Generation Of Mesodermal Cells From Pluripotent Stem Cells

Generation of mesodermal cells from pluripotent stem cells FIELD OF THE INVENTION

The invention relates to in vitro methods for producing mesodermal cells, more particularly cardiac cells, even more particularly cardiomyocytes, from mammalian pluripotent stem cells. The invention further encompasses so-obtained mesodermal or cardiac cells; cell populations comprising these cells; compositions comprising the mesodermal or cardiac cells or the cell populations; and downstream uses thereof, for example, for medicinal applications such as cell therapy, drug screening or research purposes.

BACKGROUND OF THE INVENTION

Readily accessible source of mesodermal cells such as cardiac cells, such as more particularly cardiomyocytes, is desirable in among others clinical and research settings, e.g., in the study of the pathogenic mechanisms underlying cardiac or cardiovascular diseases, in cellular therapies of cardiac or cardiovascular diseases, or in various cell- based assays e.g., for drug screening, cardiotoxicity screening of drugs or other agents, etc.

Currently, cardiac cells such as cardiomyocytes can be differentiated from pluripotent stem cells by (1 ) spontaneous embryoid body differentiation in suspension, (2) co-culture with mouse endoderm-like cells (END-2 cells), or (3) guiding the cardiac differentiation with defined growth factors or small molecules.

Embryonic stem (ES) cells including human embryonic stem (hES) cells spontaneously form spherical structures called embryoid bodies when they are cultured in suspension in serum-containing medium. Within these mixed populations of cells contracting areas with functional properties of cardiomyocytes can be found around day 8 of culture. The cardiomyocyte yield using this approach can be rather low and/or inconsistent due to the heterogeneity among the aggregates. Furthermore, this method relies on culture media containing animal serum, which is a largely undefined component subject to batch-to- batch variations. Therefore, this approach does not readily lend itself to being a clinically useful and reproducible.

While contracting cardiac cells can be enriched from the embryoid bodies by for example mechanical isolation, such enrichment protocols commonly result in inadequate cell purity, are not suitable for scaling up and are quite labour intensive.

Co-cultivating ES cells with END-2 cells and treating ES cells with growth factors such as bone morphogenetic proteins (BMP) or fibroblast growth factors (FGF) or small molecules such as dimethylsulfoxide, retinoic acid or 5-azacytidine (a DNA demethylating agent) provide alternatives to the embryoid body approach.

Hence, there remains a need in the art to develop alternative methods for obtaining mesodermal cells, particularly cardiac cells, more particularly cardiomyocytes, preferably techniques which are robust, simple, reproducible and/or which allow for consistent, and preferably comparatively high, yields of such cells.

SUMMARY OF THE INVENTION

The present invention aims to address one or more of the aforementioned disadvantages of the art. For example, the methods encompassed within the present invention achieve the production of mesodermal cells, particularly cardiac cells, more particularly cardiomyocytes, from an adherent (i.e., without the formation of embryoid bodies in suspension) culture of mammalian pluripotent stem (mPS) cells. The methods disclosed herein can allow to differentiate the mPS cells in a comparatively reductionist system, wherein the mPS cells are exposed to defined culture conditions with minimal extrinsic influences. For example, the mPS cells may be exposed to culture conditions wherein most animal polypeptides are substantially absent, such as to defined, serum-free and plasma-free conditions. For example but without limitation, such conditions may completely lack any animal proteins, or insulin, transferrin and/or albumin may be the only animal protein(s) added under such conditions. In addition, the invention allows to depart from a monoculture (i.e., without co-culture with other cell types such as END-2 cells) of mPS cells, thereby allowing to expose the mPS cells to more accurately defined culture conditions and to reduce the complexity of the method.

In particular, as shown in the experimental section illustrating an embodiment applying the principles of the present invention, when embryonic stem cells were induced to transiently express Eomesodermin (Eomes) at between day 2 and day 4 of culture, multiple beating clusters of cells staining positive for a cardiac-specific isoform of TroponinT (cTNT) surprisingly appeared in the culture, evidencing that the resulting cells had mesodermal identity, more particular cardiac mesoderm identity. In contrast, in the absence of Eomes induction, most cells generated after 10 days of culture corresponded to neural cells, identified as 3-tubulin and Sox1 -positive cells. Although Eomes was initially identified as a key early gene in Xenopus mesoderm differentiation (Ryan et al., 1996. Cell 87: 989- 1000), in mammalian systems such as murine embryos (Arnold et al., 2008. Development 135: 501 -51 1 ; Teo et al., 201 1 . Genes Dev 25: 238-250) or embryonic stem cells (David et al., 201 1 . Cardiovasc Res; Lindsley et al., 2008. Cell Stem Cell 3:55-68; Teo et al., 201 1 . Genes Dev 25: 238-250), Eomes was so far to our knowledge only shown to play a role in specification of endodermal but not mesodermal fates. In addition, the inventors also unexpectedly found a sharp decrease of cTNT positive cells and the disappearance of beating zones when culturing the embryonic stem cells in the presence of Activin or a low concentration (e.g., 2%) of serum.

Taken together, this surprisingly established that exposure of mPS cells to conditions which are permissive to differentiation of the mPS cells and in which Activin signalling is substantially absent produces mesodermal cells when Eomes activity is provided in the mPS cells.

Accordingly, in an aspect the invention provides the use of Eomes activity for producing mesodermal cells from mPS cells exposed to conditions which are permissive to differentiation of the mPS cells and in which Activin signalling is substantially absent. In a related aspect, the invention provides a method for producing mesodermal cells comprising the steps of: a) plating mPS cells onto a substrate which allows adherence of the mPS cells thereto; b) exposing the mPS cells of a) which have adhered to said substrate to conditions which are permissive to differentiation of the mPS cells and in which Activin signalling is substantially absent; c) providing Eomes activity in the mPS cells of b).

In the uses or methods disclosed herein, the Eomes activity may be provided at an early time point following exposing the mPS cells to said conditions which are permissive to differentiation of the mPS cells and in which Activin signalling is substantially absent.

In preferred embodiments of the methods or uses as taught herein, the Eomes activity may be provided at least on or before day 4, such as at least on or before day 3, or at least on or before day 2, following exposing the mPS to said conditions which are permissive to differentiation of the mPS cells and in which Activin signalling is substantially absent.

In further preferred embodiments of the methods or uses as taught herein, the Eomes activity may be provided from day 0, more preferably from day 1 , even more preferably from day 2 following exposing the mPS to said conditions which are permissive to differentiation of the mPS cells and in which Activin signalling is substantially absent. In a preferred embodiment of the methods or uses as taught herein, the Eomes activity may be provided transiently in the mPS cells. The Eomes activity may thus be present during a period (denoted in this paragraph as "p") which represents only a fraction of the whole period (denoted in this paragraph as "P") during which the mPS cells are exposed to conditions which are permissive to differentiation of the mPS cells and in which Activin signalling is substantially absent. For example but without limitation, the period "p" may be 1/2 of the period "P", such as "p" may be 2/5, 1/3, 1/4, 1/5, 1/6 or 1/10 of the period "P". Preferably, the period "p" may be a continuous period. Also preferably, the period "p" may be subsumed within the first (former, earlier) 1/2 of the period "P", such as within the first 2/5 of period "P" or first 1/3 of the period "P".

In further preferred embodiments of the methods or uses as taught herein, the Eomes activity may be provided from day 0 to day 6, or from day 0 to day 5, or from day 0 to day 4, or from day 0 to day 3, or from day 1 to day 6, or from day 1 to day 5, or from day 1 to day 4, or from day 1 to day 3, or from day 2 to day 6, or from day 2 to day 5, or from day 2 to day 4, or from day 2 to day 3 following exposing the mPS to said conditions which are permissive to differentiation of the mPS cells and in which Activin signalling is substantially absent. Very preferably, the Eomes activity may be provided from day 2 to day 4 or from day 2 to day 3 following exposing the mPS to said conditions which are permissive to differentiation of the mPS cells and in which Activin signalling is substantially absent.

In further preferred embodiments of the methods or uses as taught herein, the duration of the present methods or uses (i.e., duration thereof) for production of mesodermal such as cardiac such as cardiomyocyte cells may be between 6 and 16 days, preferably between 8 and 12 days, more preferably between 9 and 1 1 days, even more preferably about 10 days following exposing the mPS cells to said conditions which are permissive to differentiation of the mPS cells and in which Activin signalling is substantially absent.

In further preferred embodiments of the methods or uses as taught herein, the Eomes activity may be provided from day 0 to day 6, or from day 0 to day 5, or from day 0 to day 4, or from day 0 to day 3, or from day 1 to day 6, or from day 1 to day 5, or from day 1 to day 4, or from day 1 to day 3, or from day 2 to day 6, or from day 2 to day 5, or from day 2 to day 4, or from day 2 to day 3 following exposing the mPS to said conditions which are permissive to differentiation of the mPS cells and in which Activin signalling is substantially absent, and the duration of the methods may be between 6 and 16 days, preferably between 8 and 12 days, more preferably between 9 and 1 1 days, even more preferably about 10 days following exposing the mPS cells to said conditions which are permissive to differentiation of the mPS cells and in which Activin signalling is substantially absent.. Very preferably, the Eomes activity may be provided from day 2 to day 4 or from day 2 to day 3 following exposing the mPS to said conditions which are permissive to differentiation of the mPS cells and in which Activin signalling is substantially absent, and the duration of the methods may be between 9 and 1 1 days, even more preferably about 10 days following exposing the mPS cells to said conditions which are permissive to differentiation of the mPS cells and in which Activin signalling is substantially absent.

The start of said exposure, in other words the time corresponding to day 0 (t = day 0 ), as intended throughout this specification denotes the time when the mPS cells are first exposed to said conditions which are permissive to differentiation of the mPS cells and in which Activin signalling is substantially absent. Thus, by means of illustration but without limitation, providing Eomes activity in the mPS cells at t = day 1 or t = day 2 would denote that, respectively, at about 24 hours or at about 48 hours following the time t = day 0 the act providing the Eomes activity is performed.

In further preferred embodiments of the methods or uses as taught herein, the mPS cells may be exposed to conditions in which Activin signalling is substantially absent (i.e., Activin signalling may be substantially absent) at least before and during providing Eomes activity in said cells. Whereas the term "before" is clear per se, it may particularly denote in this context the period from t = day 0 to the start of the provision of the Eomes activity in said cells. Whereas the term "during" is clear per se, it may particularly denote in this context the period from the start of the provision of the Eomes activity in said cells to the end of the provision of the Eomes activity in said cells or to a time point preceding said end of the provision of the Eomes activity, insofar observing such time point conforms with the purpose of the present methods and uses (i.e., in particular obtaining mesodermal cells, particularly cardiac cells, more particularly cardiomyocyte cells). Hence, the mPS cells may but may need not be exposed to conditions in which Activin signalling is substantially absent (i.e., Activin signalling may be substantially absent) after providing Eomes activity in said cells.

In exemplary non-limiting embodiments the methods may entail:

- in one embodiment: exposing the mPS cells to conditions permissive to differentiation of the mPS cells and in which Activin signalling is substantially absent from t = day 0 to t = day (end), and providing Eomes activity in the mPS cells from t = day 1 or 2 (preferably t = day 2) to t = day 4; - in another embodiment: exposing the mPS cells to conditions permissive to differentiation of the mPS cells and in which Activin signalling is substantially absent from t = day 0 to t = day (end), and providing Eomes activity in the mPS cells from t = day 1 or 2 (preferably t = day 2) to t = day 3;

- in a further embodiment: exposing the mPS cells to conditions permissive to differentiation of the mPS cells from t = day 0 to t = day (end), exposing the mPS cells to conditions in which Activin signalling is substantially absent from t = day 0 to t = day 4, whereby Activin signalling may be present or may be substantially absent from t = day 4 to t = day (end), and providing Eomes activity in the mPS cells from t = day 1 or 2 (preferably t = day 2) to t = day 4;

- in another embodiment: exposing the mPS cells to conditions permissive to differentiation of the mPS cells from t = day 0 to t = day (end), exposing the mPS cells to conditions in which Activin signalling is substantially absent from t = day 0 to t = day 3, whereby Activin signalling may be present or may be substantially absent from t = day 3 to t = day (end), and providing Eomes activity in the mPS cells from t = day 1 or 2 (preferably t = day 2) to t = day 3;

- in one embodiment: exposing the mPS cells to conditions permissive to differentiation of the mPS cells and in which Activin signalling is substantially absent from t = day 0 to t = day (end), and providing Eomes activity in the mPS cells from t = day 0 to t = day 4;

- in a further embodiment: exposing the mPS cells to conditions permissive to differentiation of the mPS cells and in which Activin signalling is substantially absent from t = day 0 to t = day (end), and providing Eomes activity in the mPS cells from t = day 0 to t = day 3;

- in another embodiment: exposing the mPS cells to conditions permissive to differentiation of the mPS cells from t = day 0 to t = day (end), exposing the mPS cells to conditions in which Activin signalling is substantially absent from t = day 0 to t = day 4, whereby Activin signalling may be present or may be substantially absent from t = day 4 to t = day (end), and providing Eomes activity in the mPS cells from t = day 0 to t = day 4;

- in another embodiment: exposing the mPS cells to conditions permissive to differentiation of the mPS cells from t = day 0 to t = day (end), exposing the mPS cells to conditions in which Activin signalling is substantially absent from t = day 0 to t = day 3, whereby Activin signalling may be present or may be substantially absent from t = day 3 to t = day (end), and providing Eomes activity in the mPS cells from t = day 0 to t = day 3; - in a further embodiment: exposing the mPS cells to conditions permissive to differentiation of the mPS cells and in which Activin signalling is substantially absent from t = day 0 to t = day (end), and providing Eomes activity in the mPS cells from t = day 0, 1 or 2 (preferably t = day 1 or 2, more preferably t = day 2) to t = day (end).

