WO2006021460A1 - Means and methods for generating cardiomyocytes and tissue and uses thereof - Google Patents

Abstract

Provided are stem cell-derived cardiac cells and tissue. In particular, systems for the de novo generation of pacemaker-like cardiomyocytes are described as well as the use of such systems in transplantation and drug development.

Description

Means and methods for generating cardiomyocytes and tissue and uses thereof

Field of the invention The present invention is concerned generally with the use of stem cell-derived cell types suitable for use in cardiac tissue regeneration, and non-therapeutic applications such as drug screening. In particular, the present invention relates to systems for the de novo generation of pacemaker-like cardiomyocytes as well as to the use of such systems in transplantation and drug development.

Background art

Cardiac pathologies are still the leading cause of death in most industrialized countries. Defining the molecular pathways underlying normal heart development is a prerequisite step for understanding the molecular basis of congenital heart malformations and postnatal cardiac diseases in order to develop novel therapeutic strategies. However, currently only limited insight in the inductive clues that lead to specification and terminal differentiation of cardiomyocytes is available (Wobus et al., J. MoI. Cell. Cardiol. 29 (1997), 1525-1539; Drab et al., FASEB J. 11 (1997), 905-915). Because cardiomyocytes of early stages cannot be obtained in vivo, embryonic stem (ES) cells provide a valuable model for the investigation of mechanisms in early cardiac lineage commitment, differentiation, and maturation (Hescheler et al., Cardiovasc. Res. 36 (1997), 149-162; Maltsev et al., Mech. Dev. 44 (1993), 41-50, Meyer et al., FEBS Lett. 478 (2000), 151-158; Zhang et al., Circulation 106 (2002), 1294-1299). ES cells are characterized by their capacity for prolonged undifferentiated proliferation in culture while maintaining the potential to differentiate into derivatives of all three germ layers. During in vitro differentiation, ES cells can develop into specialized somatic cells, including cardiomyocytes, and can recapitulate many processes of early embryonic development (Wobus et al., J. MoI. Cell. Cardiol. 29 (1997), 1525-1539; Xu et al., Circ. Res. 91 (2002), 501-508; Kehat et al., J. Clin. Invest. 108 (2001), 407-414).

Many cardiac diseases are characterized by a loss of functional myocardium. Because the conduction system is critical for generating and synchronizing the heart beat, dysfunction of this essential tissue may lead to arrhythmias and conduction disturbances causing sudden cardiac death. Recent work suggests the feasibility of transplantational repair of contracting myocardium by ES cell-derived cardiocytes (Robbins et al., J. Biol. Chem. 265 (1990), 1 1905-11909; Klug et al., J. Clin. Invest. 98 (1996), 216-224; Min et al., J. Appl. Physiol. 92 (2002), 288-296). It has been postulated that pacemaker cells and the specific cardiac conduction tissue origin also from cardiac precursor cells (Moorman et al., Circ. Res. 82 (1998), 629-644). Hence, selection of ES cell-derived cardiomyocytes with a pacemaker-like phenotype might prove useful in the development of cell-therapeutic strategies for the regeneration and/or repair of the cardiac conduction system after heart injury or in congenital disease.

The solution to said technical problem is achieved by providing the embodiments characterized in the claims, and described further below.

Summary of the invention

This invention is directed towards methods of providing protocols and methods for providing de novo cardiac cells, tissue and organs, in particular such that display a pacemaker-like phenotype, which are useful for transplantation and other purposes.

In accordance with the present invention, a murine embryonic stem (ES) cell line stably expressing the enhanced green fluorescent protein (EGFP) under the transcriptional control of the human atrial natriuretic peptide (ANP) promoter was established to further characterize the development of very early stages of the mammalian cardiac conduction tissue. The cardiac nature of ANP-EGFP-positive cells was confirmed by immunostaining. Surprisingly, in ANP- EGFP-expressing ES cell-derived cardiomyocytes, a distinct sub-lineage of pacemaker cells could be identified. Pacemaker cells displayed a spindle shape and exhibited a higher spontaneous beating rate, faster I_f current activation and larger I_f current densities compared with triangular atrial-like cardiocytes.

Further experiments could demonstrate that exposure to endothelin-1 significantly increased the percentage of pacemaker-like cells without affecting their electrophysiological properties. These findings were corroborated by immunostaining with antibodies against connexin 40 and connexin 45, known markers for cardiac conduction tissue. Conversely, treatment of ANP-EGFP-expressing ES cells with neuregulin-1 exhibited no effect on differentiation. These results indicate that ANP-EGFP expression enables the identification of ES cell- derived pacemaker cells by their fluorescence and morphology and that endothelin-1 promotes the development of ANP-EGFP-positive cardiomyocytes to a pacemaker-like phenotype. Those ES cell-derived cardiomyocytes thus display morphology of different cardiac subtypes featuring a well-developed contractile apparatus.

Hence, a high efficiency system of selection and quality control of transgenic ES cell-derived cell types such as cardiomyocytes has been established, which allows the generation of cardiac native tissue and tissue-like structures in vitro and in vivo.

The techniques of this invention are designed in part to provide cell populations with improved characteristics for human therapy. In addition, cell populations of different embryonic and ES cell-derived cell types developing into cardiac tissue are more closely related to the in vivo situation, which provides a distinct advantage for non-therapeutic applications such as screening drug candidates.

Other embodiments of the invention will be apparent from the description that follows.

Brief description of the drawings

Fig. 1: Typical pattern of EGFP expression under the transcriptional control of the hANP promoter in a spontaneously beating EB (6+22 d). A) EB under transmission light. B) The same EB under combined fluorescence and transmission light. Images were taken with an Axiophot microscope (Zeiss,

Jena, Germany).

Fig. 2: Immunohistochemistry corroborated the cardiac nature of ANP-EGFP- expressing EBs. A-B) Isolated ANP-EGFP-positive cells (6+9 d) expressed α- actinin visualized by anti-α-actinin and secondary Alexa Fluor 568 labeled anti-mouse antibody (B, red). Expression of troponin I in ANP-EGFP-positive cells of day 6+24 (C) was confirmed by staining with anti-cardiac troponin I and secondary R-Phycoerythrin-conjugated anti-goat antibody (D, red). Images were taken with a confocal microscope (Leica Microsystems, Heidelberg,

Germany).

Fig. 3: Typical morphological and electrophysiological characteristics of ES cell- derived cardiocytes isolated from ANP-EGFP-expressing EBs. All spindle- shaped cells (A) showed pacemaker-like action potential configurations (C). while tri-/multiangular ES cells (B) typically displayed an atrial-like action potential pattern (D). (E) Spindle-shaped cells exhibited large I_f-densities (34.5±2.4 pA/pF at -150 mV) and fast current activation kinetics (τ395.3±30.7 ms at -150 mV). F) I_f density was significantly smaller in the tri-/multiangular cell population (12.8±0.7 pA/pF at -150 pA/pF) with significantly slower current activation kinetics (τ681.1±30.3 pA/pF ms at -150 mV; PO.001). Images were taken with a confocal microscope (Leica Microsystems, Heidelberg, Germany).

Fig. 4: Dose-dependent effect of ET-I on the differentiation into pacermaker-like cells and ET-I effect on protein levels of connexins and the K⁺ channel modulators minK and MiRPl in ANP-EGFP-positive cells. A) Dose response relation of the ET-I effect on the percentage of pacemaker-like cells revealed an EC_5O of 1.1 x 10^'9 M. All values are shown as the relative effect compared with the maximal response with 10^'6 M ET-I, which was set to 100 %. B) Western blot analysis demonstrated an ET-I -mediated increase of the protein amounts of connexin 40 and connexin 45, whereas the protein level of connexin 43 was unchanged by ET-I. The effect of ET-I on connexin 40 and connexin 45 was blocked by BQ123 and BQ788. C) Immunoblotting of the cell lysates with anti-minK and anti-MiRPl antibodies demonstrated no significant effects of ET-I and ET-I with additional BQ123 or BQ788 on the protein amounts of minK and MiRPl.

Fig. 5: Effect of endothelin-1 on connexin expression. Exposure of ANP-EGFP- expressing EBs (6+16 d) to endothelin-1 resulted in prominent connexin 40 expression, a known marker of the cardiac conduction system, visualized by an anti-connexin 40 and secondary R-Phycoerythrin-conjugated anti-goat antibody (B), while untreated control ANPEGFP-positive EBs displayed only weak anti-connexin 40 staining (A). In addition, ET-I increased the intensity of anticonnexin 45 staining, a marker of the mouse sinus node and conduction system, visualized by a secondary Alexa Fluor 555 anti-rabbit antibody (F) compared with untreated EBs (E). Conversely, ET-I exposure exhibited no effect on the expression level of connexin 43, a marker of the working myocardium, labeled by an anti-connexin 43 and secondary RPhycoerythrin- conjugated anti-mouse antibody (D) compared with control (C). Images were taken with a confocal microscope (Leica Microsystems, Heidelberg, Germany).

Fig- 6: Effect of endothelin-1 (ET-I) and neuregulin-1 (NRG) on the differentiation of

ANP-EGFP-expressing ES cells. A) The percentage of spindle-shaped cells significantly increased upon exposure to ET-I compared with control. This effect was blocked by BQ123 and BQ788. Conversely, NRG did not affect the differentiation of ANP-EGFP-positive cells. Treatment with the combination of ET-I and NRG resulted in a similar shift of differentiation toward pacemaker cells compared with ET-I alone. *P < 0.05 vs control. B) Western blot analysis revealed no effect of NRG on the protein amounts of connexin 40 and connexin 45. Treatment with the combination of ET-I and NRG increased the protein levels of connexin 40 and connexin 45 similar to ET-I alone.

Definitions

For the purposes of this description, the term "stem cell" can refer to either stem cell or germ cell, for example embryonic stem (ES) and germ (EG) cell, respectively. Minimally, a stem cell has the ability to proliferate and form cells of more than one different phenotype, and is also capable of self renewal - either as part of the same culture, or when cultured under different conditions. Embryonic stem cells are also typically telomerase-positive and OCT-4 positive. Telomerase activity can be determined using TRAP activity assay (Kim et al., Science 266 (1997), 2011), using a commercially available kit (TRAPeze(R) XK Telomerase Detection Kit, Cat. s7707; Intergen Co., Purchase N.Y.; or TeIoTAGGG(TM) Telomerase PCR ELISAplus, Cat. 2,013,89; Roche Diagnostics, Indianapolis). hTERT expression can. also be evaluated at the mRNA level by RT-PCR. The LightCycler TeIoTAGGG(TM) hTERT quantification kit (Cat. 3,012,344; Roche Diagnostics) is available commercially for research purposes.

In accordance with the present invention, the term embryonic stem (ES) cell includes any multi- or pluripotent stem cell derived from pre-embryonic, embryonic, or fetal tissue at any time after fertilization, and have the characteristic of being capable under appropriate conditions of producing progeny of several different cell types that are derivatives of all of the three germinal layers (endoderm, mesoderm, and ectoderm), according to a standard art- accepted test, such as the ability to form a teratoma in 8-12 week old SCID mice. "Embryonic germ cells" or "EG cells" are cells derived from primordial germ cells. The term "embryonic germ cell" is used to describe cells of the present invention that exhibit an embryonic pluripotent cell phenotype. The terms "human embryonic germ cell (EG)" or "embryonic germ cell" can be used interchangeably herein to describe mammalian, preferably human cells, or cell lines thereof, of the present invention that exhibit a pluripotent embryonic stem cell phenotype as defined herein. Thus, EG cells are capable of differentiation into cells of ectodermal, endodermal, and mesodermal germ layers! EG cells can also be characterized by the presence or absence of markers associated with specific epitope sites identified by the binding of particular antibodies and the absence of certain markers as identified by the lack of bindin *-go of certain antibodies.

"Pluripotent" refers to cells that retain the developmental potential to differentiate into a wide range of cell lineages including the germ line. The terms "embryonic stem cell phenotype" and "embryonic stem-like cell" also are used interchangeably herein to describe cells that are undifferentiated and thus are pluripotent cells and that are capable of being visually distinguished from other adult cells of the same animal.

Included in the definition of ES cells are embryonic cells of various types, exemplified by human embryonic stem cells, described by Thomson et al. (Science 282 (1998), 1145): embryonic stem cells from other primates, such as Rhesus stem cells (Thomson et al., Proc. Natl. Acad. Sci. USA 92 (1995), 7844), marmoset stem cells (Thomson et al., Biol. Reprod. 55 (1996), 254) and human embryonic germ (hEG) cells (Shamblott et al., Proc. Natl. Acad. Sci. USA 95 (1998), 13726). Other types of pluripotent cells are also included in the term. Any cells of mammalian origin that are capable of producing progeny that are derivatives of all three germinal layers are included, regardless of whether they were derived from embryonic tissue, fetal tissue, or other sources. The stem cells employed in accordance with the present invention that are preferably (but not always necessary) karyotypically normal. However, it is preferred not to use ES cells that are derived from a malignant source.

