentration of about 5 ng/ml.
  • the cardiogenic culture medium includes interleukin- 1 .
  • the cardiogenic culture medium can include interleukin-1 at a concentration of at least about 0.5 ng/ml.
  • the cardiogenic culture medium can include interleukin-1 at a concentration of up to about 50 ng/ml.
  • the cardiogenic culture medium can include interleukin-1 at a concentration of at least about 0.5 ng/ml up to about 50 ng/ml.
  • the cardiogenic culture medium can include interleukin-1 at a concentration of about 0.5 ng/ml, about 0.6 ng/ml, about 0.7 ng/ml, about 0.8 ng/ml, about 0.9 ng/ml, about 1 ng/ml, about 2 ng/ml, about 3 ng/ml, about 4 ng/ml, about 5 ng/ml, about 6 ng/ml, about 7 ng/ml, about 8 ng/ml, about 9 ng/ml, about 10 ng/ml, about 20 ng/ml, about 30 ng/ml, about 40 ng/ml, or about 50 ng/ml.
  • the cardiogenic culture medium can include interleukin-1 at a concentration of about 5 ng/ml.
  • the cardiogenic culture medium includes insulin.
  • the cardiogenic culture medium can include insulin at a concentration of at least about 1 ng/ml.
  • the cardiogenic culture medium can include insulin at a concentration of up to about 100 ng/ml.
  • the cardiogenic culture medium can include insulin at a concentration of at least about 1 ng/ml up to about 100 ng/ml.
  • the cardiogenic culture medium can include insulin at a concentration of about 1 ng/ml, about 2 ng/ml, about 3 ng/ml, about 4 ng/ml, about 5 ng/ml, about 6 ng/ml, about 7 ng/ml, about 8 ng/ml, about 9 ng/ml, about 10 ng/ml, about 1 1 ng/ml, about 12 ng/ml, about 13 ng/ml, about 14 ng/ml, about 15 ng/ml, about 16 ng/ml, about 17 ng/ml, about 18 ng/ml, about 19 ng/ml, about 20 ng/ml, about 30 ng/ml, about 40 ng/ml, about 50 ng/ml, about 60 ng/ml, about 70 ng/ml, about 80 ng/ml, about 90 ng/ml, or about 100 ng/ml.
  • the cardiogenic culture medium can include insulin at a concentration of
  • the cardiogenic culture medium includes transferin.
  • the cardiogenic culture medium can include transferin at a concentration of at least about 20 ng/ml.
  • the cardiogenic culture medium can include transferin at a
  • the cardiogenic culture medium can include transferin at a concentration of at least about 20 ng/ml up to about 2 g/ml.
  • the cardiogenic culture medium can include transferin at a concentration of about 20 ng/ml, about 30 ng/ml, about 40 ng/ml, about 50 ng/ml, about 60 ng/ml, about 70 ng/ml, about 80 ng/ml, about 90 ng/ml, about 100 ng/ml, about 1 10 ng/ml, about 120 ng/ml, about 130 ng/ml, about 140 ng/ml, about 150 ng/ml, about 160 ng/ml, about 170 ng/ml, about 180 ng/ml, about 190 ng/ml, about 200 ng/ml, about 210 ng/ml, about 220 ng/ml, about 230 ng/ml, about 240 ng/ml, about 250
  • the cardiogenic culture medium includes
  • the cardiogenic culture medium can include monothioglycerol at a concentration of at least about 10 ⁇ .
  • the cardiogenic culture medium can include monothioglycerolat a concentration of up to about 1 mM.
  • the cardiogenic culture medium can include monothioglycerolat a concentration of at least about 10 ⁇ up to about 1 mM.
  • the cardiogenic culture medium can include
  • monothioglycerolat a concentration of about 10 ⁇ , about 20 ⁇ , about 30 ⁇ , about 40 ⁇ , about 50 ⁇ , about 60 ⁇ , about 70 ⁇ , about 80 ⁇ , about 90 ⁇ , about 100 ⁇ , about 1 10 ⁇ , about 120 ⁇ , about 130 ⁇ , about 140 ⁇ , about 150 ⁇ , about 160 ⁇ , about 170 ⁇ , about 180 ⁇ , about 190 ⁇ , about 200 ⁇ , about 300 ⁇ , about 400 ⁇ , about 500 ⁇ , about 600 ⁇ , about 700 ⁇ , about 800 ⁇ , about 900 ⁇ , or about 1 mM.