In the preceding paragraph, reference to "t = day (end)" denotes the end of the duration of the respective method, such as for example t = day (end) may refer to between 6 and 16 days, preferably between 8 and 12 days, more preferably between 9 and 1 1 days, even more preferably about 10 days following t = day 0.

As intended herein, the term "providing Eomes activity in mPS cells" particularly denotes an act which provides, such as adds or increases, the Eomes activity ("gain-of-function") beyond or above the Eomes activity (if any) endogenously present in the mPS cells before said act was performed, to ensure Eomes activity in the mPS cells adequate to achieve the purpose of the herein described methods or uses (i.e., in particular obtaining mesodermal cells, particularly cardiac cells, more particularly cardiomyocyte cells).

In further preferred embodiments of the methods or uses as taught herein, the Eomes activity may be provided by increasing the amount of Eomes in the mPS cells, preferably by overexpressing Eomes in the mPS cells, more preferably by inducibly overexpressing Eomes in the mPS cells.

In further preferred embodiments of the methods or uses as taught herein, said mesodermal cells may comprise cardiac cells. Preferably, said cardiac cells are typified by comprising expression of cardiac Troponin T (cTNT) and/or forming beating clusters of cells. More preferably, said cardiac cells may be cardiomyocytes.

The methods or uses as taught herein may commonly achieve cell populations comprising or enriched for said mesodermal or cardiac cells, and optionally comprising other cell types. Accordingly, in further preferred embodiments the present uses or methods produce a cell population comprising said mesodermal or cardiac cells.

The methods or uses as taught herein thus allow to produce enriched populations of mesodermal or cardiac cells, particularly cardiomyocytes, using simple and robust techniques. If desired, such cell populations may be collected or harvested and said mesodermal or cardiac cells may be further enriched or isolated there from on the basis of their distinctive characteristics (such as, for example, their marker expression as defined above) using methods generally known in the art (e.g., FACS, clonal culture). Such cells may be employed in various applications, including medicinal (e.g., preventative or therapeutic) applications, such as without limitation cell therapy of cardiovascular or cardiac diseases; cell-based drug screening or cardiotoxicity assays; or they may be used as a model for studying pathology of said cardiovascular or cardiac diseases or cardiogenesis.

Accordingly, in further aspects the invention provides the mesodermal cells or cardiac cells or the cell population comprising said mesodermal or cardiac cells, obtainable or directly obtained using the methods as disclosed herein.

In related aspects, the invention provides compositions, including pharmaceutical compositions, comprising the mesodermal cells or cardiac cells or cell populations as disclosed herein.

In other aspects, the invention provides the mesodermal cells or cardiac cells or the cell population comprising said mesodermal or cardiac cells as disclosed herein for use in medicine; preferably the cardiac cells or the cell population comprising said cardiac cells as disclosed herein or the pharmaceutical composition comprising the cardiac cells for use in the treatment of cardiac or cardiovascular diseases.

A related aspect provides the use of the cardiac cells or the cell population comprising said cardiac cells as disclosed herein for the preparation of a medicament for treating cardiac or cardiovascular diseases.

A related aspect provides a method for treating cardiac or cardiovascular diseases in a patient in need of such treatment, comprising administering a therapeutically or prophylactically effective amount of the cardiac cells or the cell population comprising said cardiac cells as disclosed herein to said patient.

In further aspects, the invention provides uses of the mesodermal cells or cardiac cells or the cell population comprising said mesodermal cells or cardiac cells as taught herein for cell-based assays, such as drug screening or cardiotoxicity assays, or as a model for studying cardiovascular or cardiac diseases or cardiogenesis.

These and further aspects and preferred embodiments of the invention are described in the following sections and in the appended claims. The subject matter of appended claims is hereby specifically incorporated in this specification.

BRIEF DESCRIPTION OF FIGURES

Figure 1 : Generation of a doxycylin-inducible MycEomes expressing ESC cell line.

MycEomes expression in A2lox.Cre induced for 24 hours with Dox (+Dox) or not, as revealed by immunofluoresence for the N-terminal Myc-tag. Nuclei are counterstained for Hoechst. Scale bars, 100 μηι. Figure 2: Induction of Eomes during ESC differentiation promotes development of cardiac mesoderm. (A,B) Doxycyclin-inducible MycEomes mouse embryonic stem cells (ESC) were cultured in defined default medium (DDM, consisting of DMEM/F12 + GlutaMAX supplemented with N2 supplement (1 x), 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 500 μg ml BSA, 0.1 mM β-mercaptoethanol and 50 U/ml penicillin/streptomycin) for 10 days, in the presence or absence of doxycyclin (Dox) from day 2-3, and immunostained for β-tubulin III and cTnT (A) or Sox17 and Smooth muscle actin (SMA) (B). Nuclei stained with Hoechst dye. Scale bars, 100 μηι. (C) Higher magnification shows cTnt-positive striated myofibrils. Scale bar, 10 μηη. (D) qRT-PCR for Tubb3, Sox17, Sox1 and Tnnt2 at day 10 of differentiation in DDM with or without Dox. Data are presented as mean expression normalized to TBP + SEM.

Figure 3: High concentrations of Activin prevent cells overexpressing Eomes from differentiating into cardiac mesoderm. (A) ESC were cultured in defined default medium (DDM) for 10 days, in the presence or absence of Activin (100 ng/ml) from day 0- 4 and/or doxycyclin (Dox) from day 2-3 and stained for β-tubulin III and cTnT. Scale bars, 100 μηη. (B) Flow cytometric analysis of cTnT expressing cells at day 10 of ESC differentiation with or without Dox as in (A). During the first 4 days, cells were cultured in the presence of increasing concentrations of Activin (0, 1 , 10 and 100 ng/ml), or with 2% serum. Data are presented as mean proportion of positive cells + SEM. DETAILED DESCRIPTION OF THE INVENTION

As used herein, the singular forms "a", "an", and "the" include both singular and plural referents unless the context clearly dictates otherwise.

The terms "comprising", "comprises" and "comprised of" as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The term also encompasses "consisting of" and "consisting essentially of".

The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

The term "about" as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of and from the specified value, in particular variations of +/-10% or less, preferably +/-5% or less, more preferably +/-1 % or less, and still more preferably +/-0.1 % or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier "about" refers is itself also specifically, and preferably, disclosed.

Whereas the term "one or more", such as one or more members of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any 3, 4, 5, 6 or 7 etc. of said members, and up to all said members.

All documents cited in the present specification are hereby incorporated by reference in their entirety.

Unless otherwise specified, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions may be included to better appreciate the teaching of the present invention. When certain terms are explained or defined in connection with a particular aspect or embodiment, such connotation is meant to apply throughout this specification, i.e., also for other aspects or embodiments, unless otherwise specified or unless the context clearly dictates otherwise.

For general methods relating to the invention, reference is made inter alia to well-known textbooks, including, e.g., "Molecular Cloning: A Laboratory Manual, 2nd Ed." (Sambrook et al., 1989), Animal Cell Culture (R. I. Freshney, ed., 1987), the series Methods in Enzymology (Academic Press), Gene Transfer Vectors for Mammalian Cells (J. M. Miller & M. P. Calos, eds., 1987); "Current Protocols in Molecular Biology and Short Protocols in Molecular Biology, 3rd Ed." (F. M. Ausubel et al., eds., 1987 & 1995); Recombinant DNA Methodology II (R. Wu ed., Academic Press 1995).

General techniques in cell culture and media uses are outlined inter alia in Large Scale Mammalian Cell Culture (Hu et al. 1997. Curr Opin Biotechnol 8: 148); Serum-free Media (K. Kitano. 1991 . Biotechnology 17: 73); or Large Scale Mammalian Cell Culture (Curr Opin Biotechnol 2: 375, 1991 ).

For further elaboration of general techniques useful in the practice of this invention, the practitioner can refer to standard textbooks and reviews in cell biology, tissue culture, and embryology. Included are inter alia "Teratocarcinomas and embryonic stem cells: A practical approach" (E. J. Robertson, ed., IRL Press Ltd. 1987); "Guide to Techniques in Mouse Development" (P. M. Wasserman et al. eds., Academic Press 1993); "Embryonic Stem Cells: Methods and Protocols" (Kursad Turksen, ed., Humana Press, Totowa N.J., 2001 ); "Embryonic Stem Cell Differentiation in Vitro" (M. V. Wiles, Meth. Enzymol. 225: 900, 1993); "Properties and uses of Embryonic Stem Cells: Prospects for Application to Human Biology and Gene Therapy" (P. D. Rathjen et al., al.,1993). Differentiation of stem cells is reviewed, e.g., in Robertson. 1997. Meth Cell Biol 75: 173; Roach and McNeish. 2002. Methods Mol Biol 185: 1 -16; and Pedersen. 1998. Reprod Fertil Dev 10: 31.

As noted, the inventors surprisingly realised that exposure of mPS cells to conditions which are permissive to differentiation of the mPS cells and in which Activin signalling is substantially absent produces mesodermal cells when Eomes activity is provided in the mPS cells.

The methods and uses disclosed herein for producing mesodermal cells, such as particularly cardiac cells, may be denoted as in vitro methods. The term "in vitro" generally denotes outside, or external to, a body, e.g., an animal or human body. The term "ex vivo" typically refers to tissues or cells removed from a body, e.g., an animal or human body, and maintained or propagated outside the body, e.g., in a culture vessel. The term "in vitro" as used herein should be understood to include "ex vivo".

As noted, the methods and uses as taught herein can produce mesodermal cells, such as particularly cardiac cells, from undifferentiated mammalian pluripotent stem (mPS) cells. Hence, such methods or uses may also be denoted as being for differentiation of mPS cells into mesodermal cells, such as particularly cardiac cells.

The term "stem cell" generally refers to a progenitor cell capable of self-renewal, i.e., which can under appropriate conditions proliferate without differentiation. The term encompasses stem cells capable of substantially unlimited self-renewal, i.e., wherein at least a portion of the stem cell's progeny substantially retains the unspecialised or relatively less specialised phenotype, the differentiation potential, and the proliferation capacity of the mother stem cell; as well as stem cells which display limited self-renewal, i.e., wherein the capacity of the stem cell's progeny for further proliferation and/or differentiation is demonstrably reduced compared to the mother cell.

With "progenitor cell" or "progenitor" is meant herein an unspecialised or relatively less specialised and proliferation-competent cell which can under appropriate conditions give rise to at least one relatively more specialised cell type, such as inter alia to relatively more specialised progenitor cells or eventually to terminally differentiated cells, i.e., fully specialised cells that may be post-mitotic. A progenitor cell may "give rise" to another, relatively more specialised cell when, for example, the progenitor cell differentiates to become said other cell without previously undergoing cell division, or if said other cell is produced after one or more rounds of cell division and/or differentiation of the progenitor cell. As used herein, the qualifier "pluripotent" denotes the capacity of a cell to give rise to cell types originating from all three germ layers of an organism, i.e., mesoderm, endoderm, and ectoderm, and potentially capable of giving rise to any and all cell types of an organism, although not able of growing into the whole organism.

The term "mammalian pluripotent stem cell" or "mPS" cell generally refers to a pluripotent stem cell of mammalian origin. The terms "mammal" and "mammalian" refer to any animal classified as such, including, but not limited to, humans, domestic and farm animals, zoo animals, sport animals, pet animals, companion animals and experimental animals, such as, for example, mice, rats, hamsters, rabbits, dogs, cats, guinea pigs, cattle, cows, sheep, horses, pigs and primates, e.g., monkeys and apes. In an embodiment, the mPS cells may be derived from a non-human mammal. For example, in an embodiment the mPS cells may be derived from a laboratory animal, e.g., laboratory mammal, preferably from mouse, rat, hamster or rabbit, more preferably mouse. In another preferred embodiment, the mPS cells may be derived from pig. In yet another preferred embodiment, the mPS cells may be derived from primate, such as from non-human primate. In yet another preferred embodiment, the mPS cells may be derived from human (hPS cells).