"Feeder cells" or "feeders" are terms used to describe cells of one type that are co-cultured with cells of another type, to provide an environment in which the cells of the second type can grow. The feeder cells are optionally from a different species as the cells they are supporting. For example, certain types of ES cells can be supported by primary mouse embryonic fibroblasts, immortalized mouse embryonic fibroblasts (such as murine STO cells, e.g., Martin and Evans, Proc. Natl. Acad. Sci. USA 72 (1975), 1441-1445), or human fibroblast- like ceils differentiated from human ES cells, as described later in this disclosure. The term "STO cell" refers to embryonic fibroblast mouse cells such as are commercially available and include those deposited as ATCC CRL 1503.

The term "embryoid bodies" (EBs) is a term of art synonymous with "aggregate bodies". The terms refer to aggregates of differentiated and undifferentiated cells that appear when ES cells overgrow in monolayer cultures, or are maintained in suspension cultures. Embryoid bodies are a mixture of different cell types, typically from several germ layers, distinguishable by morphological criteria; see also infra.

The terms "polynucleotide" and "nucleic acid molecule" refer to a polymer of nucleotides of any length. Included are genes and gene fragments, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA and

RNA, nucleic acid probes, and primers. As used in this disclosure, the term polynucleotides refers interchangeably to double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention that is a polynucleotide encompasses both a double-stranded form, and each of the two complementary single-stranded forms known or predicted to make up the double-stranded form. Included are nucleic acid analogs such as phosporamidates and thiophosporamidates.

A cell is said to be "genetically altered", "transfected", or "genetically transformed" _.when a polynucleotide has been transferred into the cell by any suitable means of artificial manipulation, or where the cell is a progeny of the originally altered cell that has inherited the polynucleotide. The polynucleotide will often comprise a transcribable sequence encoding a protein of interest, which enables the cell to express the protein at an elevated level. The genetic alteration is said to be "inheritable" if progeny of the altered cell have the same alteration.

A "regulatory sequence" or "control sequence" is a nucleotide sequence involved in an interaction of molecules that contributes to the functional regulation of a polynucleotide, such as replication, duplication, transcription, splicing, translation, or degradation of the polynucleotide. Transcriptional control elements include promoters, enhancers, and repressors.

Particular gene sequences referred to as promoters, like the "ANP" promoter, are polynucleotide sequences derived from the gene referred to that promote transcription of an operatively linked gene expression product. It is recognized that various portions of the upstream and intron untranslated gene sequence may in some instances contribute to promoter activity, and that all or any subset of these portions may be present in the genetically engineered construct referred to. The promoter may be based on the gene sequence of any species having the gene, unless explicitly restricted, and may incorporate any additions, substitutions or deletions desirable, as long as the ability to promote transcription in the target tissue. Genetic constructs designed for treatment of humans typically comprise a segment that is at least 90 % identical to a promoter sequence of a human gene. A particular sequence can be tested for activity and specificity, for example, by operatively linking to a reporter gene; see also the Examples.

Genetic elements are said to be "operatively linked" if they are in a structural relationship permitting them to operate in a manner according to their expected function. For instance, if a promoter helps to initiate transcription of the coding sequence, the coding sequence can be referred to as operatively linked to (or under control of) the promoter. There may be intervening sequences between the promoter and coding region so long as this functional relationship is maintained.

In the context of encoding sequences, promoters, and other genetic elements, the term "heterologous" indicates that the element is derived from a genotypically distinct entity from that of the rest of the entity to which it is being compared. For example, a promoter or gene introduced by genetic engineering techniques into an animal of a different species is said to be a heterologous polynucleotide. An "endogenous" genetic element is an element that is in the same place in the chromosome where it occurs in nature, although other elements may be artificially introduced into a neighboring position.

The terms "polypeptide", "peptide" and "protein" are used interchangeably in this disclosure to refer to polymers of amino acids of any length. The polymer may comprise modified amino acids, it may be linear or branched, and it may be interrupted by non-amino acids. Detailed description of the embodiments of the present invention

In one aspect, the present invention relates to a method of providing in vitro differentiated pacemaker-like cardiomyocytes comprising culturing stem cells under conditions allowing differentiation of said cells into cardiomyocytes, wherein said stem cells comprise a recombinant nucleic acid molecule comprising a selectable marker and/or reporter gene operably linked to a regulatory sequence which is derived from the gene encoding atrial natriuretic peptide (ANP) or from a gene which exhibits substantially the same expression pattern as the ANP gene.

As demonstrated in the examples, transgenic ES cell lines have been established expressing the enhanced version of the green fluorescent protein (EGFP) under the transcriptional control of the human atrial natriuretic peptide (ANP) promoter. In these transgenic ES cells cardiac- specific EGFP expression occurred during early development. Surprisingly, an EGFP -positive sub-lineage displaying distinct morphological, immunocytochemical, and electrophysiological features characteristic of pacemaker-like cardiomyocytes could be identified.

Initially, atrial natriuretic peptide (ANP), also known as cardionatrin, atrionatriuretic factor, Pronatriodilatin (PND), atriopeptin and atrial natriuretic factor (ANF), has been identified from human as well as rat atrial tissue as peptide of natriuretic-diuretic activity and implicated in the control of extracellular fluid volume and electrolyte homeostasis. There are multiple forms of these ANP, ranging in molecular weight from 3,000 to 13,000, and it has been suggested that all may derive from the same precursor. Working from established amino acid sequence of human alpha- ANP, a 28-residue peptide with potent natriuretic action, Oikawa et al., Nature 309 (1984), 724-726, elucidated the structure of its precursor and the gene encoding it.

As mentioned above and described in the Examples, it could surprisingly be demonstrated in accordance with the present invention for the first time that the human ANP gene promoter is capable of driving the expression of a heterologous polypeptide, here EGFP, in a specific subset of in vitro differentiated cardiomyocytes, i.e. pacemaker-like cardiomyocytes. Thus, an ANP gene regulatory sequence can advantageously be used for the construction of selection marker and reporter gene constructs, respectively, particularly useful for following the fate of cardiomyocytes during development and/or enrichment of in vitro differentiated cardiac tissue such as embryoid bodies (EBs) with pacemaker-like cardiomyocytes.

Suitable regulatory sequences derived from the ANP gene that can be used in accordance with the present invention can be obtained from a public database, such as those maintained by The Institute for Genomic Research (TIGR) (www.tigr.org) and/or the National Center for Biotechnology Information (NCBI) (www.ncbi.nlm.nih.gov). For example, the regulatory sequence used in the vector pANPEGFP employed in the appended Examples is described in LaPointe et al., J. Bioi. Chem. 263 (1988), 9075-9078, which also describes reporter gene constructs and experimental setups for testing the activity of appropriate regulatory sequences. For example, a 192-base pair PvuII fragment (-400 to -208) associated with a tissue-specific expression of the ANP gene is described, indicating that the DNA sequence between -409 and -332 in the hANF gene harbors a tissue-specific element whose activity may involve association with a cardiac-specific nuclear protein. For genomic sequences of the human ANF gene see, e.g., Genbank accession no. X01471 and Greenberg et al., Nature 312 (1984), 656-658. Likewise, Maki et al., Biochem. Biophys. Res. Cornmun 125 (1984), 797- 802, describe the cloning of genomic DNA for human atrial natriuretic factor. Furthermore, see Seidman et al., Science 226 (1984), 1206-1209 for the nucleotide sequences of the human and mouse atrial natriuretic factor genes, the latter being available under Genbank accession no. K02781. In adition, Mayer et al., J. Hypertens. 20 (2002), 219-228 describe the molecular cloning and functional characterization of the upstream rat atrial natriuretic peptide promoter.

In a preferred embodiment, said regulatory sequence is or is derived from the human ANP promoter. Most preferably, the regulatory sequence substantially comprises or consists of sufficient 5 '-regulatory sequence derived from the a 4.6-kilobase EcoRI fragment from the 16.6-kilobase hANF genomic clone (Greenberg et al., 1984) which contains an additional 2000 bp of 5 '-flanking sequences (FS) upstream from those sequences previously described (Greenberg et al., 1984). The 2500 bp of 5 'FS (Pstl to the HaeIII site at +18) can be subcloned into for example a promoterless reporter and/or marker gene vector. For technical details and characterization of the mentioned regulatory sequences see LaPointe et al., J. Biol. Chem. 263 (1988), 9075-9078.

The invention can be practiced using stem cells of any vertebrate species. Included are stem cells from humans; as well as non-human primates, domestic animals, livestock, and other non-human mammals. Amongst the stem cells suitable for use in this invention are primate pluripotent stem cells derived from tissue formed after gestation, such as a blastocyst, or fetal or embryonic tissue taken any time during gestation. Non-limiting examples are primary cultures, or established lines of embryonic stem cells. The invention is also applicable to adult stem cells. It is referred to the literature of Anderson et al., Nat. Med. 7 (2001). 393-395 and Anderson et al., 2001, Gage, F.H., 200 and Prockop, Science 276 (1997), 71-74, wherein the extraction and culture of those cells is described. For research purposes the use of stem cells from mice and rat are preferred.

Media for isolating and propagating stem cells can have any of several different formulas, as long as the cells obtained have the desired characteristics, and can be propagated further. Suitable sources include Iscove's modified Dulbecco's medium (IMDM), Gibco, #12440-053; Dulbecco's modified Eagles medium (DMEM), Gibco #11965-092; Knockout Dulbecco's modified Eagles medium (KO DMEM), Gibco #10829-018; 200 mM L- glutamine, Gibco # 15039-027; non-essential amino acid solution, Gibco 11140-050; [beta]- mercaptoethanol, Sigma # M7522; human recombinant basic fibroblast growth factor (bFGF), Gibco # 13256-029. Exemplary serum-containing ES medium and conditions for culturing stem cells are known, and can be optimized appropriately according to the cell type. Media and culture techniques for particular cell types referred to in the previous section are provided in the references cited herein.

As mentioned before, several sources for ES cells are at the disposal of the skilled person of which human stem cells are preferred for most of the embodiments of the present invention, in particular for therapeutic purposes such as transplantation. Human embryonic stem cells and their use for preparing different cell and tissue types are also described in Reprod. Biomed. Online 4 (2002), 58-63. Embryonic stem cells can be isolated from blastocysts of members of the primate species (Thomson et al., Proc. Natl. Acad. Sci. USA 92 (1995), 7844). Human embryonic germ (EG) cells can be prepared from primordial germ cells present in human fetal material taken about 8-11 weeks after the last menstrual period. Suitable preparation methods are described in Shamblott et al., Proc. Natl. Acad. Sci. USA 95 (1998), 13726. Methods for making cells that resemble embryonic stem cells or embryonic germ cells in morphology and pluripotency derived from primordial germ cells isolated from human embryonic tissue, such as from the gonadal ridges of human embryo, are described in US patent 6,245,566. Recently, is has been reported that exfoliated human deciduous tooth, a comparable very accessible tissue, contains multipotent stem cells that were identified to be a population of highly proliferative, clonogenic cells capable of differentiating into a variety of cell types including neural cells, adipocytes, and odontoblasts; see Miura et al., Proc. Natl. Acad. Sci. USA 100 (2003), 5807-5812. After in vivo transplantation, those cells were found to be able to induce bone formation, generate dentin, and survive in mouse brain along with expression of neural markers. Furthermore, multilineage potential of homozygous stem cells derived from metaphase II oocytes has been described by Lin et al. in Stem Cells 21 (2003), 152-161. Various sources of precursor cells in postnatal muscles and the factors that may enhance stem cell participation in the formation of new skeletal and cardiac muscle in vivo are reviewed in Grounds et al., J. Histochem. Cytochem. 50 (2002), 589-610. Purification of rare hematopoietic stem cell(s) (HSC) to homogeneity that home to bone marrow is described in US application US2003/0032185. These adult bone marrow cells are described to have tremendous differentiative capacity as they can also differentiate into epithelial cells of the liver, lung, GI tract, and skin. This finding may contribute to clinical treatment of genetic disease or tissue repair. Furthermore, techniques such as nuclear transfer for embryo reconstruction may be employed wherein diploid donor nuclei are transplanted into enucleated Mil oocytes. This technology along with other procedures that aid in the establishment of customized embryonic stem (ES) cell lines that are genetically identical to those of the recipient have been reviewed by Colman and Kind, Trends Biotechnol. 18 (2000), 192-196. In order to avoid graft rejection associated with allogenic or xenogenic cells in transplantation syngenic or autologous cells and recipients are preferably used in the corresponding embodiments of the invention. In view of the recently discovered sources of stem cells such as from the bone marrow and tooth it should be possible to accomplish this demand without the need to resort to embryonic cells and tissue. Alternatively, cells may be genetically manipulated to suppress relevant transplantation antigens, see also infra, immunosuppressive agents may be used. The field of stem cell technology is being reviewed by Kiessling and Anderson, Harvard Medical School, in Human Embryonic Stem Cells: An Introduction to the Science and Therapeutic Potential; (2003) Jones and Bartlett Publishers; ISBN: 076372341X.