  • the cardiogenic culture medium includes FBS.
  • the cardiogenic culture medium can include FBS at a concentration of at least about 1 %.
  • the cardiogenic culture medium can include FBS at a concentration of up to about 30%.
  • the cardiogenic culture medium can include FBS at a concentration of at least about 1 % up to about 30%.
  • the cardiogenic culture medium can include FBS at a concentration of about 1 %, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 1 1 %, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21 %, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, or about 30%.
  • the cardiogenic culture medium can be a DMEM medium supplemented with about 15% FBS, interleukin-3 (about 5 ng/ml), interleukin-1 (about 5 ng/ml) insulin (about 10 ng/ml), transferrin (about 200 ng/ml) or monothioglycerol (about 100 ⁇ ).
  • the cardiogenic culture medium can be a DMEM medium supplemented with about 15% FBS, interleukin-3 (about 5 ng/ml), interleukin-1 (about 5 ng/ml) insulin (about 10 ng/ml), transferrin (about 200 ng/ml) or monothioglycerol (about 100 ⁇ ).
  • the cardiogenic culture medium can be a DMEM medium supplemented with about 15% FBS, interleukin-3 (about 5 ng/ml), interleukin-1 (about 5 ng/ml) insulin (about 10 ng/ml), transferrin (about 200 ng/ml) or monothiogly
  • methylcellulose medium supplemented with about 15% FBS, interleukin-3 (about 5 ng/ml), interleukin-1 (about 5 ng/ml) insulin (about 10 ng/ml), transferrin (about 200 ng/ml) or monothioglycerol (about 100 ⁇ ).
  • a cardiogenic culture medium described herein can increase differentiation of cardiac progenitor cells from progenitor cells.
  • a cardiogenic culture medium described herein can increase differentiation of cardiac progenitor cells from progenitor cells by at least about 10%.
  • a cardiogenic culture medium described herein can increase differentiation of cardiac progenitor cells from progenitor cells by at least about 50%.
  • a cardiogenic culture medium described herein can increase differentiation of cardiac progenitor cells from progenitor cells by at least about 100%.
  • a cardiogenic culture medium described herein can increase differentiation of cardiac progenitor cells from progenitor cells by at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 450%, at least about 500%, at least about 550%, at least about 600%, at least about 650%, at least about 700%, at least about 750%, at least about 800%, at least about 850%, at least about 900%, at least about 950%, or at least about 1 ,000%.
  • compositions and methods of the invention generally employ progenitor cells so as to form a cardiac progenitor cell.
  • the cardiac progenitor cell is understood to be a more differentiated cell than the starting progenitor cell.
  • a progenitor cell can differentiate into, or otherwise form, a cardiac progenitor cell.
  • a progenitor cell can be a pluripotent cell.
  • a progenitor cell can be a multipotent cell.
  • a progenitor cell can be self-renewing.
  • a progenitor cell can be an embryonic stem cell (ESC), such as a human embryonic stem cell (hESC).
  • ESCs can be derived from epiblast tissue of the inner cell mass (ICM) of a blastocyst or earlier morula stage embryo.
  • a progenitor cell can be an induced pluripotent stem cell, such as a human induced pluripotent stem cell (hiPSC).
  • the progenitor cell is substantially less differentiated than a cardiac progenitor cell.
  • a progenitor cell can be freshly isolated or not pre- treated with growth factors before being further cultured with a cardiogenic culture medium described herein or exposed to increased levels of Oct-4.
  • a progenitor cell is a precursor to a cardiac progenitor cell and differentiates under culture conditions including the cardiogenic culture medium described herein. In various embodiments, a progenitor cell is a precursor to a cardiac progenitor cell and differentiates under culture conditions including certain levels of exogenous or endogenous Oct-4 at one or more stages of differentiation. In some embodiments, the progenitor cell is an Oct-4-induced
  • the progenitor cell is an Oct-4-induced pluripotent embryonic stem cell. In some embodiments, the progenitor cell does not display a cardiac-specific marker, such as Nkx2.5, Tnnt2, Myh7, and Myh6.
  • Progenitor cells can be isolated, purified, or cultured by a variety of means known to the art Methods for the isolation and culture of progenitor cells are discussed in, for example, Vunjak-Novakovic and Freshney (2006) Culture of Cells for Tissue Engineering, Wiley-Liss, ISBN-10 0471629359.