Prototype mPS cell is a pluripotent stem cell derived from any kind of mammalian embryonic tissue, e.g., embryonic, foetal or pre-foetal tissue, the cell being capable under appropriate conditions of producing progeny of different cell types that are derivatives of all three germ layers, i.e., endoderm, mesoderm, and ectoderm, according to a standard art-accepted test, such as inter alia the ability to form a teratoma in SCID mice, or the ability to form identifiable cells of all three germ layers in tissue culture.

Included in the definition of mPS cells are without limitation embryonic stem cells of various types, exemplified without limitation by murine embryonic stem cells, e.g., as described by Evans & Kaufman 1981 (Nature 292: 154-6) and Martin 1981 (PNAS 78: 7634-8); rat pluripotent stem cells, e.g., as described by lannaccone et al. 1994 (Dev Biol 163: 288-292); hamster embryonic stem cells, e.g., as described by Doetschman et al. 1988 (Dev Biol 127: 224-227); rabbit embryonic stem cells, e.g., as described by Graves et al. 1993 (Mol Reprod Dev 36: 424-433); porcine pluripotent stem cells, e.g., as described by Notarianni et al. 1991 (J Reprod Fertil Suppl 43: 255-60) and Wheeler 1994 (Reprod Fertil Dev 6: 563-8); sheep embryonic stem cells, e.g., as described by Notarianni et al. 1991 (supra); bovine embryonic stem cells, e.g., as described by Roach et al. 2006 (Methods Enzymol 418: 21 -37); human embryonic stem (hES) cells, e.g., as described by Thomson et al. 1998 (Science 282: 1 145-1 147); human embryonic germ (hEG) cells, e.g., as described by Shamblott et al. 1998 (PNAS 95: 13726); embryonic stem cells from other primates such as Rhesus stem cells, e.g., as described by Thomson et al. 1995 (PNAS 92:7844-7848) or marmoset stem cells, e.g., as described by Thomson et al. 1996 (Biol Reprod 55: 254-259).

Other types of mPS cells are also included in the term as are any cells of mammalian origin capable of producing progeny that includes derivatives of all three germ layers, regardless of whether they were derived from embryonic tissue, foetal tissue or other sources. mPS cells are preferably not derived from a malignant source. A cell or cell line is from a "non-malignant source" if it was established from primary tissue that is not cancerous, nor altered with a known oncogene. It may be desirable that the mPS maintain a normal karyotype throughout prolonged culture under appropriate conditions. It may also be desirable, but not always necessary, that the mPS maintain substantially indefinite self- renewal potential under appropriate in vitro conditions.

As noted, prototype "human ES cells" are described by Thomson et al. 1998 {supra) and in US 6,200,806. The scope of the term covers pluripotent stem cells that are derived from a human embryo at the blastocyst stage, or before substantial differentiation of the cells into the three germ layers. ES cells, in particular hES cells, are typically derived from the inner cell mass of blastocysts or from whole blastocysts. Derivation of hES cell lines from the morula stage has been documented and ES cells so obtained can also be used in the invention (Strelchenko et al. 2004. Reproductive BioMedicine Online 9: 623-629). As noted, prototype "human EG cells" are described by Shamblott et al. 1998 {supra). Such cells may be derived, e.g., from gonadal ridges and mesenteries containing primordial germ cells from foetuses. In humans, the foetuses may be typically 5-1 1 weeks post- fertilisation.

Those skilled in the art will appreciate that, except where explicitly required otherwise, the term mPS cells may include primary tissue cells and established lines that bear phenotypic characteristics of the respective cells, and derivatives of such primary cells or cell lines that still have the capacity of producing progeny of each of the three germ layers.

Exemplary but non-limiting established lines of human ES cells include lines which are listed in the NIH Human Embryonic Stem Cell Registry (http://stemcells.nih.gov/research/registry), and sub-lines thereof, such as, lines hESBGN- 01 , hESBGN-02, hESBGN-03 and hESBGN-04 from Bresagen Inc. (Athens, GA), lines Sahlgrenska 1 and Sahlgrenska 2 from Cellartis AB (Goteborg, Sweden), lines HES-1 , HES-2, HES-3, HES-4, HES-5 and HES-6 from ES Cell International (Singapore), line Miz-hES1 from MizMedi Hospital (Seoul, Korea), lines I 3, I 3.2, I 3.3, I 4, I 6, I 6.2, J 3 and J 3.2 from Technion - Israel Institute of Technology (Haifa, Israel), lines HSF-1 and HSF-6 from University of California (San Francisco, CA), lines H 1 , H7, H9, H13, H14 of Wisconsin Alumni Research Foundation / WiCell Research Institute (Madison, Wl), lines CHA-hES-1 and CHA-hES-2 from Cell & Gene Therapy Research Institute / Pochon CHA University College of Medicine (Seoul, Korea), lines H1 , H7, H9, H13, H14, H9.1 and H9.2 from Geron Corporation (Menlo Park, CA), lines Sahlgrenska 4 to Sahlgrenska 19 from Goteborg University (Goteborg, Sweden), lines MB01 , MB02, MB03 from Maria Biotech Co. Ltd. (Seoul, Korea), lines FCNCBS1 , FCNCBS2 and FCNCBS3 from National Centre for Biological Sciences (Bangalore, India), and lines RLS ES 05, RLS ES 07, RLS ES 10, RLS ES 13, RLS ES 15, RLS ES 20 and RLS ES 21 of Reliance Life Sciences (Mumbai, India). Other exemplary established hES cell lines include those deposited at the UK Stem Cell Bank (http://www.ukstemcellbank.org.uk/), and sub-lines thereof, e.g., line WT3 from King's College London (London, UK) and line hES-NCL1 from University of Newcastle (Newcastle, UK) (Strojkovic et al. 2004. Stem Cells 22: 790-7). Further exemplary ES cell lines include lines FC018, AS034, AS034.1 , AS038, SA1 1 1 , SA121 , SA142, SA167, SA181 , SA191 , SA196, SA203 and SA204, and sub-lines thereof, from Cellartis AB (Goteborg, Sweden).

Further within the term mammalian pluripotent stem cells are such mPS cells obtainable by manipulation, such as inter alia genetic and/or growth factor and/or small molecule mediated manipulation, of non-pluripotent mammalian cells, such as somatic and particularly adult somatic mammalian cells, including the use of induced pluripotent stem (iPS) cells, as taught inter alia by Yamanaka et al. 2006 (Cell 126: 663-676), Yamanaka et al. 2007 (Cell 131 : 861 -872) and Lin et al. 2009 (Nature Methods 6: 805-808).

A skilled person will appreciate that further cell lines having characteristics of mammalian, esp. mouse or human, pluripotent cells, esp. ES cells or EG cells, may be established in the future, and these may too be suitable in the present invention. A skilled person can also use techniques known in the art to verify that any established or yet to be established mPS cell lines, or sub-lines thereof, show desirable cell characteristics, such as expansion in vitro in undifferentiated state, preferably normal karyotype and ability of pluripotent differentiation.

The present methods and protocols may preferably depart from pluripotent stem cell populations (e.g., mPS or hPS cell populations) which are "undifferentiated", i.e., wherein a substantial proportion (for example, at least about 60%, preferably at least about 70%, even more preferably at least about 80%, still more preferably at least about 90% and up to 100%) of cells in the stem cell population display characteristics (e.g., morphological features and/or markers) of undifferentiated mPS cells, clearly distinguishing them from cells undergoing differentiation.

Undifferentiated mPS cells are generally easily recognised by those skilled in the art, and may appear in the two dimensions of a microscopic view with high nuclear/cytoplasmic ratios and prominent nucleoli, may grow as compact colonies with sharp borders. It is understood that colonies of undifferentiated cells within the population may often be surrounded by neighbouring cells that are more differentiated. Nevertheless, the undifferentiated colonies persist when the population is cultured or passaged under appropriate conditions known per se, and individual undifferentiated cells constitute a substantial proportion of the cell population. Undifferentiated mPS cells may express the stage-specific embryonic antigens (SSEA) 3 and 4, and markers detectable using antibodies designated Tra-1 -60 and Tra-1 -81 (Thomson et al. 1998, supra). Undifferentiated mPS cells may also typically express Nanog, Oct-4 and TERT. Undifferentiated mPS cells may also comprise expression of alkaline phosphatase (AP) (e.g., as determined by a suitable AP activity assay).

Culture conditions which allow for continuous proliferation of pluripotent stem cells, mPS or hPS cells in culture substantially without inducing differentiation are generally known in the art.

By means of example and not limitation, serum-containing hES cell medium may be made with 80% DMEM (such as Knock-Out DMEM, Gibco), 20% of either defined foetal bovine serum (FBS, Hyclone) or serum replacement (e.g., WO 98/30679, i.e., serum-free conditions), 1 % non-essential amino acids, 1 mM L-glutamine, and 0.1 mM β- mercaptoethanol. Just before use, human b-FGF may be added to 4ng/ml_ (WO 99/20741 ).

Conventionally, albeit in no way limiting, ES cells may be cultured on a layer of feeder cells, typically fibroblasts derived from embryonic or foetal tissue, typically mouse tissue. In a non-limiting example, embryos may be harvested from a CF1 mouse at 13 days of pregnancy, transferred to 15 mL trypsin/EDTA, finely minced, and incubated 25 min at 37°C. Medium comprising 90% DMEM, 10% FBS, 1 % non essential amino acids and 2 mM glutamine was added, the debris is allowed to settle, and the cells are propagated in the same medium. To prepare a feeder cell layer, cells are treated to inhibit proliferation but permit synthesis of factors that support ES cells. Culture plates are coated with 0.1 % gelatine for 2 h, plated with 375,000 mitomycine-treated (10 g/ml) mEF per well, and used up to 4 days after plating. The medium is replaced with fresh hES cell medium just before seeding the pluripotent stem cells, mPS or hPS. Alternatively, pluripotent stem cells, mPS or hPS cells may be cultured on mammalian, e.g., primate or human fibroblasts. For example, Genbacev et al. 2005 (Fertil Steril. 83: 1517-29) described culturing of hES cells on pathogen-free human placental fibroblast feeders, and in serum-free conditions, Lee et al. 2005 (Biol Reprod 72: 42-9) and Lee et al. 2004 (Reproduction 128: 727-35) disclosed culturing of hES cells on mitotically inactivated human uterine endometrial cells, and Richards et al. 2002 (Nat Biotechnol 20: 933-6) described culturing of hES cells on human foetal and adult fibroblast feeders. Furthermore, pluripotent stem cells, mPS or hPS cells may be propagated in the absence of a feeder cell layer. For example, Xu et al. 2001 (Nature Biotechnology 19: 971 ) discloses culturing of pluripotent stem cells in the absence of feeder cells, wherein the cells are cultured on an extracellular matrix in a medium conditioned by MEF cells, WO 2001/51616 discloses culturing of pluripotent stem cells in the absence of feeder cells, wherein the cells are cultured on an extracellular matrix, e.g. , Matrigel ® Basement Membrane Matrix (e.g., BD Biosciences) or laminin, and in a medium conditioned by primary or permanent cell lines, e.g., primary embryonic fibroblasts, telomerised fibroblasts, and fibroblast cells differentiated and selected from cultured pluripotent stem cells, and WO 2003/020920 and WO 2006/017370 disclose feeder-free culturing of pluripotent stem cells on support structures, such as extracellular matrix, in media supplemented with sufficient fibroblast growth factor.

An initial step of the present methods, in particular step a), involves plating mPS cells onto a substrate which allows adherence of cells thereto. Hence, the methods embodying the principles of the invention involve direct differentiation of mPS cells in an adherent culture rather than initially differentiating the cells through embryoid bodies (suspension culture).

The terms "plating", "seeding" or "inoculating" generally refer to introducing a cell population into an in vitro environment capable of promoting the survival and/or growth of the introduced cells. Typically, said environment may be provided in a system suitably delimited from the surroundings, such as in a culture vessel known per se, e.g., cell culture flask, well plate or dish. Said environment comprises at least a medium, typically a liquid medium, which supports the survival and/or growth of the cells. The medium may be fresh, i.e. , not previously used for culturing of cells, or may comprise at least a portion conditioned by prior culturing of cells therein, e.g., culturing of the cells which are being plated or antecedents thereof, or culturing of cells unrelated to the cells being plated. mPS cells grown without differentiation typically form colonies on an adherent substrate. To allow for plating of so-grown mPS cells, they may be detached from said substrate and at least partly dissociated from one another, so as to obtain a dispersion of mPS cells and/or clumps or clusters thereof usually in an isotonic buffer (e.g., PBS or Hanks balanced salt solution) or medium. Appropriate ways of detaching and dissociating adherent mPS cultures are generally known in the art and may include without limitation treatment with proteolytic enzymes, chelation of bivalent ions, mechanical disintegration, or combinations of any of the above.