In order to avoid the use of for example human embryos as the donor for stem cells, which however seems to be justifiable at least under certain circumstances, it may even be possible to employ transgenic non-human animals, in particular mammals, as source for embryonic stem cells. For example, compositions and methods for making transgenic swines to be used as xenograft donors are described in US patent 5.523,226. Likewise, international application WO97/12035 describes methods of producing transgenic animals for xenotransplantation. Furthermore, immunologically compatible animal tissue, suitable for xenotransplantation into human patients, is described in international application WOO 1/88096. Methods for making embryonic germ cells from porcine are described for example in US patent 6,545,199.

In certain embodiments, the cells may be isolated from a subject. For example, for treatment of a patient suffering from a heart disease, disorder or injury, the patient's own cells may be isolated and reintroduced into the patient after exposing the cells to an ET-I agonist, so as to stimulate the cells to differentiate into cardiac cells.

Stem cells can be propagated continuously in culture, using a combination of culture conditions that promote proliferation without promoting differentiation. Traditionally, stem cells are cultured on a layer of feeder cells, typically fibroblast type cells, often derived from embryonic or fetal tissue. The cell lines are plated to near confluence, usually irradiated to prevent proliferation, and then used to support when cultured in medium conditioned by certain cells (e.g. Koopman and Cotton, Exp. Cell 154 (1984), 233-242; Smith and Hooper, Devel. Biol. 121 (1987), 1-91), or by the exogenous addition of leukemia inhibitory factor

(LIF). Such cells can be grown relatively indefinitely using the appropriate culture conditions.

International application WO03/010303 and Mummery et al., Circulation 107 (2003), 2733- 2740, disclose experiments with human embryonic stem (hES) cells differentiating to cardiomyocytes, wherein said hES cells were co-cultured with visceral-endoderm (VE)-like cells from the mouse. In those experiments the mouse endoderm cells replace the commonly used mouse fibroblast feeder cells and are used for the induction of cardiomyocyte differentiation in hES cells that do not undergo spontaneous cardiogenesis.

In the absence of feeder cells, exogenous leukemia inhibitory factor (LIF), or conditioned medium, ES or EG cells spontaneously differentiate into a wide variety of cell types, including cells found in each of the endoderm, mesoderm, and ectoderm germ layers. With the appropriate combinations of growth and differentiation factors, however, cell differentiation can be controlled. For example, mouse ES and EG cells can generate cells of the cardiomyocytes (heart muscle cells) (Klug et al., Am. J. Physiol. 269 (1995), H1913- H1921) and skeletal muscle cells (Rohwedel et al., Dev. Biol. 164 (1994), 87-101).

In certain embodiments of the invention, differentiation is promoted by withdrawing one or more medium component(s) that promote(s) growth of undifferentiated cells, or act(s) as an inhibitor of differentiation. Examples of such components include certain growth factors, mitogens, leukocyte inhibitory factor (LIF), and basic fibroblast growth factor (bFGF).

Differentiation may also be promoted by adding a medium component that promotes differentiation towards the desired cell lineage, or inhibits the growth of cells with undesired characteristics.

In accordance with this invention, populations of differentiated cells are preferably depleted of relatively undifferentiated cells and/or of cells of undesired cell types by using a selection system that is lethal to the undesired cells and cell types, i.e. by expressing a selectable marker gene that renders cells of a specific cell type resistant to a lethal effect of an external agent, under control of a regulatory sequence that causes the gene to be preferentially expressed in the desired cell type and/or at a certain stage of development. To accomplish this, the cells are genetically altered before the process used to differentiate the cells into the desired lineage for therapy, in a way that the cells comprise a selectable marker operably linked to a cell type-specific regulatory sequence specific for the desired cell type. Preferably, said regulatory sequence is derived from the ANF gene as described above.

Any suitable expression vector for the purposes of the present invention can be used. Suitable viral vector systems for producing stem cells altered according to this invention can be prepared using commercially available virus components. The introduction of the vector construct or constructs into the embryonic stem cells occurs in a known manner, e.g. by transfection, electroporation, lipofection or with the help of viral vectors. Viral vectors comprising effector genes are generally described in the publications referenced to in the last section. Alternatively, vector plasmids can be introduced into cells by electroporation, or using lipid/DNA complexes. Exemplary is the formulation Lipofectamine 2000(TM), available from Gibco/Life Technologies. Another exemplary reagent is FuGENE(TM) 6 Transfection Reagent, a blend of lipids in non-liposomal form and other compounds in 80 % ethanol, obtainable from Roche Diagnostics Corporation. Preferably, the vector constructs and transfection methods described in international application WO02/051987 are used, the disclosure content of which is incorporated herein by reference.

Resistance genes per se are known. Examples for these are nucleoside and aminoglycoside- antibiotic-resistance genes for, e.g. puromycin (puromycin-N-acetyltransferase), streptomycin, bleomycin, neomycin, gentamycin or hygromycin. Further examples for resistance genes are dehydrofolate-reductase, which confers a resistance against aminopterine and methotrexate, as well as multi drug resistance genes, which confer a resistance against a number of antibiotics, e.g. against vinblastin, doxorubicin and actinomycin D.

In a preferred embodiment of the invention, said stem cells or said stem cell-derived cardiac cells, i.e. cardiomyocytes comprises a reporter gene, wherein said reporter is operably linked to the mentioned ANF gene-specific regulatory sequence. This type of vector has the advantages of providing visualization of differentiation, definition of the time point for beginning of drug selection, visualization of drug selection and tracing of the fate of purified cells grafted in recipient tissue. Such vectors, which are preferably employed in accordance with the methods of the present invention, are described in international application WO02/051987. Usually, said regulatory sequence of the reporter gene is substantially the same as said regulatory sequence of the marker gene, but not necessarily. This can advantageously be achieved by putting said marker gene and said reporter gene into the same recombinant nucleic acid molecule, i.e. vector used for stem cell transfection, preferably such that said marker gene and said reporter gene are contained on the same cistron.

The reporter can be of any kind as long as it is non-damaging for the cell and confers an observable or measurable phenotype. According to the present invention, the green fluorescent protein (GFP) from the jellyfish Aequorea victoria (described in international applications WO95/07463, WO96/27675 and WO95/121191) and its derivates ^;iBlue GFP" (Heim et al., Curr. Biol. 6 (1996), 178-182 and Redshift GFP" (Muldoon et al, Biotechniques 22 (1997), 162-167) can be used. Particularly preferred is the enhanced green fluorescent protein (EGFP). Further embodiments are the enhanced yellow and cyan fluorescent proteins (EYFP and ECFP, respectively) and red fluorescent proteins (DsRed, HcRed). Further fluorescent proteins are known to the person skilled in the art and can be used according to the invention as long as they do not damage the cells. The detection of fluorescent proteins takes place through per se known fluorescence detection methods; see, e.g., Kolossov et al., J. Cell Biol. 143 (1998), 2045-2056 and the appended Examples. Alternatively to the fluorescent proteins, particularly in in vivo applications, other detectable proteins, particularly epitopes of those proteins, can also be used. Also the epitope of proteins, though able to damage the cell per se, but whose epitopes do not damage the cells, can be used; see also international application WO02/051987.

For the selection of stably transfected ES cells vector constructs contain a further selectable marker gene, which confers e.g. a resistance against an antibiotic, e.g. neomycin. Of course, other known resistance genes can be used as well, e.g. the resistance genes described above in association with the fluorescent protein encoding genes. The selection gene for the selection for stably transfected ES cells is under the control of a different promoter than that which regulates the control of the expression of the detectable protein. Often constitutively active promoters are used, e.g. the PGK-promoter. The use of a second selection gene is advantageous for the ability to identify the successfully transfected clones (efficiency is relatively low) at all. Otherwise a smothering majority of non-transfected ES cells may exist and during differentiation e.g. no EGFP -positive cells might be detected.

In a further embodiment of the invention the cells can be manipulated additionally, so that specific tissues are not formed. This can occur for instance by inserting repressor elements, e.g. a doxicyclin-inducible repressor element. Thereby, a possible contamination of the desired differentiated cells with pluripotent, potentially tumorigenic cells can be excluded.

Of course, the stem cells used in accordance with the present invention may also be transgenic because of other reasons, for example they express a therapeutically active protein and/or they have been genetically altered in order to suppress an inherited disease.

In a particular preferred embodiment of the present invention, said stem cells are transgenic for a recombinant nucleic acid molecule comprising a fluorescent reporter gene, preferably an EFGP gene, operably linked to the regulatory sequence which is derived from the ANP gene. Accordingly, it is likewise preferred to determine the status of the differentiation of the cells and to identify pacemaker-like cells by determining reporter gene activity, most preferably by detection of fluorescence; see also Example 3. As demonstrated in the appended examples, exposure of the transgenic ES cell lines established in accordance with the present invention to endothelin-1 but not neuregulin-1 markedly directed the differentiation of the stem cells, and later the cardiac precursor cells towards, a pacemaker-like phenotype, suggesting an important paracrine role of endothelin-1 in the development of the mammalian cardiac conduction^'system.

Thus, in a particularly preferred embodiment of the invention, the method further comprises stimulating and/or inducing the differentiation of the stem cells into pacemaker-like cardiomyocytes by exposing said cells to endothelin-1 (ET-I) or to a compound which has substantially the same biological activity as endothelin-1, i.e. an ET-I agonist.

The term "endothelin-1" (ET-I, ETl) refers to a particular member of the endothelins (ET), a family of structurally and pharmacologically distinct peptides which has been identified and sequenced in humans (Inoue et al, Proc. Nat. Acad. Sci. 86 (1989), 2863-2867). Three isoforms of human endothelin have been identified: endothelins- 1, -2, and -3. Endothelin-1 (ET-I) is a bicyclic 21-amino-acid vasoconstrictor peptide causing a potent and sustained vasoconstriction, mainly through the ET(A) receptor subtype. Inoue et al., J. Biol. Chem. 264 (1989), 14954-14959, cloned the full length of the human preproendothelin-1 gene and the corresponding cDNA and determined the complete nucleotide sequence. The human preproendothelin-1 mRNA consists of 2,026 nucleotides, excluding the poly(A) tail. Endothelin-1 was originally isolated from the supernatant of porcine aortic endothelial cell cultures and is the most potent vasoconstrictor known. The subsequent cloning and sequence analysis from a human placental cDNA library showed that human endothelin-1 is identical to porcine endothelin. Furthermore, Maemura et al., Genomics 31 (1996), 177-184 describe sequence analysis, chromosomal location, and developmental expression of the mouse preproendothelin-1 gene.

Endothelin-1 (ET-I) is proteolytically generated from its inactive precursor by endothelin- converting enzyme- 1 (ECE-I) and acts on the endothelin- A (ETA) receptor; see, e.g., Yanagisawa et al., J. Clin. Invest. 102 (1998), 22-33, which report on the role of endothelin- 1 /endothelin- A receptor-mediated signaling pathway in the aortic arch patterning in mice.

Benatti et al., J. Clin. Invest. 91 (1993), 1 149-1156 demonstrated that at least 2 preproendothelin-1 mRNAs are produced from a single gene by use of different promoters; the 2 molecules share the same coding sequence but differ in the 5 -prime untranslated region. Analysis of the tissue distribution of the 2 mRNAs showed a tissue-type specificity for one raRNA in brain and heart tissues. Furthermore, the influence of pregnancy-specific hormonal environment on expression of ETl and the ETl receptor (EDNR) has been described by Bourgeois et al., J. Clin. Endocr. Metab. 82 (1997), 3116-3123.