  • a progenitor cell can be derived from an animal, including, but not limited to, mammals, reptiles, and avians, more preferably horses, cows, dogs, cats, sheep, pigs, and chickens, and most preferably human.
  • a progenitor cell can be derived from the same or different species as an intended transplant recipient.
  • a cardiac progenitor cell is understood to be more differentiated than a progenitor cell but less differentiated than a cardiac cell.
  • a cardiac progenitor cell can be a multipotent cell.
  • a cardiac progenitor cell can be an oligopotent cell.
  • a cardiac progenitor cell can be unipotent.
  • a cardiac progenitor cell can have limited self-renewal ability.
  • a cardiac progenitor cell can be a cell that displays one or more cardiac- specific markers.
  • Cardiac-specific markers include, but are not limited to, Nkx2.5, Tnnt2, Myh7, and Myh6 (see Example 6).
  • Nkx 2.5, Tnnt2 and Myh7 are understood to be early or intermediate cardiogenic markers.
  • Myh6 is understood to be a late cardiac marker.
  • a cardiac progenitor cell can be a hematopoietic embryoid bodies (EBs).
  • EBs hematopoietic embryoid bodies
  • a cardiac progenitor cell can be a contracting embryoid body or a beating embryoid body.
  • a cardiac progenitor cell can be a spontaneously beating embryoid body.
  • a cardiac progenitor cell can be a hematopoietic embryoid body.
  • a progenitor cell or a cardiac progenitor cell can be transformed with a heterologous nucleic acid so as to express a bioactive molecule, or heterologous protein or to overexpress an endogenous protein.
  • a progenitor cell or a cardiac progenitor cell can be genetically modified to expresses a fluorescent protein marker. Exemplary markers include GFP, EGFP, BFP, CFP, YFP, and RFP.
  • a progenitor cell or a cardiac progenitor cell can be genetically modified to express an angiogenesis-related factor, such as activin A, adrenomedullin, aFGF, ALK1 , ALK5, ANF, angiogenin, angiopoietin-1 , angiopoietin-2, angiopoietin-3, angiopoietin-4, angiostatin, angiotropin, angiotensin-2, AtT20-ECGF, betacellulin, bFGF, B61 , bFGF inducing activity, cadherins, CAM-RF, cGMP analogs, ChDI, CLAF, claudins, collagen, collagen receptors ⁇ ⁇ and ⁇ 3 ⁇ 4 ⁇ , connexins, Cox-2, ECDGF (endothelial cell-derived growth factor), ECG, ECI, EDM, EGF, EMAP, endoglin, endo
  • a progenitor cell or a cardiac progenitor cell can be transfected with genetic sequences that are capable of reducing or eliminating an immune response in a host ⁇ e.g., expression of cell surface antigens such as class I and class II histocompatibility antigens can be suppressed). This can allow the transplanted cells to have reduced chance of rejection by the host.
  • Embryoid bodies can be harvested at about day 2 of development.
  • Harvested EBs can be transferred to a plate.
  • the plate can contain a suitable medium, such as a semi-solid
  • the method of forming a cardiac progenitor cell can include culturing a progenitor cell in a cardiogenic culture medium described herein so as to form a cardiac progenitor cell.
  • the method of forming a cardiac progenitor cell can include increasing a level of Oct-4 in a progenitor cell and culturing the progenitor cell so as to form a cardiac progenitor cell.
  • the method of forming a cardiac progenitor cell can include increasing a level of Oct-4 in a progenitor cell and culturing the progenitor cell in a cardiogenic culture medium described herein so as to form a cardiac progenitor cell.
  • the level of Oct-4 in the progenitor cell is detected. If the level of Oct-4 is lower than a pre-determined value, then Oct-4 can be increased accordingly. For example, if the level of Oct-4 is lower than about the level of Oct-4 in embryonic stem cell line CGR8 cultured under equivalent conditions, then Oct-4 can be increased accordingly.
  • the level of Oct-4 in the progenitor cell can be increased so as to increase the expression of cellular markers.
  • the level of Oct-4 in the progenitor cell can be increased so as to increase the expression of early or intermediate cardiogenic markers, such as Nkx 2.5, Tnnt2 or Myh7.
  • the level of Oct-4 in the progenitor cell can be increased so as to increase the expression a late cardiac marker, such as Myh6.