Exemplary proteolytic enzymes encompass, e.g., trypsin, collagenase (e.g., collagenase type I, collagenase type II, collagenase type III, or collagenase type IV), elastase, Accutase™ (Innovative Cell Technologies), dispase, pronase, papain, plasmin or plasminogen (WO 1994/03586), which may be used in quantities and at conditions known per se in the art. Trypsin or collagenase may be preferred. Chelation of bivalent ions, primarily of Ca2+ and Mg2+, may be effected using chelators, such as, e.g., EDTA (ethylenediamine tetraacetic acid) or a di-sodium salt thereof, or EGTA (ethyleneglycerol tetraacetic acid) or a di-sodium salt thereof, using concentrations and conditions known per se. EDTA may be preferred. Exemplary mechanical dissociation of cells may involve repeated passing of cell colonies, clumps or clusters through a small bore pipette (e.g., a 1000μΙ micropipette tip) to shear the cell associations. Mechanical cell dissociation may, when used in isolation, lead to cell damage and may thus be advantageously combined with a prior treatment with proteolytic enzymes and/or chelators. A suitable method of cell detachment and dissociation should preserve viability of the cells; preferably, a cell suspension obtained following detachment and dissociation may comprise at least 60% of viable cells, e.g., > 70%, more preferably > 80%, and most preferably > 90% or up to 100% of viable cells.

The detachment and dissociation of mPS cells for subsequent plating can yield a cell suspension comprising individual mPS cells and/or clumps or clusters of mPS cells. For example, the conditions of detachment and dissociation may be such as to provide a cell suspension comprising at least 10%, e.g., at least 20%, at least 30%, at least 40%, preferably at least 50%, e.g., at least 60%, and more preferably at least 70%, e.g., at least 80%, at least 90% or up to 100% of mPS cells as individual cells. Clumps or clusters of mPS cells present in such cell suspension may contain on average, e.g., between >1 and 1000 cells, between 1 and 500 cells, between 1 and 100 cells, between 1 and 50 cells or between 1 and 20 cells, e.g., about 5 cells, about 10 cells or about 15 cells.

Preferably, the mPS cells may be plated at a comparably low density, such as between about 1x101 and about 1x105 cells/cm2, more preferably between about 1x102 and about 5x104 cells/cm2, even more preferably between about 1x103 and about 1x104 cells/cm2, e.g., at about 1 x103, about 2x103, about 3x103, about 4x103, about 5x103, about 6x103, about 7x103, about 8x103, or about 9x103 cells/cm2.

As noted, the mPS cells are plated onto a substrate which allows adherence of cells thereto. Hence, the culture system whereto the mPS cells are plated may comprise a surface compatible with cell adhesion, whereby the so-plated mPS cells contact and attach to said substrate surface.

In general, a substrate which allows adherence of cells thereto may be any substantially hydrophilic substrate. In an embodiment, a suitable adherent substrate may be surface- treated (e.g., treated by atmospheric corona discharge, radio frequency vacuum plasma treatment, or DC glow discharge or plasma treatment, as known in the art) tissue culture plastic, which may typically display polar and/or hydrophilic chemical moieties, such as, e.g., amines, amides, carbonyls, carboxylates, esters, hydroxyls, sulfhydryls and the like. In an alternative embodiment, a suitable adherent substrate may be glass, optionally surface-treated to introduce functional groups such as listed above to increase the hydrophilicity. Further adherent substrates may be generated via surface-coating of, for example, tissue-culture plastic or glass, with hydrophilic substances. In an example, said coating may involve suitable poly-cations, such as, e.g., poly-ornithine or poly-lysine. In other examples, preferred coating may comprise one or more components of extracellular matrix, e.g., the ECM proteins fibrin, laminin, collagen (preferably collagen type 1 ), gelatine, glycosaminoglycans (e.g., heparin or heparan sulphate), fibronectin, vitronectin, elastin, tenascin, aggrecan, agrin, bone sialoprotein, cartilage matrix protein, fibrinogen, fibulin, mucins, entactin, osteopontin, plasminogen, restrictin, serglycin, SPARC/osteonectin, versican, thrombospondin 1 , or cell adhesion molecules including cadherins, connexins, selectins, by themselves or in various combinations.

A particularly preferred example of adherent substrate surface for plating mPS cells according to the invention comprises or consists of gelatine. The term "gelatine" as used herein refers to a heterogeneous mixture of water-soluble proteins of high average molecular weight derived from the collagen-containing parts of animals, such as skin, bone and ossein by hydrolytic action, usually either acid hydrolysis or alkaline hydrolysis. The term "gelatine" also encompasses suitable chemical derivatives thereof such as acetylated gelatine or cross-linked gelatine. Protocols for surface treatment of tissue culture surfaces with gelatine are known in the art. By means of illustration and not limitation, culture vessels may be treated for 2 hours or longer, e.g., for 24 hours, with 0.02%-1 % (w/v), typically with about 0.1 % (w/v) gelatine in, e.g., distilled and preferably sterilised water. Typically, after plating of the mPS cells, the cell suspension is left in contact with the adherent surface to allow for adhesion of mPS cells from the plated cell population to said substrate. In embodiments, the mPS cell suspension may be contacted with the adherent surface for at least about 0.5h, e.g., for about >1 h, preferably for about >2h, for about >4h, more preferably for about >8h, e.g., for about >12h, even more preferably for about >16h, e.g., for about >20h, and most preferably for about 24h. In further preferred embodiments, the mPS cell suspension may be contacted with the adherent surface for between about 2h and about 48h, e.g., for about 12h or about 24h. Although longer contacting times (before removal of the non-adherent matter) are possible, they are in general not necessary.

After mPS cells are allowed to attach to the adherent substrate, non-adherent matter is typically removed from the culture system. Non-adherent matter may comprise, for example, cells that have not attached to the adherent substrate, non-viable or dead cells, cell debris, etc. Non-adherent matter may be suitably removed by exchanging medium within the culture system, optionally including one or more washes of the attached cells with suitable medium or isotonic buffer. Hereby, cells from the mPS suspension which have adhered to the substrate surface are selected for further culturing.

The present methods typically involve culturing (e.g., maintaining and/or propagating and/or differentiating) the cells and cell populations taught herein in the presence of cell or tissue culture media, such as for example using liquid or semi-solid (e.g., gelatinous), and preferably liquid cell or tissue culture media. Such culture media can desirably sustain the maintenance (e.g., survival, genotypic, phenotypic and/or functional stability) and/or propagation of the cells or cell populations.

In particular, the present methods comprise as a further step exposing the mPS cells that have attached to the adherent substrate to conditions (e.g., culturing the mPS cells that have attached to the adherent substrate in a medium) "permissive to differentiation of the mPS cells", which means that the medium may for example not contain components, in sufficient quantity, which would suppress mPS differentiation or would cause maintenance and/or proliferation of the mPS cells in undifferentiated or substantially undifferentiated state. By means of illustration, such components absent from the medium may include leukaemia inhibitory factor (LIF), basic fibroblast growth factor (b-FGF), and/or embryonic fibroblast feeders or conditioned medium of such feeders, depending on the particular mPS cell type.

As noted, said conditions (e.g., medium) are such in which Activin signalling is substantially absent, e.g., is completely absent or is present only to a degree which does not prevent or significantly diminish the differentiation of the mPS cells to mesodermal, cardiac or cardiomyocyte cells.

For example, "substantially absent" Activin signalling may correspond to a degree of Activin signalling in which the present methods or uses are > 60%, preferably >70%, more preferably > 80% and even more preferably > 90% or >95% effective compared to conditions in which Activin signalling is completely absent. The effectiveness may be expressed as a proportion of cells which attain mesodermal or cardiac (e.g., cTNT positive) phenotype.

For example, "substantially absent" Activin signalling may correspond to a degree of Activin signalling comparable to exposing the mPS cells to 5 ng/ml or less, preferably 4 ng/ml or less, more preferably 3 ng/ml or less, even more preferably 2 ng/ml or less and most preferably 1 ng/ml or less of Activin A.

The presence of Activin signalling may as well be suitably assessed by inspecting the extent of phosphorylation of Smad 2/3 and/or and its association with Smad4 and/or the translocation of this complex to the cell nucleus. Hence, the conditions may lack any components that may otherwise induce these changes in the mPS cells, preferably phosphorylation of Smad 2/3 in the mPS cells.

In a preferred example, the conditions to which the mPS cells are exposed may substantially lack animal serum, animal plasma, Activin and Nodal. It shall be appreciated that said conditions may entirely lack such factors or may comprise trace amounts thereof below concentrations at which they would exhibit a (significant) effect on the differentiation of the mPS cells.

Typically, animal serum and animal plasma comprise factors that may induce Activin signalling. Accordingly, the conditions may be preferably serum- or plasma-free or may be very low-serum or very low-plasma (e.g., serum or plasma 0.5% vol/vol or lower, preferably about 0.4% vol/vol or lower, more preferably about 0.3% vol/vol or lower, yet more preferably about 0.2% vol/vol, still more preferably about 0.1 % vol/vol). Serum- or plasma-free media may be typically supplemented with defined serum-replacements, which are known per se (e.g., WO 98/30679) and are commercially available.

In an embodiment the medium may lack one, preferably any two or more, and most preferably all of the following: animal (e.g., mammalian) serum, animal (e.g., mammalian) plasma; any members of the transforming growth factor beta (TGF ) family of proteins, such as bone morphogenetic proteins (BMPs), growth and differentiation factors (GDFs), anti-mullerian hormone (AMH), Activin, Nodal and TGF 's. It shall be appreciated that the culture medium may entirely lack such factors or may comprise trace amounts thereof below concentrations at which they would exhibit a (significant) effect on the differentiation of the mPS cells.

The term "serum" is as conventionally defined. Serum can be usually obtained from an isolated sample of whole blood by first allowing clotting to take place in the sample and subsequently separating the so formed clot and cellular components of the blood sample from the liquid component (serum) by an appropriate technique, typically by centrifugation. Clotting can be facilitated by an inert catalyst, e.g., glass beads or powder. Alternatively, serum can be obtained from plasma by removing the anticoagulant and fibrin.

The term "plasma" is as conventionally defined. Plasma is usually obtained from an isolated sample of whole blood, provided or contacted with an anticoagulant, (e.g., heparin, citrate, oxalate or EDTA). Subsequently, cellular components of the blood sample are separated from the liquid component (plasma) by an appropriate technique, typically by centrifugation.

Typically, a medium may comprise a basal medium formulation as known in the art. Various basal media formulations are available, e.g., from the American Type Culture Collection (ATCC) or from Invitrogen (Carlsbad, California). By means of example and not limitation, basal media formulations may include Eagle's Minimum Essential Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), alpha modified Minimum Essential Medium (alpha-MEM), Basal Medium Essential (BME), F12 Nutrient Mixture (Ham), Iscove's Modified Dulbecco's Medium (IMDM), RPMI 1640 Medium, and modifications and/or combinations thereof. Compositions of basal media are generally known in the art and it is within the skill of one in the art to modify or modulate concentrations of media and/or media supplements as necessary for the cells cultured.

Such basal media formulations contain ingredients necessary for mammalian cell development, which are known per se. By means of illustration and not limitation, these ingredients may include inorganic salts (in particular salts containing Na, K, Mg, Ca, CI, P and possibly Cu, Fe, Se and Zn), physiological buffers (e.g., HEPES, bicarbonate or phosphate buffers), pH indicators, sources of carbon (e.g., glucose, sodium pyruvate, sodium acetate), and may further also comprise reducing agents or antioxidants (e.g., glutathione), vitamins, nucleotides, nucleosides and/or nucleic acid bases, ribose, deoxyribose, amino acids, etc.

In a preferred embodiment, basal medium employed in culturing the mPS cells may be chosen from DMEM, or F12 or a mixture thereof. In a particularly preferred embodiment, basal medium employed to culture the mPS cells is DMEM/F12 mixture, more preferably 1 :1 , vol/vol.