In accordance with the present invention the terms "endothelin-1", "endothelin-1 agonist", "agonist of endothelin- 1 " and "a compound having substantially the same biological activity of endothelin-1" are used interchangeably herein unless indicated otherwise and include any compound that is capable of stimulating or inducing differentiation of stem cells into cardiomyocytes with a pacemaker-like phenotype, for example as described in Example 3, which is structurally related to ET-I and/or is capable of binding to a receptor of ET-I, and wherein preferably the biological activity as regards the capability of inducing differentiation of ES cells towards a pacemaker phenotype is substantially similar or even better than that for observed for human endothelin-1 under otherwise substantially identical culture conditions. For example, stem cells are incubated in the presence of an endothelin-1 agonist polypeptide, or fragment, or homolog, or peptidomimetic thereof, and differentiation is monitored. ET-I agonists may also be referred to as ET-I receptor agonists. Such agonists are described in the literature; see for example Langlois et al., Br. J. Pharmacol. 139 (2003), 616-622, which describes the development of agonists of endothelin-1 exhibiting selectivity towards ETA receptors.

As mentioned above, endothelins mediate their actions via only two receptor types that have been cloned and classified as the ET(A) and ET(B) receptors. For review on endothelin receptor nomenclature including sources for their nucleic and amino acid sequences, functional characterization, agonists, antagonists, etc. see Davenport, Pharmacol. Rev. 54 (2002), 219-226, the disclosure content of which is incorporated herein by reference.

Thus, in principle, ET-I is a dual agonist for both endothelin type A and B receptors. As demonstrated in the Example 3, the effect of ET-I on cardiac differentiation could be prevented by both the selective ET(A) receptor antagonist BQ 123 and the selective ET(B) receptor antagonist BQ788. Without intending to be bound by theory it is therefore believed that ET-I stimulates or induces ES cell-derived cardiocytes towards pacemaker cells in an endothelin receptor-dependent manner without affecting electrophysiological properties. Accordingly, and again without intending to be bound by theory, it is hypothesized in accordance with the present invention that for the purpose of stimulating or inducing the differentiation of stem cells towards a pacemaker phenotype ET-I acts directly or indirectly as an endothelin receptor agonist for both ET(A) and ET(B) receptors. In this respect the experiments performed in accordance with present invention fit in the observation by reported Ozaki et al, J. Biochem. 121 (1997), 440-447, describing coexpression studies with endothelin receptor subtypes, which indicate the existence of intracellular cross-talk between ET(A) and ET(B) receptors.

For the above reasons, the term "ET-I agonist" and ET-I receptor agonist", respectively, as well as the synonymous terms mentioned above include compounds that act as agonist on either or both the ET(A) and ET(B) receptors. For example, selective agonists for endothelin B receptors such as sarafotoxin 6c (Granstrδm et al., Pharmacol. & Toxicol. 95 (2004), 43-48) or BQ-3020 (Ozaki et al., 1997) may be used as in accordance with the present as well. Further ET(B) receptor agonists that may be employed in accordance with the present invention are described for example in international application WO2004/037235. Common endothelin agonists are also disclosed in international application WOO 1/28509. In addition, Chinsese patent application CN1453265 describes N-(trans-4-isopropyl heterocycle or cyclohexyl-l-formyl)-alpha-substituted amino acid compounds with endothelin receptor agonist-like effects and thus may be used for the purposes of the present invention. The mentioned national and international applications also provide useful information concerning the pharmaceutical formulation of those agonists, which information is incorporated herein by reference.

Hence, in accordance with the present invention endothelin- 1 may be used in embodiments of inducing external oriented differentiation of embryonic stem cells similar as described for icariin in Chinese patent application CN1425763. Thus, previously described compositions and methods for modulating cell differentiation, in particular towards cardiac cells and tissue may be used and adapted in accordance with the teaching of the present invention; see for example US patent application US2004/014209.

ET-I agonist for use in accordance with the present invention can be produced according to the methods described in the literature cited above but are also commercially available, for example from Sigma-Aldrich. 3050 Spruce St., St. Louis, MO 63103, USA, which offer Endothelin 1 human, porcine minimum 97% (HPLC), Powder, #E7764; Endothelin 2 minimum 97% (HPLC), Powder, #E9012; Endothelin 3 human, rat minimum 97% (HPLC), Powder, #E9137; [Ala^u'^{1 U5}]-Endothelin 1, #E6877; BQ-3020, #E-139; IRL-1620, #E-137; Sarafotoxin S6al minimum 97% (HPLC), #S1522; Sarafotoxin S6b Atractaspis engaddensis sequence minimum 90% (HPLC), #S4146 and Sarafotoxin S6c minimum 97% (HPLC), #S6545,

Preferably, the ET-I agonist is added to the culture medium in a concentration of aboutlO^"5 to 10^~9 M, more preferably in a concentration of aboutlO^"6 to 10^"8 M, and most preferably in a concentration of about 10^~7 M; see also the Examples. The cells or cell aggregates, i.e. embryoid bodies (EBs) may be contacted with the ET-I agonist for about 1 to 30 days, preferably 7 to 21 days, and most preferably for about 14 days; see also the Example. The appropriate concentration of the ET-I agonist and time of exposure may dependent on the potency of the compound used and/or the indented goal of the investigator. Of course, the person skilled in the art may test and adjust concentration of the ET-I agonist and time of exposure in routine experiments, for example adapting the experiments described in the Examples accordingly.

In a preferred embodiment of the present invention said ET-I or ET-I agonist is ET-I itself or a derivative thereof. Most preferably, said ET-I or ET-I agonist is human ET-I.

The term "stimulating" and "inducing" with reference to differentiation of a stem cell into a cardiac cell, i.e. pacemaker-like cardiomyocytes is meant to encompass any change in a stem cell which increases the likelihood that the cell will progress toward becoming a pacemaker or pacemaker-like cell as compared to what would occur in the absence of such changes. Such differentiation may be monitored by a variety of means, including, for example, visually (e.g., by inspecting the cell, cell population, or tissue under a microscope), electrically (e.g., by measuring changes in electrical potential of the cell or cell surface), mechanically (e.g., by measuring changes in cell length or volume), or biochemically (e.g., by assaying for the presence of one or more gene and/or protein markers). In certain embodiments, stimulation of differentiation will have the effect of priming the cell or causing a partial differentiation of the cell toward a cardiac cell which differentiation may be completed upon exposure to another factor. In other embodiments, stimulation of differentiation will lead to full differentiation of at least a portion of the stem cells in a cell population into cardiac cells or cardiomyocytes, i.e. pacemaker-like cardiomyocytes. As described in the to test the effect of endothelin-1 (ET-I) on the differentiation of the stem cells, the cells were cultured after the phase of forming ES cell aggregates, i.e. embryoid bodies (EBs) from the beginning of plating for 14 days in the presence of ET-L Accordingly, in a preferred embodiment of the method of the present invention the stem cells are cultured under conditions such "hanging drops" or "mass culture" in order to form embryoid bodies (EBs) at which stage the cells are preferably exposed to the ET-I agonist.

The examples and international application WO02/051987 provide protocols to obtain embryoid bodies. The manufacturing takes place preferably with the "hanging drop" method or by methylcellulose culture (Wobus et al, Differentiation 48 (1991), 172-182). Alternatively to this, spinner flasks (stirring cultures) can be used as culture method. Therefore, the undifferentiated ES cells are introduced into stirring cultures and are mixed permanently according to an established procedure. For example, 10 million ES cells are introduced into 150 ml medium with 20 % FCS and are stirred constantly with the rate of 20 rpm., wherein the direction of the stirring motion is changed regularly. 24 hours after introduction of the ES cells an extra 100 ml medium with serum is added and thereupon 100 - 150 ml of the medium is exchanged every day (Wartenberg et al., FASEB J. 15 (2001), 995- 1005). Under these culture conditions large amounts of ES cell-derived cells, i.e. cardiomyocytes, endothelial cells, neurons etc., depending on the composition of the medium, can be obtained. The cells are selected by means of the resistance gene either still within the stirring culture or after plating, respectively.

Alternatively to this, the EBs differentiated in the hanging drop might be not plated, but kept simply in suspension. Even under these conditions a progression of a differentiation could be observed experimentally. The washing off of the non-desired cell types can be done with mechanical mixing alone and addition of low concentration of enzyme (e.g. collagenase, trypsin); a single cell suspension is achieved with easy washing off of the non-desired cell types.

In a preferred embodiment of the present invention the stem cells are substantially differentiated into atrial cardiomyocytes or precursors thereof. Advantageously, said pacemaker cells are identified by their morphology and/or electrophysiological properties; see also the Examples. Preferably, the cardiac cells or cardiomyocytes, i.e. pacemaker-like cardiomyocytes produced in accordance with a method of the present invention display one or more of the following features:

(a) they are spindle shape and exhibited a high spontaneous beating rate of at least about 100 bpm, preferably at least about 150 bpm or more;

(b) they display faster I_f current activation and larger I_f current densities compared with triangular atrial-like cardiocytes;

(c) immuno staining with antibodies against connexin 40 and connexin 45 show marked enhanced expression of these known markers for cardiac conduction tissue; (d) in an in vitro differentiated stem cell population, e.g. embryoid bodies, at least 10% of the cells, preferably at least 15%, more preferably at least 20%, and advantageously at least 25% or more of the differentiated cells display a pacemaker-like phenotype. For a more detailed description see also the Examples.

Muller et al. describe the selection of ventricular-like cardiomyocytes from ES cells in vitro by use of enhanced green fluorescent protein (EGFP) under transcriptional control of the ventricular-specific 2.1 kb myosin light chain-2v (MLC-2v) promoter and the 0.5 kb enhancer element of the cytomegalovirus (CMV(enh)); see Muller et al., FASEB J. 14 (2000), 2540- 2548. This publication also describes electrophysiological studies which may be similarly performed with the in vitro-generated cardiomyocytes of the present invention.

In a further aspect, the present invention relates to a vector or a composition of vectors comprising the recombinant nucleic acid molecules as defined in context with the methods of the present invention hereinbefore. Those vectors or vector compositions may be substantially isolated or may be present in a sample or, e.g., in one or more host cells useful for, e.g., propagation of the vectors. Preferably, the vector is plasmid p ANPEGFP described in Example 1.

The present invention also relates to cells, cell aggregates and tissue obtainable by the above described methods, comprising cells which are capable of differentiating into pacemaker-like cardiomyocytes. Hence, said cells are preferably embryonic cardiac cells. Likewise, organs constituted from those cells, cell aggregates and tissue are subject of the present invention as well as implants or transplants comprising such cells, cell aggregates, tissue or organs. AU of those can be used in a method of treatment of damaged tissue or organs in a subject comprising implanting or transplanting to the subject in need thereof. Hence, compositions such as pharmaceutical compositions comprising any one of those recombinant nucleic acid molecules, cells, cell aggregates, or tissue of the present invention as described herein are encompassed in the scope of the present invention. As described before, those compositions and methods of the invention can be used for a variety of purposes, for example for analyzing early steps of tissue formation during embryonic development or the influence of factors and compounds on this process.

In one particular aspect, the present invention relates to a stem cell as defined above, preferably embryonic stem cell, comprising stably integrated into its genome a recombinant nucleic acid molecule comprising a fluorescent reporter gene, preferably an EFGP gene, operably linked to a regulatory sequence which is derived from the ANP gene. Preferably, said stem cell is stem cell as described and produced in Example 1.

In a further aspect, the present invention relates to isolated cardiomyocytes obtained according to a method of the present invention, which are capable of differentiating into pacemaker-like cardiomyocytes.. In a related aspect, the present invention is directed to an isolated population of in vitro differentiated cardiomyocytes, wherein at least about 15% of the cells display a pacemaker phenotype, preferably at least about 25%. As shown and discussed in the Examples, the present invention for the first time enables the provision of in vitro differentiated cardiac cell populations which are comprised of a substantially greater amount of pacemaker-like cardiomyocytes or precursors thereof compared to previous attempts without addition of a corresponding differentiation factor for a pacemaker phenotype; see Example 3.

One main object of the present invention is the provision of cells and tissue for use in transplantation For example, differentiated cells of this invention can also be used for tissue reconstitution or regeneration in a human patient in need thereof. The cells are administered in a manner that permits them to graft to the intended tissue site and reconstitute or regenerate the functionally deficient area. Thus, the present invention particularly concerns a method of improving cardiac tissue repair and/or organ function of the heart in a mammal comprising the steps of:

(a) introducing a cellular inoculum comprising a culture of preferably transgenic stem cells of the present invention in which differentiation has been initiated or corresponding tissue to at least a portion of the previously damaged area of the tissue; and (b) allowing said introduced cellular inoculum to engraft in situ as viable cells or tissue situated within the previously damaged area of the tissue, wherein the engraftment results in improved tissue and/or organ function in said mammal.