  • the level of Oct-4 in the progenitor cell can be increased so as to increase the expression an early mesodermal marker, such as Brachyury.
  • the level of Oct-4 in the progenitor cell can be increased at a
  • the level of Oct-4 in the progenitor cell can be increased prior to 1 1 days of culturing the progenitor cell.
  • the level of Oct-4 in the progenitor cell can be increased prior to expression of early mesodermal marker Brachyury.
  • Oct-4 can be increased in a progenitor cell by supplying exogenous Oct-4.
  • Oct-4 can be increased in a progenitor cell by increasing expression of endogenous Oct-4 in the progenitor cell.
  • Methods described herein can increase the number of formed cardiac progenitor cells as compared to conventional methods.
  • culture methods described herein can increase differentiation of cardiac progenitor cells from progenitor cells by at least about 10%.
  • culture methods described herein can increase differentiation of cardiac progenitor cells from progenitor cells by at least about 50%.
  • culture methods described herein can increase differentiation of cardiac progenitor cells from progenitor cells by at least about 100%.
  • culture methods described herein can increase differentiation of cardiac progenitor cells from progenitor cells by at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 450%, at least about 500%, at least about 550%, at least about 600%, at least about 650%, at least about 700%, at least about 750%, at least about 800%, at least about 850%, at least about 900%, at least about 950%, or at least about 1 ,000%.
  • a progenitor cell or a cardiac progenitor cell can be co-cultured with one or more additional cell types.
  • additional cell types can include (but are not limited to) skin cells, liver cells, heart cells, kidney cells, pancreatic cells, lung cells, bladder cells, stomach cells, intestinal cells, cells of the urogenital tract, breast cells, skeletal muscle cells, skin cells, bone cells, cartilage cells, keratinocytes, hepatocytes, gastro-intestinal cells, epithelial cells, endothelial cells, mammary cells, skeletal muscle cells, smooth muscle cells, parenchymal cells, osteoclasts, or chondrocytes.
  • a first progenitor cell can be cultured with a second progenitor cell under conditions described herein.
  • an embryonic progenitor cell can be cultured with an endothelial stem/progenitor cell (HUVEC).
  • HUVEC endothelial stem/progenitor cell
  • co-culture of ESCs and HUVECs can increase cardiogenic potential of ESCs differentiating into cardiac progenitor cells (see Example 15). It is presently thought that vasculogenesis associated with HUVECs facilitates cardiogenesis associated with ESCs.
  • a co-culture of ESCs and HUVECs can include about 99% ESCs, about 98% ESCs, about 97% ESCs, about 96% ESCs, about 95% ESCs, about 94% ESCs, about 93% ESCs, about 92% ESCs, about 91 % ESCs, about 90% ESCs, about 85% ESCs, or about 80% ESCs.
  • a co-culture of ESCs and HUVECs can include about 1 % HUVECs, about 2% HUVECs, about 3% HUVECs, about 4% HUVECs, about 5% HUVECs, about 6% HUVECs, about 7% HUVECs, about 8% HUVECs, about 9% HUVECs, about 10% HUVECs, about 15% HUVECs, or about 20% HUVECs.
  • a co-culture of ESCs and HUVECs can include about 98% ESCs and about 2% HUVECs.
  • a co-culture of ESCs and HUVECs can include about 95% ESCs and about 5% HUVECs.
  • nucleotide and/or polypeptide variants having, for example, at least 95-99% identity to the reference sequence described herein and screen such for desired phenotypes according to methods routine in the art.
  • conservative substitutions can be made at any position so long as the required activity is retained.
  • Nucleotide and/or amino acid sequence identity percent is understood as the percentage of nucleotide or amino acid residues that are identical with nucleotide or amino acid residues in a candidate sequence in comparison to a reference sequence when the two sequences are aligned. To determine percent identity, sequences are aligned and if necessary, gaps are introduced to achieve the maximum percent sequence identity. Sequence alignment procedures to determine percent identity are well known to those of skill in the art. Often publicly available computer software such as BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR) software is used to align sequences. Those skilled in the art can determine appropriate parameters for
  • Highly stringent hybridization conditions are defined as hybridization at 65 °C in a 6 X SSC buffer (i.e., 0.9 M sodium chloride and 0.09 M sodium citrate). Given these conditions, a determination can be made as to whether a given set of sequences will hybridize by calculating the melting temperature (T m ) of a DNA duplex between the two sequences. If a particular duplex has a melting temperature lower than 65°C in the salt conditions of a 6 X SSC, then the two sequences will not hybridize. On the other hand, if the melting temperature is above 65 °C in the same salt conditions, then the sequences will hybridize. In general, the melting temperature for any hybridized
  • T m of a DNA:DNA hybrid is decreased by 1 -1 .5°C for every 1 % decrease in nucleotide identity (see e.g., Sambrook and Russel, 2006).