For use in culture, basal media can be supplied with one or more further components. For example, additional supplements can be used to supply the cells with further necessary trace elements and substances for optimal growth and expansion. Further antioxidant supplements may be added, e.g., β-mercaptoethanol. While many basal media already contain amino acids, some amino acids may be supplemented later, e.g., L-glutamine, which is known to be less stable when in solution, or a stabilized form dipeptide from L- glutamine, i.e. L-alanyl-L-glutamine (such as sold under the trade name GlutaMAX™). A medium may be further supplied with antibiotic and/or antimycotic compounds, such as, typically, mixtures of penicillin and streptomycin, and/or other compounds, exemplified but not limited to, amphotericin, ampicillin, gentamicin, bleomycin, hygromycin, kanamycin, mitomycin, mycophenolic acid, nalidixic acid, neomycin, nystatin, paromomycin, polymyxin, puromycin, rifampicin, spectinomycin, tetracycline, tylosin, and zeocin. Protein factors such as insulin and transferrin may also be used to supplement culture media.

Lipids and lipid carriers can also be used to supplement cell culture media. Such lipids and carriers can include, but are not limited to cyclodextrin, cholesterol, linoleic acid conjugated to albumin, linoleic acid and oleic acid conjugated to albumin, unconjugated linoleic acid, linoleic-oleic-arachidonic acid conjugated to albumin, oleic acid unconjugated and conjugated to albumin, among others. Albumin can similarly be used in fatty-acid free formulations.

In a preferred embodiment, a suitable basal medium, such as preferably DMEM/F12, may be supplemented with one or more animal polypeptides selected from the group consisting of insulin, transferrin and albumin, preferably my be supplemented with at least insulin, or with at least insulin and transferrin, or may be supplemented with each insulin, transferrin and albumin. In such embodiments, the recited protein(s) may be the only animal proteins with which the basal medium is supplemented.

In a preferred embodiment, a suitable basal medium, such as preferably DMEM/F12, may further comprise any one, preferably any two or more, and more preferably all components chosen from: L-alanyl-L-glutamine, insulin, transferrin, progesterone, putrescine, selenite, non-essential amino acids, sodium pyruvate, beta-mercaptoethanol, bovine serum albumin (BSA) and a mixture of penicillin and streptomycin. Such components may be preferably present as follows: L-alanyl-L-glutamine - usually at final concentration between about 0,5 mM and about 10 mM, preferably between about 1 mM and about 5 mM, more preferably about 2 mM; non-essential amino acids - usually at final concentration between about 0,01 mM and about 1 mM, preferably between about 0,05 mM and about 0,5 mM, more preferably between about 0,08 mM and about 0.12 mM, such as at about 0,1 mM; sodium pyruvate - usually at final concentration between about 0,1 mM and about 10 mM, preferably between about 0,5 mM and about 5 mM, more preferably between about 0,8 mM and about 1 ,2 mM, such as at about 1 mM; beta- mercaptoethanol - usually at final concentration between about 10 μΜ and about 1 mM, preferably between about 50 μΜ and about 500 μΜ, more preferably between about 80 μΜ and about 120 μΜ, such as at about 100 μΜ; BSA - usually at final concentration between about 50 μg/ml and about 5 mg/ml, preferably between about 100 μg/ml and about 1 mg/ml, more preferably between about 250 μg/ml and about 750 μg/ml, such as at about 500 μg/ml; penicillin/streptomycin - usually at final concentration between about 5 U/ml and about 500 U/ml, preferably between about 10 U/ml and about 100 U/ml, more preferably between about 25 U/ml and about 75 U/ml, such as at about 50 U/ml; a mixture of insulin, transferrin, progesterone, putrescine, selenite such as sold as N-2 Supplement - usually at final concentration of 1 x (transferrin - usually at final concentration between about 10 mg/L and about 1 g/L, preferably between about 20 mg/L and about 500 mg/L, more preferably between about 50 mg/L and about 200 mg/L, such as at about 100 mg/L; insulin - usually at final concentration between about 100 μg/L and about 50 mg/L, preferably between about 500 μg/L and about 20 mg/L, more preferably between about 1 mg/L and 10 mg/L, such as about 5 mg/L; progesterone - usually at final concentration between about 1 ng/L and about 10 ng/L, preferably between about 5 ng/L and 7.5 ng/L about, more preferably between about 6.0 ng/L and about 6.5 ng/L, such as at about 6.3 ng/L; putrescine - usually at final concentration between about and about, such as at about 16.1 1 mg/L; selenite - usually at final concentration between about 1 ng/L and about 10 ng/L, preferably between about 2.5 ng/L and about 7.5 ng/L, more preferably between about 5 ng/L and about 5.5 ng/L, such as at about 5.2 ng/L; or such components may be added each separately or in any combinations).

The methods and uses disclosed herein rely on providing Eomes activity in the mPS cells.

The terms "Eomes", "Eomesodermin" and "Tbr2" are synonymous and refer to a T-box transcription factor of the T-brain subfamily known as such in the art.

The terms encompass Eomes of any organism where found, and particularly of animals, preferably warm-blooded animals, more preferably vertebrates, yet more preferably mammals, including humans and non-human mammals, still more preferably of humans. The terms particularly encompass Eomes with a native sequence, i.e., one of which the primary sequence is the same as that of Eomes found in or derived from nature. A skilled person understands that native sequences of Eomes may differ between different species due to genetic divergence between such species. Moreover, native sequences of Eomes may differ between or within different individuals of the same species due to normal genetic diversity (variation) within a given species. Also, native sequences of Eomes may differ between or even within different individuals of the same species due to post- transcriptional or post-translational modifications. Any such variants or isoforms of Eomes are intended herein. Accordingly, all sequences of Eomes found in or derived from nature are considered "native". The terms encompass Eomes when forming a part of a living organism, organ, tissue or cell, when forming a part of a biological sample, as well as when at least partly isolated from such sources. The terms also encompass Eomes when produced by recombinant or (semi-)synthetic means.

Exemplary human Eomes protein sequence may be as annotated under NCBI Genbank (http://www.ncbi.nlm.nih.gov/) accession number NP_005433.2 (sequence version 2, entered August 29, 2002). Exemplary human Eomes mRNA (cDNA) sequence may be as annotated under NCBI Genbank accession number NM_005442.2 (sequence version 2, entered August 29, 2002). Exemplary mouse Eomes protein sequence may be as annotated under NCBI Genbank accession number NP_034266.2 (isoform 1 ) (sequence version 2, entered December 23, 2005) or NP_001 158261 .1 (isoform 2) (sequence version 1 , entered September 18, 2009). Exemplary mouse Eomes mRNA (cDNA) sequence may be as annotated under NCBI Genbank accession number NM_010136.3 (transcript variant 1 ) (sequence version 3, entered September 18, 2009) or NM_001 164789.1 (transcript variant 2) or (sequence version 1 , entered September 18, 2009).

The provision of Eomes activity particularly denotes provision of biological activity of Eomes polypeptide (protein) in the mPS cells, thereby achieving the desired differentiation of mPS cells.

Without limitation, an agent able to provide Eomes activity may be able to effect or increase the expression of Eomes nucleic acid or polypeptide in the mPS cells (Eomes "overexpression"). For example, such agent may comprise, consist essentially of or consist of a recombinant nucleic acid comprising a sequence encoding Eomes polypeptide operably linked to one or more regulatory sequences allowing for expression of said sequence encoding Eomes polypeptide in the mPS cells (expression construct). Introduction (e.g., by transfection or transduction) of such agent to mPS cells shall effect the expression of Eomes polypeptide in the cells. Such recombinant nucleic acid may be comprised in a suitable vector. By "encoding" is meant that a nucleic acid sequence or part(s) thereof corresponds, by virtue of the genetic code of an organism in question to a particular amino acid sequence, e.g., the amino acid sequence of one or more desired proteins or polypeptides.

Preferably, a nucleic acid encoding one or more proteins, polypeptides or peptides may comprise one or more open reading frames (ORF) encoding said one or more proteins, polypeptides or peptides. An "open reading frame" or "ORF" refers to a succession of coding nucleotide triplets (codons) starting with a translation initiation codon and closing with a translation termination codon known per se, and not containing any internal in- frame translation termination codon, and potentially capable of encoding a protein, polypeptide or peptide. Hence, the term may be synonymous with "coding sequence" as used in the art.

An "operable linkage" is a linkage in which regulatory sequences and sequences sought to be expressed are connected in such a way as to permit said expression. For example, sequences, such as, e.g., a promoter and an ORF, may be said to be operably linked if the nature of the linkage between said sequences does not: (1 ) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter to direct the transcription of the ORF, (3) interfere with the ability of the ORF to be transcribed from the promoter sequence.

The precise nature of regulatory sequences or elements required for expression may vary between expression environments, but typically include a promoter and a transcription terminator, and optionally an enhancer.

Reference to a "promoter" or "enhancer" is to be taken in its broadest context and includes transcriptional regulatory sequences required for accurate transcription initiation and where applicable accurate spatial and/or temporal control of gene expression or its response to, e.g., internal or external (e.g., exogenous) stimuli. More particularly, "promoter" may depict a region on a nucleic acid molecule, preferably DNA molecule, to which an RNA polymerase binds and initiates transcription. A promoter is preferably, but not necessarily, positioned upstream, i.e., 5', of the sequence the transcription of which it controls. Typically, in prokaryotes a promoter region may contain both the promoter per se and sequences which, when transcribed into RNA, will signal the initiation of protein synthesis (e.g., Shine-Dalgarno sequence).

In embodiments, promoters contemplated herein may be constitutive or inducible.

The terms "terminator" or "transcription terminator" refer generally to a sequence element at the end of a transcriptional unit which signals termination of transcription. For example, a terminator is usually positioned downstream of, i.e., 3' of ORF(s) encoding a polypeptide of interest. For instance, where a recombinant nucleic acid contains two or more ORFs, e.g., successively ordered and forming together a multi-cistronic transcription unit, a transcription terminator may be advantageously positioned 3' to the most downstream ORF.

The term "vector" generally refers to a nucleic acid molecule, typically DNA, to which nucleic acid segments may be inserted and cloned, i.e., propagated. Hence, a vector will typically contain one or more unique restriction sites, and may be capable of autonomous replication in a defined host or vehicle organism such that the cloned sequence is reproducible. Vectors may include, without limitation, plasmids, phagemids, bacteriophages, bacteriophage-derived vectors, PAC, BAC, linear nucleic acids, e.g., linear DNA, viral vectors, etc., as appropriate. Expression vectors are generally configured to allow for and/or effect the expression of nucleic acids or ORFs introduced thereto in a desired expression system, e.g., in vitro, in a host cell, host organ and/or host organism. For example, expression vectors may advantageously comprise suitable regulatory sequences.

A vector such as an expression vector as intended herein may for example be an autonomously replicating vector (i.e., a vector which exists as an extra chromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid) or a vector which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated, depending on whether transient or stable (e.g., constitutive or inducible) transfection of the sequence of interest into the host cell is pursued.

As used herein, the term "transfection" refers to the introduction of a foreign material like exogenous nucleic acids, typically DNA, into eukaryotic cells by any means of transfer. Different methods of transfection are known in the art and include, but are not limited to, calcium phosphate transfection, electroporation, lipofectamine transfection, DEAE- Dextran transfection, microinjection or virally mediated transfection, i.e. transduction. "Transient transfection" refers to methods of transfection in which the exogenous nucleic acid is not stably incorporated into the recipient host cell's chromosomal DNA and functions for only a limited time. Stably transfection refers to the permanent expression of the transgene due to the integration of the transgene into the genome of the host cell.

In a preferred embodiment, provision of Eomes activity may be performed by stably transfecting said mPS cells with an inducible expression vector comprising the Eomes gene sequence. With "inducible expression vector" is meant herein an expression vector wherein the transgene is under the control of an inducible promoter, i.e. a promoter which activation requires either the presence of a particular compound, i.e. the inducer, or a defined external condition, e.g. elevated temperature. Transient expression of Eomes may then be achieved by e.g. adding a suitable inducer such as doxycyclin to the medium wherein the mPS cells are cultured. Non-limiting examples of such inducible expression vectors include, but are not limited to, the tetracyclin or doxycyclin induced expression systems, Rheo switch systems, CRE-LOX inducible systems, FRT system, IPTG-LAC inducible systems, ecdysone inducible systems, or the cumate repressor/operator systems.

In another example, an agent able to provide Eomes activity may suitably comprise, consist essentially of or consist of Eomes polypeptide, such as preferably isolated or recombinant Eomes polypeptide, for example suitably formulated for introduction to the mPS cells. Such may be suitably obtained through expression by host cells or host organisms, transformed with an expression construct encoding and configured for expression of said protein, polypeptide or peptide in said host cells or host organisms, followed by purification of the protein, polypeptide or peptide. Expression constructs are discussed above. In this context, the terms "host cell" and "host organism" may suitably refer to cells or organisms encompassing both prokaryotes, such as bacteria, and eukaryotes, such as yeast, fungi, protozoan, plants and animals. Contemplated as host cells are inter alia unicellular organisms, such as bacteria (e.g., E. coli, Salmonella tymphimurium, Serratia marcescens, or Bacillus subtilis), yeast (e.g., Saccharomyces cerevisiae or Pichia pastoris), (cultured) plant cells (e.g., from Arabidopsis thaliana or Nicotiana tobaccum) and (cultured) animal cells (e.g., vertebrate animal cells, mammalian cells, primate cells, human cells or insect cells). Contemplated as host organisms are inter alia multi-cellular organisms, such as plants and animals, preferably animals, more preferably warm-blooded animals, even more preferably vertebrate animals, still more preferably mammals, yet more preferably primates; particularly contemplated are such animals and animal categories which are non-human.