Previous means and methods of generating human cardiac cells and tissue may be used and adapted in accordance with the teaching of the present invention; see for example international application WO03/008535. Likewise, known methods of preparing cells for transplantation such as described in international application WO2004/065589 can be implemented in the teaching of the present invention.

In a particular aspect, the present invention relates to a method for markedly improving cardiac function and repairing heart tissue in a living mammalian subject after the occurrence of a myocardial infarction or tissue damage. The method is a surgical technique which introduces and implants embryonic stem cells, i.e. mammalian embryonic stem cell-derived cardiomyocytes into the infarcted or damaged area of the myocardium. After implantation, the cells form stable grafts and survive indefinitely within the infarcted or damaged area of the heart in the living host..

Hence, the instant invention also concerns a method for improving the cardiac function in a mammal after a myocardial infarct, said method comprising the steps of:

(a) culturing undifferentiated mammalian embryonic stem (ES) cells comprising a resistance gene and/or a reporter gene under the control of any one of the above described regulatory sequence in vitro in a culture medium, optionally containing the selective agent for the resistance gene, under conditions allowing differentiation of said ES cells into cardiomyocytes or precursors thereof;

(b) isolating said differentiated cardiomyocytes or precursors thereof and/or eliminating non-differentiated cells, optionally along with cells differentiating towards irrelevant cell types from said cardiomyocytes in the course of differentiation; (c) subsequently co-transplanting said cardiomyocytes or precursors thereof to at least a portion of the previously infarcted area of the heart tissue; and

(d) allowing said introduced cellular inoculum to engraft in situ as viable cells situated within the previously infarcted area of the heart tissue, wherein the engraftment results in improved cardiac function in said mammal. Implantation of embryonic stem cells in which differentiation has been initiated and determining cardiac function can be done as described in the cited references, or, e.g., as described in US patent 6,534,052.

In one embodiment, the fate of the cell types and formation of cell aggregates and cardiomyocytes, in particular those of the pacemaker phenotype as well as the physiological and/or developmental status of the cells or cell aggregates are analyzed, for example by isometric tension measurements, echocardiography and the like. Preferably, the status of the cells or cell aggregates is analyzed by monitoring the differentiation of electrical activity of the cells on an array, for example by recording the extracellular field potentials with a microelectrode array (MEA). For example, electrophysiological properties during the ongoing differentiation process of embryonic stem cells differentiating into cardiac myocytes can be followed by recordings of extracellular field potentials with microelectrode arrays (MEA) consisting of, e.g., 60 substrate-integrated electrodes; see Banach et al. Am. J. Physiol. Heart Circ. Physiol. 284 (2003), H2114-H2123. Multiple arrays of tungsten microelectrodes were used to record the concurrent responses of brain stem neurons that contribute to respiratory motor pattern generation; see Morris et al., Respir. Physiol. 121 (2000), 119-133.

In a still further embodiment, the present invention relates to transgenic non-human animals which can be generated from the mentioned ES cells and ES cell-derived cell types and cell aggregates; see supra. The generation of transgenic animals from ES cells is known in the art; see, e.g., A. L. Joyner Ed., Gene Targeting, A Practical Approach (1993), Oxford University Press. A general method for making transgenic non-human animals is described in the art, see for example international application WO94/24274.

The cells, cell aggregates, tissue, organ and methods of the present invention are particularly suited for use in drug screening and therapeutic applications. For example, differentiated stem cells of this invention can be used to screen for factors (such as solvents, small molecules, drugs, peptides, polynucleotides, and the like) or environmental conditions (such as culture conditions or manipulation) that affect the characteristics of differentiated cells. Particular screening applications of this invention relate to the testing of pharmaceutical compounds in drug research. It is referred generally to the standard textbook "In vitro Methods in Pharmaceutical Research", Academic Press, 1997, and US patent 5,030,015. Assessment of the activity of candidate pharmaceutical compounds generally involves combining the differentiated cells of this invention with the candidate compound, determining any change in the morphology, marker phenotype, or metabolic activity of the cells that is attributable to the compound (compared with untreated cells or cells treated with an inert compound), and then correlating the effect of the compound with the observed change. The screening may be done, for example, either because the compound is designed to have a pharmacological effect on certain cell types, or because a compound designed to have effects elsewhere may have unintended side effects. Two or more drugs can be tested in combination (by combining with the cells either simultaneously or sequentially) to detect possible drug-drug interaction effects. In some applications, compounds are screened initially for potential toxicity (Castell et al., pp. 375-410 in "In vitro Methods in Pharmaceutical Research," Academic Press, 1997). Cytotoxicity can be determined in the first instance by the effect on cell viability, survival, morphology, and expression or release of certain markers, receptors or enzymes. Effects of a drug on chromosomal DNA can be determined by measuring DNA synthesis or repair. [HJthymidine or BrdU incorporation, especially at unscheduled times in the cell cycle, or above the level required for cell replication, is consistent with a drug effect. Unwanted effects can also include unusual rates of sister chromatid exchange, determined by metaphase spread. It is referred to A. Vickers (pp 375-410 in "In vitro Methods in Pharmaceutical Research," Academic Press, 1997) for further elaboration.

Thus, in a further embodiment the present invention relates to methods for obtaining and/or profiling a test substance capable of influencing cardiac cell, i.e. cardiomyocyte development and/or tissue structure formation comprising the steps of:

(a) contacting a test sample comprising a cell, a cell aggregate, a tissue or an organ prepared or differentiating according to a method of the present invention, with a test substance; and

(b) determining a phenotypic response in said test sample compared to a control sample, wherein a change in the phenotypic response in said test sample compared to the control sample is an indication that said test substance has an effect on cell development and/or tissue structure formation. These methods can replace various animal models, and form novel human-based tests and extreme environment biosensors. In particular, the methods of the invention can be used for toxicological, mutagenic, and/or teratogenic in vitro tests. Since the cardiomyocytes obtained in accordance with the present invention more closely resemble the in vivo myocard situation, the results obtained by the toxicological assays of the present invention are expected to correlate to in vivo teratogenicity of the tested compounds as well.

For example, compounds, in particular cardiac-active compounds can be tested in accordance with methods described in DE 195 25 285 Al; Seiler et al., ALTEX 19 Suppl. 1 (2002), 55- 63; Takahashi et al., Circulation 107 (2003), 1912-1916, and Schmidt et al., Int. J. Dev. Biol. 45 (2001), 421-429; the latter describing an ES cell test (EST) used in a European Union validation study for the screening of embryotoxic agents by determining concentration- dependently the differentiation of ES cells into cardiac and myogenic cells.

Preferred compound formulations for testing do not include additional components such as preservatives, that have a significant effect on the overall formulation. Thus preferred formulations consist essentially of a biologically active compound and a physiologically acceptable carrier, e.g. water, ethanol, DMSO, etc. However, if a compound is liquid without an excipient the formulation may consist essentially of the compound itself.

Furthermore, a plurality of assays may be run in parallel with different compound concentrations to obtain a differential response to the various concentrations. As known in the art, determining the effective concentration of a compound typically uses a range of concentrations resulting from 1 :10, or other log scale, dilutions. The concentrations may be further refined with a second series of dilutions, if necessary. Typically, one of these concentrations serves as a negative control, i.e. at zero concentration or below the level of detection.

Preferably, the test substance is added to the culture medium in a concentration of aboutlO^" to 10^"9 M, more preferably in a concentration of about 10^~6 to 10^~8 M, and most preferably in a concentration of about 10^~7 M, thus similar to the ET-I agonist antagonists, respectively, in Example 3. The cells or cell aggregates or in vitro differentiated cardiomyocytes may be contacted with the test substance for about 1 to 30 days, preferably 7 to 21 days, and most preferably for about 14 days, for example if the identification of ET-I antagonists or agonists is desired. The appropriate concentration of the compounds and time of exposure may dependent on the potency of the compound used and/or the indented goal of the investigator. Furthermore, the test substance may be added to the cells, cell aggregates or in vitro differentiated cardiomyocytes prior, concomitantly or after their exposure to the ET-I agonist. Of course, the person skilled in the art may test and adjust concentration of the ET-I agonist, of the test substance the and time of joint or individual exposure in routine experiments, for example adapting the experiments described in the Examples accordingly.

Compounds of interest encompass numerous chemical classes, though typically they are organic molecules. Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including peptides, nucleic acids, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.

Compounds and candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. For example, inhibition of tumor-induced angiogenesis and matrix-metalloproteinase expression in confrontation cultures of embryoid bodies and tumor spheroids by plant ingredients used in traditional Chinese medicine has been described by

Wartenberg et al, Lab. Invest. 83 (2003), 87-98.

Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce, combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.

The compounds may also be included in a sample including fluids to which additional components have been added, for example components that affect the ionic strength, pH, total protein concentration, etc. In addition, the samples may be treated to achieve at least partial fractionation or concentration. Biological samples may be stored if care is taken to reduce degradation of the compound, e.g. under nitrogen, frozen, or a combination thereof. The volume of the sample used is sufficient to allow for measurable detection, usually from about 0.1 μl to 1 ml of a biological sample is sufficient.

Test compounds include all of the classes of molecules described above, and may further comprise samples of unknown content. While many samples will comprise compounds in solution, solid samples that can be dissolved in a suitable solvent may also be assayed.

Samples of interest include environmental samples, e.g. ground water, sea water, mining waste, etc.; biological samples, e.g. lysates prepared from crops, tissue samples, etc.; manufacturing samples, e.g. time course during preparation of pharmaceuticals; as well as libraries of compounds prepared for analysis; and the like. Samples of interest compounds are being assessed for potential therapeutic value, i.e. drug candidates.

The test compound may optionally be a combinatorial library for screening a plurality of compounds. Such a collection of test substances can have a diversity of about 10³ to about 10⁵, is usually successively reduced in running the method, optionally combined with others twice or more. Compounds identified in the method of the invention can be further evaluated, detected, cloned, sequenced, and the like, either in solution or after binding to a solid support, by any method usually applied to the detection of a specific DNA sequence such as PCR, oligomer restriction (Saiki et al., Bio/Technology 3 (1985), 1008-1012), allele-specific oligonucleotide (ASO) probe analysis (Conner et al., Proc. Natl. Acad. Sci. USA 80 (1983), 278), oligonucleotide ligation assays (OLAs) (Landegren et al., Science 241 (1988), 1077), and the like. Molecular techniques for DNA analysis have been reviewed (Landegren et al., Science 242 (1988), 229-237). Hence, the method of the present invention can also be used for transcriptional profiling of embryonic and adult stem cells; see, e.g., Ramalho-Santos et al., Science 298 (2002), 597-600; Tanaka et al., Genome Res. 12 (2002), 1921-1928.

Incubating includes conditions which allow contact between the test compound and the ES cells or ES-derived cells. Contacting can be done under both in vitro and in vivo conditions. For example, it may be desirable to test an array of compounds or small molecules on a single or few ES cells on a "chip" or other solid support; see supra. For example, cardiomyocytes on chips would give a readout of the rate of contraction or number of firings, respectively, in response to a compound and for the detection of harmful or at least biologically active environmental agents. The electrical activity of cardiomyocytes can be monitored by plating the cells on an array of extracellular microelectrodes (Connolly et al., Biosens. Biores. 5 (1990), 223-234). The cells show regular contractions, and the extracellular signal recorded shows a relationship to intracellular voltage recordings (Connolly et al., supra). This non-invasive method allows long-term monitoring and is simpler and more robust than typical whole cell patch clamp techniques.

Hence, in a preferred method of the present invention, the phenotypic response to be determined comprises electrophysiological properties, preferably determined during the ongoing differentiation process. This embodiment is particularly suited to provide modulation reference patterns and databases of modulation reference patterns for a wide range of biologically active compounds. The reference patterns are then used for the identification and classification of test compounds. Evaluation of test compounds may be used to achieve different results.

Methods for the classification of biological agents according to the spectral density signature of evoked changes in cellular electric potential are known to the person skilled in the art; see, e.g., US patent 6,377,057. Thus, biologically active compounds are classified according to their effect on ion channels, changes in membrane potential and ionic currents, and the frequency content of action potentials that the compound(s) evoke in excitable cells. The spectral density changes of such evoked membrane potential or action potential are a characteristic for each channel type that is modulated by the test compound. A pattern of spectral changes in membrane potential is determined by contacting a responsive cell with a compound, and monitoring the membrane potential or ionic currents over time. These changes correlate with the effect of that compound, or class of compounds, on the ion channels of the responding cell. This pattern of spectral changes provides a unique signature for the compound, and provides a useful method for characterization of channel modulating agents. The effect of a compound on ion channels, and on the action potential of a living cell, can provide useful information about the classification and identity of the compound. Methods and means for extracting such information are of particular interest for the analysis of biologically active compounds, with specific applications in pharmaceutical screening, drug discovery, environmental monitoring, biowarfare detection and classification, and the like. Examples of whole cell-based biosensors are described in Gross et al., Biosensors and Bioelectronics 10 (1995), 553-567; Hickman et al. Abstracts of Papers American Chemical Society 207 (1994), BTEC 76; and Israel et al., American Journal of Physiology: Heart and Circulatory Physiology 27 (1990), H1906-H1917.