  • Host cells can be transformed using a variety of standard techniques known to the art (see, e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10:
  • Host strains developed according to the approaches described herein can be evaluated by a number of means known in the art (see e.g., Studier (2005) Protein Expr Purif. 41 (1 ), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10:
  • expressed protein activity can be down-regulated or eliminated using antisense oligonucleotides, protein aptamers, nucleotide aptamers, and RNA
  • RNAi small interfering RNAs
  • shRNA short hairpin RNA
  • miRNA micro RNAs
  • RNAi molecules are commercially available from a variety of sources ⁇ e.g., Ambion, TX; Sigma Aldrich, MO; Invitrogen).
  • sources e.g., Ambion, TX; Sigma Aldrich, MO; Invitrogen.
  • siRNA molecule design programs using a variety of algorithms are known to the art (see e.g., Cenix algorithm, Ambion; BLOCK-iTTM RNAi Designer, Invitrogen; siRNA Whitehead Institute Design Tools, Bioinofrmatics & Research Computing).
  • Traits influential in defining optimal siRNA sequences include G/C content at the termini of the siRNAs, Tm of specific internal domains of the siRNA, siRNA length, position of the target sequence within the CDS (coding region), and nucleotide content of the 3' overhangs.
  • numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term "about.”
  • the term “about” is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value.
  • the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment.
  • the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
  • composition or device that "comprises,” “has” or “includes” one or more features is not limited to possessing only those one or more features and can cover other unlisted features.
  • One or more members of a group can be included in, or deleted from, a group for reasons of convenience or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.
  • Embryonic stem cell (ESCs) lines used were CGR8, ZHBTc4 and ZHTc6.
  • ZHBTc4 is an ES cell line that has had both endogenous Pou5f1 (Oct-4) alleles deleted, and an exogenous tetracycline-repressible Oct-4 vector introduced, along with an expression vector producing the tetracycline (tet) activator. In the absence of tet, these cells express Oct-4 and maintain an undifferentiated state. In the presence of 10 g/ml tet (Sigma, St. Louis, MO), which binds to and represses the tet activator, Oct-4 expression is completely repressed in these cells within 24 hours.
  • 10 g/ml tet Sigma, St. Louis, MO
  • ZHTc6 is an ES cell line that had one allele of Pou5f1 inactivated by targeted integration of an IRESzeopA cassette, and contains a tet-repressed Oct-4 transgene. This transgene can be fully activated when removed from tet for 48 hours, resulting in over-expression of Oct-4.
  • CGR8 cells are the parental wild-type (wt) ES cell line from which ZHBTc4 and ZHTc6 cells were derived.
  • ESCs CGR8, ZHTc6 and ZHBTc4 were maintained in Dulbecco's Modified Eagle's Medium (DMEM, Hyclone, Thermo Fisher Scientific, Pittsburgh, PA) containing 15% fetal bovine serum (FBS, embryonic stem cell grade, Hyclone), 1 % sodium pyruvate (Invitrogen, Carlsbad, CA), 2mM L-glutamine (StemCell Technologies, Vancouver, BC), 0.1 mM nonessential amino acids (NEAA) (Invitrogen), 50 U/ml penicillin, 50 g/ml streptomycin (Invitrogen), leukemia inhibitory factor (LIF) (1000U/ml) (Chemicon, Temecula, CA) and 55 ⁇ ⁇ -mercaptoethanol ( ⁇ , Sigma).
  • DMEM Dulbecco's Modified Eagle's Medium
  • FBS fetal bovine serum
  • FBS embryonic stem cell grade, Hyclone
  • NEAA no
  • ESCs were cultured either with the hanging drop method in DMEM supplemented with 20% fetal calf serum (FCS, Hyclone), 2 mM L-glutamine, 0.1 mM NEAA and 0.1 mM ⁇ , or directly cultured in 10 cm bacteriological culture plates with 10 ml DMEM supplemented with 15% FBS, 2mM L-glutamine, 0.1 mM NEAA, 50 U/ml penicillin, 50 ⁇ g ml streptomycin and 0.1 mM ⁇ for 2 days.