Alternatively, an agent able to provide Eomes activity may comprise, consist essentially of or consist of a factor, preferably a transcription factor, that stimulates the expression of endogenous Eomes.

Where a reference is made herein to certain peptides, polypeptides or proteins (such as, e.g., Eomes polypeptide or protein or agents for use herein), such peptides, polypeptides or proteins may be preferably of animal origin, more preferably of mammalian origin, such as of non-human mammalian or human origin, preferably may be of same origin as the cells (mPS cells) being treated. By means of example and without limitation, human mPS cells may be exposed to human Eomes polypeptide, whereas mouse mPS cells may be exposed to mouse Eomes polypeptide. Said peptides, polypeptides or proteins may be, e.g., isolated from biological sources, produced by recombinant means, or produced by synthetic means.

Where a reference is made herein to certain peptides, polypeptides or proteins (such as, e.g., Eomes polypeptide or protein or agents for use herein), such reference is to be understood as also encompassing functional fragments and/or variants of said peptides, polypeptides or proteins.

The term "fragment" generally denotes a N- and/or C-terminally truncated form of a peptide, polypeptide or proteins. Preferably, a fragment may comprise at least about 30%, e.g., at least 50% or at least 70%, preferably at least 80%, e.g., at least 85%, more preferably at least 90%, and yet more preferably at least 95% or even about 99% of the amino acid sequence length of said peptide, polypeptide or protein.

The term "variant" of a recited given peptide, polypeptide or protein to peptides, polypeptides or proteins the amino acid sequence of which is substantially identical (i.e., largely but not wholly identical) to the sequence of said recited peptide, polypeptide or protein, e.g., at least about 85% identical, e.g., preferably at least about 90% identical, e.g., at least 91 % identical, 92% identical, more preferably at least about 93% identical, e.g., 94% identical, even more preferably at least about 95% identical, e.g., at least 96% identical, yet more preferably at least about 97% identical, e.g., at least 98% identical, and most preferably at least 99% identical.

Sequence identity may be determined using suitable algorithms for performing sequence alignments and determination of sequence identity as know per se. Exemplary but non- limiting algorithms include those based on the Basic Local Alignment Search Tool (BLAST) originally described by Altschul et al. 1990 (J Mol Biol 215: 403-10), such as the "Blast 2 sequences" algorithm described by Tatusova and Madden 1999 (FEMS Microbiol Lett 174: 247-250), for example using the published default settings or other suitable settings (such as, e.g., for the BLASTN algorithm: cost to open a gap = 5, cost to extend a gap = 2, penalty for a mismatch = -2, reward for a match = 1 , gap x_dropoff = 50, expectation value = 10.0, word size = 28; or for the BLASTP algorithm: matrix = Blosum62, cost to open a gap = 1 1 , cost to extend a gap = 1 , expectation value = 10.0, word size = 3). The term "functional" denotes that fragments and/or variants at least partly retain the biological activity or functionality of the recited peptides, polypeptides or proteins. Preferably, such functional fragments and/or variants may retain at least about 20%, e.g., at least 30%, or at least 40%, or at least 50%, e.g., at least 60%, more preferably at least 70%, e.g., at least 80%, yet more preferably at least 85%, still more preferably at least 90%, and most preferably at least 95% or even 100% or higher of the activity (such as, e.g., ability to induce or inhibit a pathway or signalling) of the corresponding peptides, polypeptides or proteins. Particularly, a functional fragment or variant of Eomes would retain, to at least a certain degree, the ability to stimulate mesodermal and/or cardiac and/or cardiomyocyte differentiation of mPS cells in the present methods or uses.

Where a reference is made herein to certain substances or molecules or biological molecules such as peptides, polypeptides or proteins (such as, e.g., Eomes polypeptide or protein or agents for use herein), such reference is to be understood as also encompassing functional (i.e., adequately achieving the desired effect or function) derivatives and analogues of substances or molecules or biological molecules. For example, such derivatives or analogues may encompass chemical modifications (e.g., additions, omissions or substitutions of atoms and/or moieties) , and/or biological modifications (e.g., post-expression modifications including, for example, phosphorylation, glycosylation, lipidation, methylation, cysteinylation, sulphonation, glutathionylation, acetylation, oxidation of methionine to methionine sulphoxide or methionine sulphone, and the like).

As noted, the present specification concerns mesodermal cells, particularly cardiac cells, more particularly cardiomyocytes.

The term "mesodermal cell" generally denotes any cell originating from the mesodermal germ layer.

In an embodiment, said mesodermal cells may be positive for smooth muscle actin. In a further embodiment, said mesodermal cells may be positive for at least one vascular marker, indicating that said mesodermal cells may have vascular smooth muscle cell identity.

In a preferred embodiment, said mesodermal cells may comprise cardiac cells.

The term "cardiac cell" as used herein refers to a cell having at least one characteristic associated with the phenotype of a native, specialised (i.e. mature) cardiac cell type such as cardiomyocyte, cardiac vascular smooth muscle cell, cardiac fibroblast or cardiac endothelial cell. In a preferred embodiment, said cardiac cells are beating and are positive for cardiac TroponinT, indicating that said cardiac cells may have cardiomyocyte identity. In a further embodiment, said cardiac cells have a striated pattern of cardiac TroponinT staining.

The expression - for example, the presence or absence or quantity - of markers as discussed throughout this specification by cells or cell populations can be detected and/or measured using any suitable technique known in the art, such as without limitation immunological techniques including immunocytochemistry, immunofluorescence, flow cytometry and fluorescence activated cell sorting (FACS), immunoblotting including inter alia Western blots, dot blots and slot blots, immunoassays including inter alia ELISA (enzyme-linked immunosorbent assay) and RIA (radioimmunoassay) or by any suitable biochemical assay of enzyme activity, or by any suitable technique of detecting and/or measuring the marker mRNA including Northern blots, semi-quantitative or quantitative RT-PCR, array or microarray expression analysis, and so forth. Principles of the above assays are known in the art, and are further set out in the methodology guides cited elsewhere in this specification, and further inter alia in Ed Harlow and David Lane, "Antibodies - A Laboratory Manual", 1st ed., Cold Spring Harbor Laboratory Press 1988, ISBN 0879693142; J.M. Polak, "Introduction to Immunocytochemistry" 3rd ed., Garland Science 2003, ISBN 1859962084; M.A. Hayat, "Microscopy, Immunohistochemistry, and Antigen Retrieval Methods: For Light and Electron Microscopy", 1 st ed., Springer 2002, ISBN 0306467704; John R. Crowther, "The ELISA Guidebook", 1 st ed., Humana Press 2000, ISBN 0896037282; Stephen A. Bustin, "A-Z of Quantitative PCR", 1 st ed., International University Line 2004, ISBN 0963681788; Anton Yuryev, "PCR Primer Design", 1st ed., Humana Press 2007, ISBN 158829725X; and others. Sequence data including gene, transcript and protein sequence data for markers mentioned in this disclosure are generally known and can be retrieved from public databases such as for example GenBank (http://www.ncbi.nlm.nih.gov/entrez) and UniProtKB/Swiss-Prot (http://www.expasy.org).

Where a cell is said to be positive for or to express a particular marker, this means that a skilled person will conclude the presence or evidence of a distinct signal (e.g., antibody- detectable or detection by reverse transcription polymerase chain reaction) for that marker when carrying out the appropriate measurement compared to suitable controls (e.g., cells known or expected not to express the marker, i.e., to be negative for said marker, i.e., "negative control"). Where the method allows for quantitative assessment of the marker, positive or expressing cells may on average generate a signal that is significantly different (e.g., higher) from such negative control, e.g., but without limitation, at least 1 .5-fold higher than such signal generated by the negative control, e.g., at least 2-fold, at least 4- fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40- fold, at least about 50-fold, at least about 100-fold, or at least about 200-fold higher or even higher.

Where a cell is said to be negative for or to not express (or substantially not express) a particular marker, this means that a skilled person will conclude the absence of a distinct signal (e.g., antibody-detectable or detection by reverse transcription polymerase chain reaction) for that marker when carrying out the appropriate measurement compared to suitable controls (e.g., cells known or expected to express the marker, i.e., "positive control"; or cells known or expected not to express the marker, i.e., to be negative for said marker, i.e., negative control). Where the method allows for quantitative assessment of the marker, negative or non-expressing (or substantially non-expressing) cells may on average generate a signal that is comparable to or is not significantly different from such negative control, e.g., but without limitation, which is less than 1.5-fold of the signal generated by the control, e.g., less than 1.4-fold, less than 1.3-fold, less than 1 .2-fold or less than 1.1 -fold or even lower than the signal generated by the control.

As can be understood, the methods or uses disclosed herein can achieve cell populations comprising or enriched for mesodermal cells, particularly cardiac cells, more particularly cardiomyocytes. For example, a cell population obtained or obtainable according to the methods disclosed herein may comprise at least 40 %, preferably at least 50 %, more preferably at least 60%, 70%, 80% or more of mesodermal cells.

In a preferred embodiment, a cell population obtained or obtainable according to the methods disclosed herein may comprise at least 4 %, such as at least 5%, preferably at least 6 %, such as at least 7%, more preferably at least 8%, such as for example at least 9% or at least 10% or more of cardiac cells, particularly cardiomyocytes, such as cells that are beating and that are expressing cardiac Troponin!".

Besides said cardiac cells, cell populations obtained or obtainable according to the methods disclosed herein may further comprise cardiac progenitors. The terms "cardiac progenitor" and "cardiac progenitor cell" generally refer to a progenitor cell that can under appropriate conditions give rise to one or more cardiac cells. For example, a cardiac progenitor cell may give rise to any one or more or all of cardiomyocytes, cardiac fibroblasts, endothelial cells or vascular smooth muscle cells.

As can be appreciated, mesodermal cells or cardiac cells or cardiomyocytes may be further enriched or isolated from cell populations obtained or obtainable according to the methods disclosed herein on the basis of their distinctive characteristics (such as, for example, their marker expression and/or other phenotypic properties taught herein) using methods generally known in the art (e.g., FACS, clonal culture, panning, immunomagnetic cell separation, eic), thereby yielding isolated mesodermal cells or cardiac cells or cardiomyocytes or substantially pure (e.g., >85% pure, preferably >90% pure, more preferably >95% pure or even >99% pure) subpopulations of mesodermal cells or cardiac cells or cardiomyocytes. Accordingly, also disclosed herein are isolated mesodermal cells or cardiac cells or cardiomyocytes and substantially pure populations of mesodermal cells or cardiac cells or cardiomyocytes.

Further contemplated are the progeny of the herein taught cardiac cells, including genetically or otherwise modified derivatives of said cells.

Also provided are downstream derivatives of the herein taught cardiac cells, including without limitation: isolated nucleic acids (e.g., DNA, total RNA or mRNA), isolated or cloned DNA or cDNA, isolated proteins or antigens, isolated lipids, or isolated extracts (e.g., nuclear, mitochondrial, microsomal, etc.) from said cardiac cells.

The invention also provides a composition, preferably a pharmaceutical composition, comprising mesodermal cells or cardiac cells or cardiomyocytes cardiac or cell populations containing such, obtained or obtainable according to the methods disclosed herein.

Apart from said mesodermal cells or cardiac cells or cardiomyocytes cardiac or cell populations, such composition may comprise one or more other components. For example, components may be included that can maintain or enhance the viability of the cells or cell populations. By means of example and without limitation, such components may include salts to ensure substantially isotonic conditions, pH stabilisers such as buffer system(s) (e.g., to ensure substantially neutral pH, such as phosphate or carbonate buffer system), carrier proteins such as for example albumin, media including basal media and/or media supplements, serum or plasma, nutrients, carbohydrate sources, preservatives, stabilisers, anti-oxidants or other materials well known to those skilled in the art.

Also disclosed are methods of producing said compositions by admixing the herein taught cells or cell populations with one or more additional components as above. The compositions may be for example liquid or may be semi-solid or solid (e.g., may be frozen compositions or may exist as gel or may exist on solid support or scaffold, eic). Cryopreservatives such as inter alia DMSO are well known in the art. In an embodiment, the composition as defined herein may be a pharmaceutical composition. Said pharmaceutical composition may thus comprise the herein taught mesodermal cells or cardiac cells or cardiomyocytes cells or cell populations as the active ingredient, and one or more pharmaceutically acceptable carrier/excipient.