Connolly et al., Biosens. Biores. 5 (1990), 223-234, describe a planar array of microelectrodes developed for monitoring the electrical activity of cells in culture. The device allows the incorporation of surface-topographical features in an insulating layer above the electrodes. Semiconductor technology is employed for the fabrication of gold electrodes and for the deposition and patterning of an insulating layer of silicon nitride. The electrodes were tested using a cardiac cell culture of chick embryo myocytes, and the physical beating of the cultured cells correlated with the simultaneous extracellular voltage measurements obtained. The molecular control of cardiac ion channels is reviewed by Clapham, Heart Vessels Suppl. 12 (1997), 168-169. Oberg and Samuelsson, J. Electrocardiol. 14 (1981), 13942, performed fourier analysis on the repolarization phases of cardiac action potentials. Rasmussen et al., American Journal of Physiology 259 (1990), H370-H389, describe a mathematical model of electrophysiological activity in bullfrog atria.

A large body of literature exists in the general area of ion channels. A review of the literature may be found in the series of books, "The Ion Channel Factsbook", volumes 1-4, by Edward C. Conley and William J. Brammar, Academic Press. An overview is provided of: extracellular ligand-gated ion channels (ISBN: 0121844501), intracellular ligand-gated channels (ISBN: 01218445 IX), inward rectifier and intercellular channels (ISBN: 0121844528), and voltage-gated channels (ISBN: 0121844536). HiIIe, B. (1992) "Ionic Channels of Excitable Membranes", 2.sup.nd Ed. Sunderland MA: Sinauer Associates.

In another aspect, cells cultured or modified using the materials and methods provided by the present invention are mounted to support surfaces to screen for bioactive substances. In one example, the cells are coupled with a substrate such that electrophysiological changes in the cells in response to external stimuli can be measured, e.g., for use as a high-throughput screen for bioactive substances. The cells can also be transfected with DNA that targets, expresses, or knocks-out specific genes or gene products in the cell. By providing such chip-mounted cells coupled with measuring devices, such as a computer, many compounds can be screened rapidly and accurately. The cells or chips could also be coupled to the measuring device in arrays for large-scale parallel screening. The assay methods of the present invention can be in conventional laboratory format or adapted for high throughput. The term "high throughput" (HTS) refers to an assay design that allows easy analysis of multiple samples simultaneously, and has capacity for robotic manipulation. Another desired feature of high throughput assays is an assay design that is optimized to reduce reagent usage, or minimize the number of manipulations in order to achieve the analysis desired. Examples of assay formats include 96- well, 384- well or more- well plates, levitating droplets, and "lab on a chip" microchannel chips used for liquid handling experiments. It is well known by those in the art that as miniaturization of plastic molds and liquid handling devices are advanced, or as improved assay devices are designed, that greater numbers of samples may be performed using the design of the present invention.

In the method of the invention, said cells are preferably contained in a container, for example in a well in a microtiter plate, which may be a 24-, 96-, 384- or 1586-well plate. Alternatively, the cells can be introduced into a microfiuidics device, such as those provided by Caliper (Newton, MA, USA). In another preferred embodiment, the method of the present invention comprises taking 2, 3, 4, 5, 7, 10 or more measurements, optionally at different positions within the container. In one embodiment of the screening methods of the present invention a compound known to activate or inhibit differentiation process and/or tissue structure formation is added to the sample or culture medium, for example retinoic acid; for appropriate compounds see also supra.

Furthermore, the above-described methods can, of course, be combined with one or more steps of any one of the above-described screening methods or other screening methods well- known in the art. Methods for clinical compound discovery comprise for example ultrahigh- throughput screening (Sundberg, Curr. Opin. Biotechnol. 11 (2000), 47-53) for lead , identification, and structure-based drug design (Verlinde and HoI, Structure 2 (1994), 577- 587) and combinatorial chemistry (Salemme et al., Structure 15 (1997), 319-324) for lead optimization. Once a drug has been selected, the method can have the additional step of repeating the method used to perform rational drug design using the modified drug and to assess whether said modified drug displays better affinity according to for example interaction/energy analysis. The method of the present invention may be repeated one or more times such that the diversity of said collection of compounds is successively reduced. Substances are metabolized after their in vivo administration in order to be eliminated either by excretion or by metabolism to one or more active or inactive metabolites (Meyer, J. Pharmacokinet. Biopharm. 24 (1996), 449-459). Thus, rather than using the actual compound or drug_^ identified and obtained in accordance with the methods of the present invention, a corresponding formulation as a pro-drug can be used which is converted into its active form in the patient by his/her metabolism. Precautionary measures that may be taken for the application of pro-drugs and drugs are described in the literature; see, for review, Ozama, J. Toxicol. Sci. 21 (1996), 323-329.

Furthermore, the present invention relates to the use of a compound identified, isolated and/or produced by any one of these methods for the preparation of a composition for the treatment of disorders related to, for example, damaged tissue or aberrant tissue or organ formation, heart insufficiency, etc.; see also supra. Preferably, the isolated compound or corresponding drug supports wound healing and/or healing of damaged cardiac tissue. As a method for treatment the identified substance or the composition containing it can be administered to a subject suffering from such a disorder. Compounds identified, isolated and/or produced by the method described above can also be used as lead compounds in drug discovery and preparation of drugs or prodrugs. This usually involves modifying the lead compound or a derivative thereof or an isolated compound as described hereinbefore such as modifying said substance to alter, eliminate and/or derivatize a portion thereof suspected causing toxicity, increasing bioavailability, solubility and/or half-life. The method may further comprise mixing the substance isolated or modified with a pharmaceutically acceptable carrier. The various steps recited above are generally known in the art. For example, computer programs for implementing these techniques are available; e.g., Rein, Computer-Assisted Modeling of Receptor-Ligand Interactions (Alan Liss, New York, 1989). Methods for the preparation of chemical derivatives and analogs are-well known to those skilled in the art and are described in, for example, Beilstein, Handbook of Organic Chemistry, Springer Edition New York Inc., 175 Fifth Avenue, New York, N. Y. 10010 U.S.A., and Organic Synthesis, Wiley, New York, USA. Furthermore, peptidomimetics and/or computer-aided design of appropriate derivatives and analogues can be used, for example, according to the methods described above. Methods for the lead generation in drug discovery also include using proteins and detection methods such as mass spectrometry (Cheng et al., J. Am. Chem. Soc. 1 17 (1995), 8859-8860) and some nuclear magnetic resonance (NMR) methods (Fejzo et al., Chem. Biol. 6 (1999), 755— 769; Lin et al., J. Org. Chem. 62 (1997), 8930-8931). They may also include or rely on quantitative structure-action relationship (QSAR) analyses (Kubinyi, J. Med. Chem. 41 (1993), 2553-2564, Kubinyi, Pharm. Unserer Zeit 23 (1994), 281-290), combinatorial biochemistry, classical chemistry and others (see, for example, Holzgrabe and Bechtold, Pharm. Acta HeIv. 74 (2000), 149-155). Furthermore, examples of carriers and methods of formulation may be found in Remington's Pharmaceutical Sciences.

Once a drug has been selected in accordance with any one of the above-described methods of the present invention, the drug or a pro-drug thereof can be synthesized in a therapeutically effective amount. As used herein, the term "therapeutically effective amount" means the total amount of the drug or pro-drug that is sufficient to show a meaningful patient benefit, i.e., treatment, healing, prevention or amelioration of damaged tissue, or an increase in the rate of treatment, healing, prevention or amelioration of such conditions. In addition or alternatively, in particular with respect to pre-clinical testing of the drug the term "therapeutically effective amount" includes the total amount of the drug or pro-drug that is sufficient to elicit a physiological response in a non-human animal test.

The present invention also relates to kit compositions containing specific reagents such as those described hereinbefore useful for conducting any one of the above-described methods of the present invention, containing the vector or the composition of vectors described hereinbefore, stem cells, ET-I and ET-I agonists, respectively, and optionally a culture medium, recombinant nucleic acid molecules, standard compounds, etc. Such a kit would typically comprise a compartmentalized carrier suitable to hold in close confinement at least one container. The carrier would further comprise reagents useful for performing said methods. The carrier may also contain a means for detection such as labeled enzyme substrates or the like.

Hence, the means and methods of the present invention described herein-before can be used in a variety of applications including, but not limited to "loss of function" assays with ES cells containing homozygous mutations of specific genes, "gain of function" assays with ES cells overexpressing exogenous genes, developmental analysis of teratogenic/embryotoxic compounds in vitro, pharmacological assays and the establishment of model systems for pathological cell functions, and application of differentiation and growth factors for induction of selectively differentiated cells which can be used as a source for tissue grafts; see for review, e.g., Guan et al., Altex 16 (1999), 135-141. In particular, the present invention relates to the use of the above-described stem cells and in- vitro differentiated cardiomyocytes, tissue and organ of claim as biological pacemaker or precursor thereof. Likewise, the present invention relates to the use of a regulatory sequence derived from the ANF gene as defined above and described herein, the mentioned recombinant nucleic acid molecule of the present invention, and vectors and composition of vectors for the staining, identification and/or (pre)selection of in vitro differentiated atrial and/or pacemaker cells.

These and other embodiments are disclosed and encompassed by the description and ex¬ amples of the present invention. Further literature concerning any one of the materials, methods, uses and compounds to be employed in accordance with the present invention may be retrieved from public libraries and databases, using for example electronic devices. For example the public database "medline" may be utilized, which is hosted by the National Center for Biotechnology Information and/or the National Library of Medicine at the National Institutes of Health. Further databases and web addresses, such as those of the European Bioinformatics Institute (EBI), which is part of the European Molecular Biology Laboratory (EMBL), are known to the person skilled in the art and can also be obtained using internet search engines. An overview of patent information in biotechnology and a survey of relevant sources of patent information useful for retrospective searching and for current awareness is given in Berks, TIBTECH 12 (1994), 352-364.

The above disclosure generally describes the present invention. A more complete under¬ standing can be obtained by reference to the following specific examples and figures which are provided herein for purposes of illustration only and are not intended to limit the scope of the invention. The contents of all cited references (including literature references, issued patents, published patent applications as cited throughout this application and manufacturer's specifications, instructions, etc.) are hereby expressly incorporated by reference; however, there is no admission that any document cited is indeed prior art as to the present invention.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. For further elaboration of general techniques concerning stem cell technology, the practitioner can refer to standard textbooks and reviews, for example 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 Cell Differentiation in Vitro (Wiles, Meth. Enzymol. 225 (1993), 90O₃); Properties and uses of Embryonic Stem Cells: Prospects for Application to Human Biology and Gene Therapy (Rathjen et al., Reprod. Fertil. Dev. 10 (1998), 31,). Differentiation of stem cells is reviewed in Robertson, Meth. Cell Biol. 75 (1997), 173; and Pedersen, Reprod. Fertil. Dev. 10 (1998), 31. Besides the sources for stem cells described already above further references are provided; see Evans and Kaufman, Nature 292 (1981), 154-156; Handyside et al., Roux's Arch. Dev. Biol, 196 (1987), 185-190; Flechon et al., J. Reprod. Fertil. Abstract Series 6 (1990), 25: Doetschman et al., Dev. Biol. 127 (1988), 224-227; Evans et al., Theriogenology 33 (1990), 125-128; Notarianni et al., J. Reprod. Fertil. SuppL, 43 (1991), 255-260; Giles et al., Biol. Reprod. 44 (SuppL 1) (1991), 57; Strelchenko et al., Theriogenology 35 (1991), 274; Sukoyan et al., MoI. Reprod. Dev. 93 (1992), 418-431; Iannaccone et al., Dev. Biol. 163 (1994), 288-292.