  • Embryoid Bodies (EBs) were collected and placed into fresh 10 cm bacteriological plates supplied with fresh differentiation medium. On the fifth day, single EBs were seeded onto 0.1 % gelatin pre- coated 24-well plates and medium was changed every other day until cells were ready for analysis.
  • EBs were collected and subsequently seeded onto 35 mm culture plates (StemCell Technologies) containing 1 .5 ml MethoCult methylcellulose (StemCell Technologies) supplemented with either (iii) 20% FBS (Differentiation Grade, StemCell Technologies), 1 %
  • NEAA nonessential amino acids
  • StemCell nonessential amino acids
  • Cardiogenic differentiating condition A used culture reagents (i) + (iii), and condition B used (ii) + (iv).
  • Total protein was extracted from undifferentiated ESCs or differentiating EBs using 1 x lysis buffer (50 mM Tris-CI, pH 7.8, 150 mM NaCI, 1 mM EDTA, 1 mM DTT, 0.5% NP-40, and 1 x protease inhibitor cocktail). Briefly, 3 volumes of lysis buffer (cell pellet size equals one volume) was added to each sample and was pipetted multiple times on ice before being subjected to centrifugation at 12,000 RPM at 4°C for 15 minutes. Supernatants were collected as total protein extracts. Thirty ⁇ g of each protein sample was used for PAGE, and separated proteins were transferred to a nitrocellulose membrane for immunoblotting.
  • 1 lysis buffer 50 mM Tris-CI, pH 7.8, 150 mM NaCI, 1 mM EDTA, 1 mM DTT, 0.5% NP-40, and 1 x protease inhibitor cocktail. Briefly, 3 volumes of lysis buffer (cell pellet size equals one volume)
  • primary antibodies used were anti-Oct-4 monoclonal antibody (1 :400, Santa Cruz Biotechnology, Santa Cruz, CA) and anti-actin monoclonal antibody (1 :10,000, Sigma). Secondary antibodies were donkey anti-mouse IgG conjugated with horse radish peroxidase (HRP, 1 :10,000, GE Healthcare, Pittsburgh, PA) and sheep anti-rabbit IgG HRP-conjugated (1 :10,000, GE Healthcare).
  • RNA Microprep Kit (Stratagene, La Jolla, CA) as described above from the indicated cell populations.
  • First strand cDNA was synthesized using the Invitrogen cDNA synthesis kit according to the manufacturer's instructions. Quantitative expression analysis was performed using SYBR Green PCR Master Mix reagents (Applied Bioscience).
  • Results are represented as means +/- SEM of at least three independent experiments.
  • EBs Hematopoietic embryoid bodies
  • fetal bovine serum (differentiation grade, Stem Cell Technologies) supplemented with 10% fetal bovine serum (differentiation grade, Stem Cell Technologies), 5% protein-free hybridoma medium-ll (Invitrogen), murine IL-1 (5ng/ml), murine IL-3 (5 ng/ml), SCF (10 ng/ml), GM-CSF (5 ng/ml) and EPO (3U/ml) at 37°C and 5% CO 2 for 12-14 days.
  • Differentiated cells and EBs from 12d cultures were collected in phosphate-buffered saline (PBS) and spun down at 1 ,200 rpm for 10 minutes. EBs were disaggregated by the addition of 3 ml 0.25% collagenase to each sample and incubated at 37°C for one hour.
  • PBS phosphate-buffered saline
  • This example demonstrates a 5-fold increase in the number of contracting embryoid bodies when cultured in a modified cardiogenic culture condition (condition B).
  • condition B a modified cardiogenic culture condition
  • CGR8 mESCs cultured in condition A results in a low-level of beating cells.
  • Oct-4 knock-in ZHTc6 ESCs failed to generate contracting EBs upon induction of differentiation. Methods are according to Example 1 , unless otherwise specified.
  • Previously published protocols for generating contracting embryoid bodies were found to be inefficient at generating cardiogenic differentiation (see e.g., FIG. 1 ).
  • CGR8 mESCs were cultured in conventional cardiac differentiation medium (non- cytokine-supplemented condition A).
  • Low-level of beating cells are formed under this culture condition (see e.g., white arrowheads in FIG. 1A and FIG. 1 B).