Also disclosed are methods of producing said pharmaceutical compositions by admixing the herein taught cells or cell populations with one or more pharmaceutically acceptable carrier/excipient.

Preferably, the pharmaceutical compositions may comprise a therapeutically effective amount of the herein taught cells or cell populations. The term "therapeutically effective amount" refers to an amount which can elicit a biological or medicinal response in a tissue, system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, and in particular can prevent or alleviate one or more of the local or systemic symptoms or features of a disease or condition being treated.

The term "pharmaceutically acceptable" as used herein is consistent with the art and means compatible with the other ingredients of a pharmaceutical composition and not deleterious to the recipient thereof.

As used herein, "carrier" or "excipient" includes any and all solvents, diluents, buffers (such as, e.g., neutral buffered saline or phosphate buffered saline), solubilisers, colloids, dispersion media, vehicles, fillers, chelating agents (such as, e.g., EDTA or glutathione), amino acids (such as, e.g., glycine), proteins, disintegrants, binders, lubricants, wetting agents, emulsifiers, sweeteners, colorants, flavourings, aromatisers, thickeners, agents for achieving a depot effect, coatings, antifungal agents, preservatives, stabilisers, antioxidants, tonicity controlling agents, absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Such materials should be non-toxic and should not interfere with the activity of the cells or cell populations. The precise nature of the carrier or excipient or other material will depend on the route of administration. For general principles in medicinal formulation, the reader is referred to Cell Therapy: Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy, by G. Morstyn & W. Sheridan eds., Cambridge University Press, 1996; and Hematopoietic Stem Cell Therapy, E. D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000.

Liquid pharmaceutical compositions may generally include a liquid carrier such as water or a pharmaceutically acceptable aqueous solution. For example, physiological saline solution, tissue or cell culture media, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

Such pharmaceutical compositions may contain further components ensuring the viability of the cells therein. For example, the compositions may comprise a suitable buffer system (e.g., phosphate or carbonate buffer system) to achieve desirable pH, more usually near neutral pH, and may comprise sufficient salt to ensure iso-osmotic conditions for the cells to prevent osmotic stress. For example, suitable solution for these purposes may be phosphate-buffered saline (PBS), sodium chloride solution, Ringer's Injection or Lactated Ringer's Injection, as known in the art. Further, the composition may comprise a carrier protein, e.g., albumin (e.g., bovine or human albumin), which may increase the viability of the cells.

Further suitably pharmaceutically acceptable carriers or additives are well known to those skilled in the art and for instance may be selected from proteins such as collagen or gelatine, carbohydrates such as starch, polysaccharides, sugars (dextrose, glucose and sucrose), cellulose derivatives like sodium or calcium carboxymethylcellulose, hydroxypropyl cellulose or hydroxypropylmethyl cellulose, pregeletanized starches, pectin agar, carrageenan, clays, hydrophilic gums (acacia gum, guar gum, arabic gum and xanthan gum), alginic acid, alginates, hyaluronic acid, polyglycolic and polylactic acid, dextran, pectins, synthetic polymers such as water-soluble acrylic polymer or polyvinylpyrrolidone, proteoglycans, calcium phosphate and the like.

If desired, the cell preparation can be administered on a support, scaffold, matrix or material to provide improved tissue regeneration. For example, the material can be a granular ceramic, or a biopolymer such as gelatine, collagen, or fibrinogen. Porous matrices can be synthesized according to standard techniques (e.g., Mikos et al., Biomaterials 14: 323, 1993; Mikos et al., Polymer 35:1068, 1994; Cook et al., J. Biomed. Mater. Res. 35:513, 1997). Such support, scaffold, matrix or material may be biodegradable or non-biodegradable.

Alternatively or in addition, the present cells or cell populations may be stably or transiently transformed with nucleic acids of interest prior to introduction to the subject. Nucleic acid sequences of interest include, but are not limited to those encoding gene products that enhance the growth and/or functioning of mesodermal cells or cardiac cells or cardiomyocytes. Methods of cell transformation are known to those skilled in the art.

By means of example and not limitation, the cells, cell populations or pharmaceutical compositions as taught herein may be administered to a subject systemically or locally, for example but without limitation, cardiac or cardiomyocyte cells or cell populations comprising such or pharmaceutical compositions thereof may be administered into the heart (e.g., into heart muscle or into vascular system of the heart), for example, by injection or by delivery by a catheter.

In a further aspect, the invention relates to an arrangement comprising a surgical instrument or device for administration of a composition to a subject, such as for example systemically or locally, for example by into the heart (e.g., into heart muscle or into vascular system of the heart), for example, by injection or by delivery by a catheter, and further comprising the cells or cell populations as taught herein, or a pharmaceutical composition comprising said cells or cell populations. For example, a suitable surgical instrument may be capable of injecting a liquid composition comprising cells of the present invention such as direct into the heart tissue or intravenously.

In an embodiment the pharmaceutical cell preparation as defined above may be administered in a form of liquid composition.

In another embodiment, the cardiac cells or cell populations may be transferred to and/or cultured on suitable substrate, such as porous or non-porous substrate, to provide for implants. For example, cells that have proliferated, or that are being differentiated in culture dishes, can be transferred onto three-dimensional solid supports in order to cause them to multiply and/or continue the differentiation process by incubating the solid support in a liquid nutrient medium of the invention, if necessary. Cells can be transferred onto a three-dimensional solid support, e.g. by impregnating said support with a liquid suspension containing said cells. The impregnated supports obtained in this way can be implanted in a human subject. Such impregnated supports can also be re-cultured by immersing them in a liquid culture medium, prior to being finally implanted. The three- dimensional solid support needs to be biocompatible so as to enable it to be implanted in a human. It may be biodegradable or non-biodegradable.

Cardiac cells and cell cultures may be implanted or transplanted into a patient by any technique known in the art, e.g., as discussed above and further explained inter alia in Sturzu et al., 201 1 . Circ Res. 180:353-364, Nelson et al., 2010. Nat Rev Cardiol 7:700- 710.

Further, cardiac cells and cell populations taught herein may be included in bio-artificial heart (see, e.g., Kobayashi N. 2008. Cell Transplant 17: 1 1 -7).

Where administration of mesodermal cells or cardiac cells or cardiomyocytes or cell populations as taught herein to a patient is contemplated, it may be preferable that the cells or cell populations are selected such as to maximise the tissue compatibility between the patient and the administered cells, thereby reducing the chance of rejection of the administered cells by patient's immune system (graft vs. host rejection). For example, advantageously the cells or cell cultures may be typically selected which have either identical HLA haplotypes (including one or preferably more HLA-A, HLA-B, HLA-C, HLA- D, HLA-DR, H LA-DP and HLA-DQ; preferably one or preferably all HLA-A, HLA-B and HLA-C) to the patient, or which have the most HLA antigen alleles common to the patient and none or the least of HLA antigens to which the patient contains pre-existing anti-HLA antibodies. In a preferred example, the cardiac cells and cell populations are obtained according to the methods of the present invention departing from autologous pluripotent stem cells, e.g. iPS cells derived from somatic cells of the patient.

The invention further contemplates the use of the herein taught mesodermal cells or cardiac cells or cardiomyocytes or cell populations, or a (pharmaceutical) composition comprising the cells or cell populations as disclosed herein, for use in therapy. In particular, the cells and cell populations are contemplated for use in the treatment of cardiovascular or cardiac diseases. Also contemplated is the use of said cells or cell populations, or said compositions, for the manufacture of a medicament for the treatment of said diseases.

As used herein, the terms "treat" or "treatment" refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the development of a cardiac disease. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilised (i.e., not worsening) state of disease, delay or slowing of disease progression and occurrence of complications, amelioration or palliation of the disease state. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

The terms "heart disease" or "cardiac disease" refer to any disease, disorder or condition that affects the heart. The terms "cardiovascular disease" refer to any disease, disorder or condition that affects the heart and/or the vascular system, such as but without limitation the vascular system supplying the heart.

Non-limiting examples include inter alia congenital heart disease, such as malformations and misplacements of cardiac structures, congestive heart failure, Long QT Syndrome and hypertrophic cardiomyopathy, acquired heart diseases, such as myocardial infarction, cardiac hypertrophy and cardiac arrhythmia and cardiac damage due to trauma. Accordingly, also disclosed herein is a method for treating a cardiac or cardiovascular disease in a patient in need of such treatment, comprising administering a therapeutically effective amount of the cardiac or cardiomyocyte cells or cell populations as taught herein or a (pharmaceutical) composition comprising the same as taught herein to said patient. Except when noted, "subject" or "patient" are used interchangeably and refer to animals, preferably vertebrates, more preferably mammals, and specifically includes human patients and non-human mammals. Accordingly, "subject" or "patient" as used herein means any animal, mammalian or human patient or subject to which the cardiac cells and cell populations of the invention can be administered. Preferred patients are human subjects.

As used herein, a phrase such as "a subject in need of treatment" includes subjects, such as mammalian or human subjects, that would benefit from treatment of a given disease, preferably a cardiac or cardiovascular disease. Such subjects will typically include, without limitation, those that have been diagnosed with the disease, those prone to have or develop the said disease and/or those in whom the disease is to be prevented.

Said cells or cell populations may be transplanted or injected to the patient as disclosed elsewhere in this specification, allowing allogeneic, autologous or xenogeneic cellular therapy. For instance, said cells and cell populations may be injected into the heart surgically, by infusion into coronary arteries or delivered with a catheter or they may be injected intravenously.

The cardiac cells or cell populations of the invention may be used alone or in combination with any of the known therapies for heart diseases. The cardiac cells or cell populations of the invention can thus be administered alone or in combination with one or more active compounds. The administration may be simultaneous or sequential in any order.

In a further aspect, the herein taught mesodermal cells or cardiac cells or cardiomyocytes or cell populations may represent in vitro models for studying (pathogenesis of) cardiovascular or cardiac diseases, such as mentioned above.

Said mPS cells, e.g. iPS cells, may be derived from patients carrying gene mutations affecting the cardiovascular system, such as for example patients with Long QT Syndrome or hypertrophic cardiomyopathy. Departing from such mPS cells, one may obtain cardiac cells and cell populations with the same mutations, which may allow pathogenesis to be followed at the cellular level in a dish and should enable molecular and genetic screens to find drugs to halt or reverse the disease phenotype. Alternatively, said mPS cells may be derived from healthy subjects and further manipulated to display a pathological phenotype of interest. For example, such manipulation may include contacting said mPS cells externally with an agent, e.g., a chemical or biological agent, known or suspected of causing a pathological phenotype of interest. Exemplary agents may include, without limitation, toxins, metabolites, drugs, antisera, viral agents etc. In another example, such manipulation may include transiently or stably transforming the cells (e.g., by transfection or transduction as known in the art) with a recombinant construct encoding an RNA or protein agent known or suspected of causing a pathological phenotype of interest, or an agent (e.g., an RNAi agent or a dominant negative variant) that can suppress the expression of an endogenous gene known or suspected to contribute to a disease of interest.

In another aspect, the cardiac cells or cardiomyocyte or cell populations may represent an in vitro model for studying cardiogenesis. For example, the methods of the present invention may be used as an assay to screen for agents or factors (e.g., small molecules, chemokines, growth factors, etc.) which modulate cardiac differentiation, or to screen for phenotypic changes (e.g., markers, gene expression, cell morphology, etc.) that typify such differentiation.

Accordingly, also disclosed herein is an in vitro screening assay for agents or factors that modulate cardiac or cardiomyocyte differentiation comprising the steps of: a) producing cardiac cells or cardiomyocyte cells or cell populations from mPS cells according to any one of the methods as taught herein; b) exposing said mPS cells to a candidate agent or factor during the differentiation; and c) analyzing or comparing cardiac differentiation (e.g. phenotypic changes such as markers expression (e.g. cTNT) and/or contraction) in the presence and absence of said agent.

In yet another aspect, the invention provides for the use of the herein taught mesodermal cells or cardiac cells or cardiomyocyte cells or cell populations, optionally wherein said cells and cell populations represent (e.g., are derived from patients carrying gene mutations, such as affecting the cardiovascular system or have been so manipulated, see above) models for conditions or diseases taught herein, particularly cardiac or cardiovascular diseases, in any variety of cell-based screening assays, particularly in vitro screening assays, such as, e.g., in assays of biological effects of candidate pharmacological substances and compositions; assays of cardiotoxicity, genotoxicity or carcinogenicity of chemical or biological agents; and the like.