Methods in molecular genetics and genetic engineering are described generally in the current editions of Molecular Cloning: A Laboratory Manual, (Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Gene Transfer Vectors for Mammalian Cells (Miller & Calos, eds.); Current Protocols in Molecular Biology and Short Protocols in. Molecular Biology, 3rd Edition (F. M. Ausubel et al., eds.); and Recombinant DNA Methodology (R. Wu ed., Academic Press). Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, vols. 154 and 155 (Wu et al. eds.); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N. Y.); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes HV (D. M. Weir and C. C. Blackwell eds., 1986). Reagents, cloning vectors, and kits for genetic manipulation referred to in this disclosure are available from commercial vendors such as BioRad, Stratagene, Invitrogen, and ClonTech. General techniques in cell culture and media collection are outlined in Large Scale Mammalian Cell Culture (Hu et al., Curr. Opin. Biotechnol. 8 (1997), 148); Serum-free Media (TCitano, Biotechnology 17 (1991), 73); Large Scale Mammalian Cell Culture (Curr. Opin. Biotechnol. 2 (1991), 375); and Suspension Culture of Mammalian Cells (Birch et al., Bioprocess Technol. 19 (1990), 251). Other observations about the media and their impact on the culture environment have been made by Marshall McLuhan and Fred Allen.

EXAMPLES

Example 1: Cardiac-specific EGFP expression driven by the human ANP promoter

D3 cells have been previously characterized as pluripotent ES cells that can develop cardiomyocytes with electrophysiological properties resembling sinus node, atrial, and ventricular cells at the terminal differentiated stage (6+ .9d) (4). The aim of the present invention was to possibly identify pacemaker-like cells by specific labeling and morphological criteria. Since it had been shown that ES cell-derived ventricular cardiomyocytes labeled by tissue-specific EGFP expression under the control of the Mlc2v promoter did not develop into cells exhibiting pacemaker properties (Muller et al., FASEB J. 14 (2000), 2540-2548), it was hypothesized in accordance with the present invention that selection of predominantly atrial cardiac precursor cells would include a sufficient number of pacemaker cells. Thus, the human ANP promoter was chosen to stably express EGFP in ES cell-derived cells.

The vector pSVOcat-2593hANP encoding the -2593 bp regulatory fragment of the hANP. gene was obtained from LaPointe et al., J. Biol. Chem. 263 (1988), 9075-9078. The -2593bp coding sequence of hANP was cloned into the multiple cloning site of the plasmid pEGFP-1 containing the enhanced version of the GFP coding sequence (Clontech Laboratories, Palo Alto, CA) to generate pANPEGFP. After linearization the plasmid was electroporated into 2.5 x 10⁷ ES cells of the line D3 (American Type Cell Culture, ATCC, Manassas, VA; (Doetschman et al., J. Embryol. Exp. Morphol. 87 (1985), 27-45)). The ES clones (ANP- EGFP) were propagated in the presence of leukemia inhibitory factor 1000 units/ml (ESGRO™, Chemicon International Inc., Temecula, CA) and selected for 10 days using G418 (250 μg/ml). Several neomycin-resistant colonies of ES cells showing the brightest fluorescence after 9 days of differentiation were further selected. No difference between selected clones was noticed.

Embryoid bodies (EBs) were generated from ES cells of the line D3 using standard protocols as described previously (Maltsev et al., Mech. Dev. 44 (1993), 41-50; Maltsev et al., Circ. Res. 75 (1994), 233-244). Briefly, cells were cultivated in hanging drops (ca. 400 cells per drop) for 2 days, afterwards kept in suspension for 4 days and finally plated on gelatinized multiwell culture plates. Three (6+3d) to four (6+4d) days after plating, green fluorescent spontaneously contracting cell clusters could be observed at the outgrowths of the EBs. Spontaneously beating fluorescent areas were dissected at a stage of 6+4 to 6+28 days as described previously (Muller et al., FASEB J. 14 (2000), 2540-2548). The percentages of different cell shapes were determined by the evaluation of 130 consecutive isolated EGFP- positive ES cell-derived cells of 12 observations in each group. The dissociated cells were plated onto glass coverslips and stored in the incubator. Within the first 12 h, the isolated cells attached to the glass surface and began spontaneous rhythmical beating. The culture medium consisted of DMEM supplemented with 20 % fetal calf serum, penicillin/streptomycin, non¬ essential amino acids, glutamax, and β-mercaptoethanol. To examine the effect of ET-I and NRG-I on cardiac differentiation, ET-I (10^"7 M), the selective endothelin A receptor antagonist BQ123 (10^"6 M), the selective endothelin B receptor antagonist BQ788 (10^'6 M) (Sigma, Taufkirchen, Germany), and the recombinant peptide containing the β variant of the epidermal growth factor-like domain of NRG-I (2.5 x 10^'9 M) (R and D Systems, Minneapolis, MN) were added to the DMEM culture medium, as indicated.

Generally, the ANP promoter was switched on at day 6+4, rarely on day 6+3, which was indicated by the formation of cell clusters featuring a bright EGFP fluorescence. All fluorescent areas within the embryoid bodies (EBs) developed spontaneous contractions 24 h later. Two-dimensional planimetric calculation revealed that the EGFP-positive area within EBs amounted to 18.0 ± 3.3 % (n=14) at the late stage of development (6+20 d) (Fig. 1). During all stages of cardiomyogenesis EGFP fluorescence was exclusively detected in these beating areas, indicating the cardiac specificity of the human ANP promoter. However, not all beating areas within the same EB displayed a prominent fluorescence, suggesting a spatial- restricted expression of the ANP promoter. To further confirm the cardiac nature of EGFP expressing cells, immuno staining was performed on EBs and single ES cell-derived cells. EB outgrowths (6+8 to 6+20 d) or single, enzymatically dissociated ES cell-derived cells (6+10 to 6+28 d) were plated on gelatin- covered glass coverslips for 48 h. Cells were fixed in a solution containing 4 % paraformaldehyde in 0.1 M PBS buffer, pH 7.4, for 20 min. Subsequently, cells were washed in 0.1 M PBS, permeabilized for 10 min with 0.4 % Triton X-IOO, and incubated with the primary antibody in a humidified chamber at 37 ⁰C for 2 h. The mouse monoclonal antibody recognizing α-actinin was purchased from Sigma (Germany), the goat polyclonal anticardiac troponin I and goat anti-connexin 40 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA), and the mouse anti-connexin 43 monoclonal antibody and rabbit anti-connexin 45 polyclonal antibody were from Chemicon International. After the cells were washed with 0.4 % Triton X-100 and PBS, secondary antibody was added and the cells were incubated overnight at 37 ⁰C. The secondary antibodies used were Alexa Fluor 568 labeled goat anti- mouse IgM (Molecular Probes, Eugene, OR), Alexa Fluor 555 goat anti-rabbit IgG (Molecular Probes), R-Phycoerythrin conjugated anti -mouse IgG (Sigma), and R- Phycoerythrin conjugated anti-goat IgG (Sigma). Finally, the cells were washed with PBS and embedded in mounting medium. Analysis was performed using a confocal microscope (Leica Microsystems, Heidelberg, Germany). Images of connexin-staining were evaluated by two blinded investigators. Of the EGFP -positive cells, 91 % stained positively with an antibody against α-actinin (Fig. 2 A, 2B). Additionally, staining with an antibody specific for cardiac troponin I resulted in positive labeling of 83 % of EGFP -positive cells isolated from 24- to 28-day-old EBs (Fig. 2C, 2D). The small number of ANP-EGFP labeled cells with negative troponin I staining most likely was due to different developmental stages of ES cell-derived cells, since troponin I is a marker for late-stage cardiomyocytes in EBs (Hidaka et al., FASEB J. 17 (2003), 740-742). Thus, immunocytochemistry corroborated the expression of cardiac- specific reporter genes in ANP-EGFP-transfected ES cells.

Example 2: ANP-EGFP cells differentiate into distinct cell populations

The hyperpolarization-activated inward current I_f is a characteristic ionic current of cardiac pacemaker cells (DiFrancesco, Annu. Rev. Physiol. 55 (1993), 455-472). I_f is essential for spontaneous beating activity and for the modulation of the pacing rate (Er et al.. Circulation 107 (2003), 485-489). Therefore, in the present study, I_f current recordings and registrations of action potentials were performed to identify and further characterize ES cell-derived pacemaker cells of the terminal differentiation stage (6+ .9d) (Maltsev et al., Mech. Dev. 44 (1993), 41-50; Abi-Gerges et al., J. Physiol. 523 (2000), 377-389). Experiments were performed by using standard microelectrode whole-cell patch clamp techniques (Hamill et al., Pflugers Arch. 391 (1981), 85-100; Hoppe et al., Proc. Natl. Sci. USA 98 (2001), 5335-5340) with an, Axopatch 200B amplifier (Axon instruments, Foster City, CA) while sampling at 10 kHz and filtering at 2 kHz. Current recordings were performed at room temperature (21 to 23 °C), while action potential measurements were taken at 36 ± 0.5 ⁰C. The recording bath solution contained (mM) NaCl 135, KCl 5, CaCl₂ 2, glucose 10, MgCl₂ 1, HEPES 10; pH was adjusted to 7.4 with NaOH. For I_f recordings [K^τ]o was increased to 100 mM; and BaCl₂ 2 mM, CdCl₂ 200 μM, and 4-aminopyridine 4 mM were added to block Iκi, Ic_aL, and I_t0, respectively. The micropipette electrode solution was composed of (mM): K-glutamate 130, KCl 15, NaCl 5, MgCl₂ 1, HEPES 10, and Mg-ATP 5; pH was adjusted to 7.3 with KOH. Borosilicate microelectrodes had tip resistances of 2-4 MΩ when filled with the internal recording solution.

I_f size was measured as the difference between the instantaneous current at the beginning of a hyperpolarizing step ranging from -50 to -150 mV in 10 mV increments and the steady-state current at the end of hyperpolarization for 2.45 to 3 s, as described previously (Hoppe et al., Circulation 97 (1998), 55-65; Er et al., Circulation 107 (2003), 485-489). Fast-current inactivation was achieved by a depolarization pulse to 20 mV. In quiescent myocytes action potentials were initiated by short depolarizing current pulses (2 ms, 500-800 pA). A xenon arc lamp was used to view EGFP at 488/530 run (excitation/emission). Pooled data are presented as mean ± SEM. Comparisons between groups were performed using one-way ANOVA. P-values < 0.05 were deemed significant.

I_f was present in 74 % of all EGFP-positive cells investigated with a mean current density of 20.4 ± 2.0 pA/pF at -150 mV (n=128). More interesting, however, distinct subpopulations of ANP-EGFP-positive cells were observed. As described previously cardiomyocytes enzymatically isolated from beating areas of embryoid body outgrowths exhibit a spindle-, round-, or tri-/multiangular-shaped morphology after dissociation (Maltsev et al., Mech. Dev. 44 (1993), 41-50). Although basic electrophysiological properties were reported to be independent of the cellular morphology in unlabeled cardiomyocytes (Maltsev et al., see supra)), in this study ANP-EGFP expressing cells with a spindle-like morphology all had a pacemaker-like phenotype (n=22) (Fig. 3A). These spindle-shaped cardiocytes exhibited vigorous spontaneous beating with a rate of 173 ± 14 bpm. All of these cells generated spontaneous sinus nodal action potentials synchronous to the contractions (resting potential -45.5 ± 2.2 mV; slow diastolic depolarization; overshoot 19 ± 3 mV, APD₉₀ 1 11.3 ± 1.7 ms; Fig. 3C). Conversely, 58 % of tri-/multiangular cells (n=77; Fig. 3B) were quiescent with the remaining beating at a significantly slower rate of 62 ± 3 bpm (PO.001). All triangle-shaped cells displayed atrial-like action potentials with more negative resting potentials (-68.7 ± 2.1 mV), more positive overshoots (37 ± 1.8 mV), and shorter durations (APDg₀ 48.0 ± 2.1 ms; Fig. 3D). Consistent with these differences in beating frequency and action potential configuration, all spindle-like cells revealed significantly larger I_f current densities (34.5 ± 2.4 pA/pF at -150 mV), earlier first I_f current activation (-50 to -60 mV) and faster current activation kinetics (τ 395.3 ± 30.7 ms at -150 mV) compared with tri- /multiangular cells (12.8 ± 0.7 pA/pF at -150 mV; first activation at -80 to -90 mV; τ 681.1 ± 30.3 ms at -150 mV; PO.001; Fig. 3E₅ 3F). These distinct I_f current properties further supported the pacemaker-like phenotype of spindle-shaped cells.