  • RT-PCR analysis show low- to moderate-expression levels of cardiac-specific markers in cells cultured in condition A (see e.g., FIG. 1 C).
  • ZHTc6 is a tet-regulated, heterozygous Oct-4 knock-in ES cell line. In these cells, one copy of endogenous Pou5f1 was replaced by a tet-regulated Oct-4 transgene. Without activation of the Oct-4 transgene, Oct-4 protein expression level in this cell line is ⁇ 70-80% of that seen in wt bi-allelic ESCs (CGR8) 6 . To address the possibility that a suboptimal expression level of Oct-4 protein in the undifferentiated ESCs contributed to the lack of contracting cardiogenic EB formation, the experiments were repeated and included conditions that restored Oct-4 protein expression to a level similar to wt expression, or to -50% higher than wt in the ZHTc6 cells.
  • This example shows high expression of cardiac-specific markers in EBs expressing wild-type levels of Oct-4, but not decreased levels of Oct-4. Methods are according to Example 1 , unless otherwise specified.
  • undifferentiated ESCs day 0
  • day 10 day 10
  • 18 differentiated EBs were harvested from CGR8 and ZHTc6 (cultured with or without tet) real-time PCR (qRT-PCR) was performed to examine the expression of cardiac-specific markers. Day 0 ESCs and the well-differentiated day 18 EBs were examined.
  • This example shows that generation of myeloid progenitors increased at a dose-dependent manner corresponding to Oct-4 protein expression levels in ZHTc6 ESCs. Methods are according to Example 1 , unless otherwise specified.
  • hematopoietic progenitor formation increased in a dose-dependent manner (see e.g., FIG. 7).
  • contracting EBs derived from CGR8 mESCs cultured in condition B demonstrate a significant increase in generation of contracting EBs.
  • RT-qPCR analysis was performed on cardiac-specific marker expression in DO undifferentiated ESCs or D18 wt CGR8- or Oct4 Knock-in ZHTc6-derived EBs (see e.g., FIG. 1 1A-D).
  • RT-qPCR analysis was performed on cardiac-specific marker expression in DO undifferentiated ESCs or D10 wt CGR8- or Oct4 Knock-in ZHTc6-derived EBs (see e.g., FIG 1 1 E-G).
  • Fluorescence microscopy illustrates that Myh6/7 is not expressed in the NBEBs regardless of ES cell line (see e.g., FIG. 12A-L).
  • Percent of Myh6/7+ve cells in BEBs and NBEBs was determined by immunofluorescence analysis of cardiac myosin heavy chain expression from CGR8 and ZHTc6 EBs.
  • Beating EBs (BEBs) and non-beating EBs (NBEBs) were collected from CGR8 ESC-derived EBs or ZHTc6 24h ECS-derived EBs at day 12 (see e.g., FIG. 13A).
  • Areas of myosin heavy chain staining (Myh6/7) and DAPI staining were measured from each EB (see e.g., FIG. 13B).
  • BEBs and BEBs derived from CGR8 were stained using MHC6/7 staining (cardiac) and CD31 (vascular) staining (see e.g., FIG. 16A-L).
  • Palmieri SL, Peter W, Hess H, et al. Oct-4 transcription factor is
  • Endoh M, Ogawa M, Orkin S, et al. SCL/tal-1 -dependent process determines a competence to select the definitive hematopoietic lineage prior to endothelial differentiation.
  • Endoh M, Ogawa M, Orkin S, et al. SCL/tal-1 -dependent process determines a competence to select the definitive hematopoietic lineage prior to endothelial differentiation.
  • Puceat M Protocols for cardiac differentiation of embryonic stem cells. Methods. 2008;45:168-171 .
  • Murry CE Keller G. Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development. Cell. 2008;132:661 -680.

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Abstract

L'invention concerne des compositions et des méthodes de production de cellules souches cardiaques à partir de cellules progénitrices, telles que des cellules souches embryonnaires. Dans un aspect de l'invention, on décrit un milieu de culture cardiogène comprenant l'interleukine-3, l'interleukine-1, de l'insuline ou de la transferrine. Dans un autre aspect, on décrit une méthode de production de cellules souches cardiaques à partir de cellules progénitrices, par mise en culture dans le milieu de culture cardiogène. Dans un autre aspect, on décrit une méthode de production de cellules souches cardiaques à partir de cellules progénitrices, par modulation des niveaux d'oct-4.
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