Cell-based in vitro screening assays can be carried out as generally known in the art. For example, cells grown in a suitable assay format (e.g., in multi-well plates or on coverslips, etc.) are contacted with a candidate agent (e.g., a potential pharmacological agent) and the effect of said agent on one or more relevant readout parameters is determined and compared to a control. Relevant readout parameters may greatly vary depending on the type of assay and may include, without limitation, survival, occurrence of apoptosis or necrosis, altered morphology, altered responsiveness to external signals or metabolites, gene expression, altered electrophysiological characteristics such as contractile function etc.

Hence, in an embodiment the invention provides an in vitro screening assay to identify pharmacological agents for the treatment of a cardiac or cardiovascular disease as discussed herein, comprising contacting the cardiac cells or cardiomyocytes or cell populations taught herein with a candidate pharmacological agent, and determining alleviation of said disease phenotype when said agent is administered. The invention also relates to so-identified pharmacological agents.

Another embodiment relates to the use of cardiac cells or cardiomyocytes or cell populations for assessing the cardiotoxicity of an agent (e.g. a drug). Accordingly, the invention also relates to an assay for assessing the toxicity of an agent (e.g. a drug) on cardiac cells or cardiomyocytes, comprising the steps of: a) producing cardiac cells or cardiomyocytes or cell populations from mPS cells according to any one of the methods as taught herein; b) subjecting said cells to an agent; and c) analysing the toxic effect (e.g. survival or occurrence of apoptosis or necrosis) of said agent on the cells.

While the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art in light of the foregoing description. Accordingly, it is intended to embrace all such alternatives, modifications, and variations as follows in the spirit and broad scope of the appended claims.

EXAMPLES

Example 1 : Experimental procedures

ESC culture and differentiation

ICE (A2lox.Cre) mouse embryonic stem cells were routinely propagated as described (Gaspard et al., 2009. Nat Protoc 4:1454-1463). For differentiation, ESC were plated at low density (20x103/ml) on gelatin-coated coverslips and after one day, medium was changed to defined default medium (DDM) (Gaspard et al., 2009). DDM consists of DMEM/F12 + GlutaMAX™ (Sigma) supplemented with N-2 Supplement (1 x, Sigma; 10 nM human transferrin, 0.861 nM insulin recombinant full chain, 0.02 nM progesterone, 0.0301 nM putrescine), 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 500μg ml BSA, 0.1 mM β-mercaptoethanol, 50U/ml penicillin/streptomycin.

DDM was changed every two days. Activin A (R&D) was added from day 0-4 at 1 , 10 or 100ng/ml. ^g ml doxycyclin (Sigma) was added from day 2-4. When specified, 2% foetal bovine serum (FBS) was added to DDM from day 0 to day 10.

Generation of a Tetracyclin-inducible MycEomes ESC line

To generate the tetracyclin-inducible MycEomes ESC line, the N-terminal Myc-tagged murine Eomes open reading frame (ORF) was amplified by polymerase chain reaction (PCR), sequence verified and cloned into the p2Lox vector (Kyba et al., 2002. Cell 109:29-37). This construct was electroporated in the A2LoxCre cells to generate the tetracyclin-inducible Myc-Eomes ESC line following a cassette exchange recombination (lacovino et al., 201 1 . Nat Cell Biol 13:72-78). Neomycin-resistant clones were screened for their expression of Myc-Eomes by immunofluorescence 24 hours after induction. Results were confirmed in 3 independent clones.

RNA isolation and qRT-PCR

RNA extraction, DNAse treatment and RT-PCR were performed as previously described (Bondue et al., 2008. Cell Stem Cell 3:69-84; Gaspard et al., 2008. Nature 455:351 -357). All quantitative PCR (qRT-PCR) were performed in duplicate using the Power SybrGreen Mix (Applied Biosystems) and a 7500 Real-Time PCR System (Applied Biosystems).

Results were normalized to the housekeeping gene TBP; primers used are summarized in Table 1 .

Table 1. qRT-PCR primers

Immunofluorescence staining and FACS analysis

Fixation, blocking, primary and secondary antibodies, as well as mounting medium used were previously described (Gaspard et al, 2008). Primary antibodies used were the following: Mouse anti cardiac isoform of TroponinT Ab1 (1/100; NeoMarkers), Rabbit anti β-tubulin III (1/2000; Covance), Rabbit anti Eomes (1/2000; Abeam), Goat anti Sox17 (1/1000; R&D), Mouse anti c-Myc (1/1000; Roche), Mouse anti Smooth muscle actin (1/200; Sigma). Secondary antibodies were donkey anti-mouse, anti-goat or anti-rabbit coupled to cyanin 3 or cyanin 5 (Jackson Immunoresearch) or to AlexaFluor 488 (Molecular Probes).

Nuclei were stained with bisbenzimide (Hoechst#33258; Sigma) and coverslips were mounted with glycergel (DAKO).

For flow cytometry, staining against cTnT (Ab1 -NeoMarkers, Fremont, CA) was performed as previously described (Bondue et al, 2008). Fluoresence activated cell sorting (FACS) analyses were performed on a FACSCalibur device (BD Biosciences) and data analyzed using CellQuest Pro (BD Biosciences).

Statistical analysis

Unless stated otherwise, data are presented as mean of at least three biologically independent experiments + standard error of the mean. qPCR data are presented as linearized Ct-values normalized to TBP (2"ACt); P-values were calculated using a two way ANOVA test with a post-hoc Tukey test for multiple comparisons. To calculate fold increase of qPCR data, TBP-normalized Ct-values of +Dox conditions were normalized to -Dox (AACt) for each independent experiment; no p-values were calculated for fold- increase. P-values for FACS-analysis were calculated using a two way ANOVA test with a post-hoc Tukey test for multiple comparisons on the proportions of troponinT-positive cells. Interaction between the effects of Dox and Activin was tested using a two way ANOVA test.

Example 2: Induction of Eomes in ESC promotes cardiogenesis in minimal culture conditions.

In order to explore the impact of Eomes on mouse embryonic stem cell (ESC) early differentiation in reductionist conditions with minimal extrinsic influence, ESC were cultured as a monolayer in a chemically defined default medium (DDM) devoid of any added exogenous morphogens, with addition of insulin. In these conditions, most of the cells generated correspond to a neural fate (Gaspard & Vanderhaeghen, 2010. Curr Opin Neurobiol 20: 37:43; Levine & Brivanlou, 2007. Dev Biol 308:247-256). To study the role of Eomes, we generated a recombinant ESC line in which the expression of a Myc-tagged version of mouse Eomes can be temporally induced upon doxycyclin (Dox) addition (lacovino et al., 201 1. Nat Cell Biol 13: 72-78; Kyba et al., 2002. Cell 109:29-37) (Figure 1 )- In the absence of Eomes induction, most cells generated after ten days corresponded to neural cells and very few cells showed expression of mesodermal or endodermal markers (Figure 2), as previously reported (Gaspard et al., 2008. Nature 455:351 :357). Following transient induction of Eomes at day 2 of differentiation, the cells revealed a different fate. Immunofluorescence and qRT-PCR analyses performed eight days after Eomes induction, revealed strong induction of a cardiac-specific isoform of TroponinT (cTNT) and concomitant reduction of 3-tubulin and Sox1 -positive neural cells (Figure 2 A,D). Closer examination of the cTNT staining revealed a striated pattern characteristic of cardiomyocytes myofibrils (Figure 2C), and the appearance of multiple beating clusters of cells. In addition, Eomes overexpression also promoted the appearance of other mesodermal derivatives such as smooth muscle actin-expressing cells, while the number of Sox17-expressing endodermal cells remained low despite high levels of Eomes expression (Figure 2 B, D).

Example 3: High levels of Activin prevents induction of cardiac mesoderm by Eomes.

W examined the effect of Eomes in our reductionist system, but in the presence of either serum or with increasing levels of Activin. In absence of Eomes induction, high levels of Activin during the first four days of differentiation resulted in a marked reduction of neural differentiation (Figure 3A). Most strikingly however, increasing the Activin concentration almost completely abolished the effect of Eomes-induction on cardiomyocyte differentiation, as reflected by a sharp decrease of cTNT positive cells detected by immunofluorescence (Figure 3A), and the disappearance of beating zones. Flow cytometric analysis confirmed these results and showed that induced expression of Eomes at day 2 lead to a >5 fold increase in the number of cTNT-positive cells in DDM. This effect was inhibited, in a dose dependent manner, by addition of Activin to the cultures (Figure 3B). Similarly, no induction of cardiac differentiation could be seen when cells were cultured in the presence of a low concentration (2%) of serum (Figure 3B).

CLAIMS

1 . Use of Eomesodermin (Eomes) activity for producing mesodermal cells from mammalian pluripotent stem (mPS) cells exposed to conditions which are permissive to differentiation of the mPS cells and in which Activin signalling is substantially absent. 2. A method for producing mesodermal cells comprising the steps of: a) plating mPS cells onto a substrate which allows adherence of the mPS cells thereto; b) exposing the mPS cells of a) which have adhered to said substrate to conditions which are permissive to differentiation of the mPS cells and in which Activin signalling is substantially absent; c) providing Eomes activity in the mPS cells of b). 3. The use according to claim 1 or the method according to claim 2, wherein the Eomes activity is provided at least on or before day 4 following exposing the mPS cells to said conditions which are permissive to differentiation of the mPS cells and in which Activin signalling is substantially absent.

4. The use according to any one of claims 1 or 3 or the method according to any one of claims 2 to 3, wherein the Eomes activity is provided from day 2 following exposing the mPS cells to said conditions which are permissive to differentiation of the mPS cells and in which Activin signalling is substantially absent.

5. The use according to any one of claims 1 , 3 or 4 or the method according to any one of claims 2 to 4, wherein the Eomes activity is provided transiently. 6. The use according to any one of claims 1 , 3 to 5 or the method according to any one of claims 2 to 5, wherein the Eomes activity is provided from day 2 to day 4 or from day 2 to day 3 following exposing the mPS cells to said conditions which are permissive to differentiation of the mPS cells and in which Activin signalling is substantially absent.

7. The use according to any one of claims 1 , 3 to 6 or the method according to any one of claims 2 to 6, wherein the duration thereof is between 8 and 12 days, preferably between 9 and 1 1 days, more preferably about 10 days, following exposing the mPS cells to said conditions which are permissive to differentiation of the mPS cells and in which Activin signalling is substantially absent.

8. The use according to any one of claims 1 , 3 to 7 or the method according to any one of claims 2 to 7, wherein the mPS cells are exposed to conditions in which Activin signalling is substantially absent at least before and during providing Eomes activity to said cells.

9. The use according to any one of claims 1 , 3 to 8 or the method according to any one of claims 2 to 8, wherein the Eomes activity is provided by increasing the amount of Eomes in the mPS cells, preferably by overexpressing Eomes in the mPS cells, more preferably by inducibly overexpressing Eomes in the mPS cells.

10. The use according to any one of claims 1 , 3 to 9 or the method according to any one of claims 2 to 9, wherein the conditions in which Activin signalling is substantially absent define conditions such that SMAD2 in the mPS cells is substantially not phosphorylated.

1 1 . The use according to any one of claims 1 , 3 to 10 or the method according to any one of claims 2 to 10, wherein the conditions in which Activin signalling is substantially absent define conditions substantially lacking animal serum, animal plasma, Activin and Nodal.

12. The use according to any one of claims 1 , 3 to 1 1 or the method according to any one of claims 2 to 1 1 , wherein the mesodermal cells comprise cardiac cells.

13. The use or the method according claim 12, wherein the cardiac cells comprise expression of cardiac Troponin T and/or form beating clusters of cells.

14. The use or the method according any one of claims 12 or 13, wherein the cardiac cells are cardiomyocytes. 15. The use according to any one of claims 1 , 3 to 14 or the method according to any one of claims 2 to 14, wherein the use or the method produce a cell population comprising said mesodermal or cardiac cells.

16. The mesodermal cells or cardiac cells or the cell population comprising said mesodermal or cardiac cells, obtainable or directly obtained using the method according to any one of claims 2 to 15.

17. A pharmaceutical composition comprising the mesodermal cells or cardiac cells or the cell population according to claim 16.

18. The mesodermal cells or cardiac cells or the cell population comprising said mesodermal or cardiac cells according to claim 16 for use in medicine; preferably the cardiac cells or the cell population comprising said cardiac cells according to claim 16 or the pharmaceutical composition comprising the cardiac cells according to claim 17 for use in the treatment of cardiac or cardiovascular diseases.

19. Use of the mesodermal cells or cardiac cells or the cell population comprising said mesodermal cells or cardiac cells according to claim 16 for cell-based assays, such as drug screening or cardiotoxicity assays, or as a model for studying cardiovascular or cardiac diseases or cardiogenesis.

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