By their morphology, 21.3 ± 3.1 % of ANP-EGFP-expressing cells could not be classified to either of these two clearly distinguishable sub-lineages. These cells exhibited a round-shaped morphology, whereas their electrophysiological properties resembled those of either spindle- shaped (33 %) or triangular-shaped (67 %) cardiocytes. It still has to be determined whether these cells were not well enough attached to the coverslip surface to show their typical morphology (i.e., various cell types are round-shaped after trypsinization before attachment), exhibit an intermediate phenotype, or are not yet fully differentiated. Based on action potential patterns, no ventricle-like cells could be identified reflecting an atrial-specific expression of the human ANP promoter in ES cells.

Example 3 Endothelin-1 directs development of ANP-EGFP ES cells toward a pacemaker phenotype

So far no developmental factors promoting the differentiation of mammalian atrial-derived, presumably pacemaker conduction tissue have been reported. To test the effect of endothelin- 1 (ET- 1) on the differentiation of the two sub-lineages which had been identified, cultured ANP-EGFP-expressing EBs were cultured from the beginning of plating for 14 days in the presence of ET-I or ET-I with additional BQ 123 or BQ788. ET-I tended to change the expression pattern of EGFP, increasing the number of small intense fluorescent areas throughout the EBs while slightly decreasing the total area of EGFP fluorescence within EBs (15.4 ± 2.2 %; n=24; P=NS vs. control). Indeed, ET- 1 significantly increased the relative percentage of spindle-shaped cells by 67 % (from 18.3 ± 1.7 % to 30.3 ± 2.1 %; PO.001), while decreasing the relative percentage of tri-/multiangular cells by 35 % (from 60.3 ± 4.2 % to 39.2 ± 3.5 %; P=O.001). ET-I tended to increase the number of undifferentiated round cells (30.5 ± 3.1 %; P=NS vs. control), but this effect was variable and not significant. The concentration-response relation for ET-I revealed an EC₅₀ of 1.1 x 10.^'9 M (Fig. 4A). All spindle-like and tri-/multiangular cells exhibited action potential configurations and I_f current properties of pacemaker and atrial cells, respectively, identical to untreated control cells with the respective morphologies. The effects of ET-I on ES cell differentiation could be prevented by BQ123 or BQ788 (Fig. 6A). This indicated that ET-I shifted differentiation of ANP-EGFP-positive ES cell-derived cardiocytes toward pacemaker cells in an endothelin receptor-dependent manner without affecting electrophysiological properties.

The effect of ET-I on the development of the cardiac conduction tissue was further confirmed by immunostaming and Western blot analyses. For Western blot analysis, single, enzymatically dissociated ANP-EGFP-positive ES cell-derived cells were lysed with Laemmli sample buffer (Tris-HCl, 62.5 mM, 2 % SDS₃ 5 % glycerol, 5 % β-mercaptoethanol, and 1 mM phenylmemylsulfonylfluoride, pH 7.5). Total protein (20 μg) was subjected to 10 % PAGE in the presence of SDS (SDS-PAGE) under reducing conditions, and the separated proteins were electrophoretically transferred to nitrocellulose membrane by using the tank blotting system (BioRad, Munich, Germany). Blots were incubated with the primary antibodies against connexin 40, connexin 43, connexin 45, minimal K⁺ channel subunit (minK; mouse anti-minK polyclonal antibody), and minK related peptide 1 (MiRP 1 ; mouse polyclonal anti-MiRPl, Alomone Labs, Israel) overnight at +4 °C, followed by incubation with peroxidase-conjugated secondary antibodies (Sigma) at the next day for 1 h at room temperature. Antibody reaction was visualized by enhanced chemiluminescence using ECL _. reagents (Amersham Corp., Arungton Heights, IL). Exposure of EBs to ET-I markedly increased the intensity of connexin 40 staining, a marker of the early differentiating conduction system in mice (Delorme et al., Dev. Dyn. 204 (1995), 358-371), compared with controls (Fig. 5A, 5B). In addition, expression of connexin 45, a marker of the mouse sinus node and conduction system (Alcolea et al., Circ. Res. 94 (2004), 100-109; Coppen et al., Dev. Genet. 24 (1999), 82-90), was increased by ET-I (Fig. 5E, 5F), while expression of connexin 43 (Alcolea et al., see supra), a marker of working myocardium, remained unaffected (Fig. 5C, 5D). Similar results were obtained by Western blot analysis. ET-I markedly increased the protein level of connexin 40 and elevated the protein amount of connexin 45 compared with control. These changes could be inhibited by treatment of EBs with BQ123 or BQ788 in the presence of ET-I . In contrast the protein level of connexin 43 was not affected by ET-I or ET-I with additional ET-I receptor antagonists (Fig. 4B).

Previous studies have shown an abundant expression of the K⁺ channel β-subunit minK in the adult cardiac conduction tissue (Pourrier et al., J. Membr. Biol. 194 (2003), 141-152; Kupershmidt et al., Circ. Res. 84 (1999), 146-152). Moreover, high transcript levels of a member of minK-related peptides, MiRPl, were demonstrated in the sinus node and MiRPl was found to be involved in the regulation of I_f current expression (Yu et al., Circ. Res. 88 (2001), E84-E87). Thus, to examine a possible effect of ET-I on these K⁺ channel modulators, minK and MiRPl protein levels were estimated in ET-I treated EGFP -positive cells compared with controls. However, no effect of ET-I or ET-I with additional BQ123 or BQ788 on minK or MiRPl protein amounts was obtained (Fig. 4C). In contrast to MiRPl minK was barely detectable in treated and untreated cells, indicating a weak expression of this protein in ES cell-derived atrial cardiomyocytes.

Example 4: Neuregulin-1 shows no inductive effect on differentiation of pacemaker cells

In addition, the effect of neuregulin-1 (NRG-I) on the development of the ANPEGFP expressing ES cells was examined. ANP-EGFP expressing EBs were cultured from the beginning of plating for 14 days in the presence of NRG-I. However, no effect of NRG-I on differentiation of ANP-EGFP-positive ES cells (spindle-shaped cells 15.1 ± 1.6 %; triangular cells 57.4 ± 2.7 %; P=NS vs. control) was observed (Fig. 6A). Similar to ET-I treated EBs, all spindle-shaped cells exhibited electrophysiological properties of pacemaker cells, while triangular cells had an atrial-like phenotype. NRG-I did not affect protein levels of connexins (Fig. 6B). Treatment with the combination of ET-I and NRG-I resulted in similar changes of cell differentiation (spindle-shaped cells 28.6 ± 3.6 %; triangular cells 37.5 ± 6.8 %; P=NS vs. ET-I) and protein amounts of connexins as ET-I alone (Fig. 6A, 6B).

Conclusion

Although studies on primary cardiac cell cultures are limited because cell properties and normal cardiogenesis apparently are disturbed during cultivation, ES cells provide a useful model for the evaluation of early differentiation and development of various tissues (Gepstein, Circ. Res. 91 (2002), 866-876; Kolossov et al., J. Cell Biol. 143 (1998), 2045-2056). Therefore, in the present study ANP-EGFP expressing ES cell lines were established to further characterize the development of very early stages of the mammalian cardiac conduction tissue. In ANP-EGFP-expressing ES cell-derived cardiomyocytes a distinct sub-lineage of pacemaker cells could be identified by morphological and electrophysiological parameters. Furthermore, the present results clearly indicate that ET-I, a vascular cytokine, induces ANP-EGFP-positive cells to develop a pacemaker-like phenotype.

The present observation was consistent with previous experiments in neonatal heart (LaPointe et al., J. Biol. Chem. 263 (1988), 9075-9078), which was an atrial-specific expression of the human ANP promoter during cardiomyogenesis in the ES cell system. Thus, ANP-EGFP expression enabled precise identification and characterization of atrial-derived cardiocytes and the exclusion of ventricular cells, which previously were demonstrated not to develop into pacemaker-like cells (Muller et al, FASEB J. 14 (2000), 2540-2548). ANP-EGFP-positive cells revealed distinct morphological and electrophysiological subpopulations. The most common cell type was tri-/multiangular-shaped with atrial electrophysiological characteristics presumably further developing into working atrial myocardium. More interesting, a second sub-lineage of cells with a spindle-like shape consistently exhibiting a pacemaker-like phenotype could be identified. Besides distinct action potential configurations, the two cellular subtypes differed significantly in I_f current size and kinetics. I_f is the characteristic ionic current of primary and secondary adult pacemaker cells (DiFrancesco, Annu. Rev. Physiol. 55 (1993), 455-472). Additionally, I_f has been recorded in working adult atrial and ventricular myocardium (Hoppe et al., Circulation 97 (1998), 55-65; Hoppe and Beuckelmann, Cardiovasc. Res. 38 (1998), 788-801). Similar to findings with adult myocytes, significantly larger I_f current densities and more positive current activation in ES cell-derived pacemaker cells compared with atrial cardiocytes we^'re observed. Consistent with previous, findings of the invention in neonatal cardiomyocytes, larger I_f current size was associated with a faster beating rate in ANP-EGFP-expressing ES cells (Er et al., Circulation 107 (2003), 485-489). The observed differences in morphology, action potentials, and I_f properties indicate a very early diversification of electrophysiological and morphological parameters between pacemaker cells vs. atrial working myocardium.

In chicken the cardiac conduction system develops in close spatial association with forming coronary arterial blood vessels, and ET-I was found to induce the differentiation of Purkinje fibers (Gourdie et al., Development 121 (1995), 1423-1431; Schiaffmo, Circ. Res. 80 (1997), 749-750). No such close regional relation has yet been reported for mammalian cardiac development. In addition, so far no studies have been performed of any inductive factors on atrial-derived cardiomyocytes. The present results now clearly demonstrate that ET-I can also promote differentiation of ANP-EGFP-expressing ES cells to pacemaker cells. ET-I exposure resulted in a shift toward spindle-shaped cells with pacemaker-like electrophysiological characteristics. Furthermore, ET-I increased the expression of connexin 40 and connexin 45. Previously, these connexin isoforms have predominantly been detected in the conduction tissue of developing mammalian hearts and in the adult murine cardiac central conduction system and sinus node (Delorme et al., Dev. Dyn. 204 (1995), 358-371; Alcolea et al, Circ. Res. 94 (2004), 100-109; Coppen et al., Dev. Genet. 24 (1999), 82-90; Coppen et al., MoL Cell Biochem. 242 (2003), 121-127). Conversely, a very low level of connexin 43 imrnunolabeling in ANP-EGFP-expressing EBs was observed, which was unaffected by ET- 1. This finding is consistent with recent reports demonstrating wide abundance of connexin 43 in adult working myocardium, while connexin 43 was only weakly expressed in the developing atria and virtually absent in the central conduction tissue (Coppen et al., MoI. Cell Biochem. 242 (2003), 121-127; Delorme et al., Circ. Res. 81 (1997), 423-437). Thus, the present results suggest a paracrine role of ET-I in the development of the conduction system.

Members of the neuregulin family encode a variety of growth and differentiation factors. The epidermal growth factor (EGF)-like domain of each NRG is sufficient for receptor binding and bioactivity (Meyer and Birchmeier, Nature 378 (1995), 386-390). NRG-I is expressed by endocardial cells in the embryonic heart and is found mainly in the ventricles (Carraway et al., Nature 387 (1997), 512-516). In embryonic mice NRG-I converted ventriculocytes into cells of the ventricular conduction system, Purkinje cells (Rentschler et al., Proc. Natl. Acad. Sci. USA 99 (2002), 10464-10469). Conversely, no effect of a recombinant peptide encompassing, the β variant of the NRG-I EGF-like domain on the differentiation of ANP-EGFP-positive ES cells was observed in the present study, indicating a local inductive role of NRG-I on the development of the cardiac conduction system restricted to the ventricles, while atrial cardiocytes physiologically hardly exposed to NRG-I show no response to this paracrine signal. These results are consistent with an early diversity of paracrine signaling in the differentiation of the atrial-derived, presumably central vs. the ventricular (Purkinje-like) conduction system. Considering the feasibility of cell therapy aiming to replace dysfunctional cardiac tissue, strategies need to be developed for directing in vitro differentiation to a specific lineage. Thus, a first step is to identify and characterize candidate cells and to determine their developmental mechanisms. The findings of the present observations give further insight into the differentiation of the cardiac conduction system. ANP-EGFP expression enabled the identification of ES cell-derived pacemaker cells only by their fluorescence and morphology, which may obviate further electrophysiological testing in the future. Since it was possible in accordance with the present invention to markedly enrich the percentage of pacemaker cells by ET-I, these results represent a valuable first step in the specific selection of pacemaker cells for the development of cell therapeutic strategies for degenerative or congenital diseases of the cardiac conduction system.

It will be recognized that the compositions and procedures provided in the description can be effectively modified by those skilled in the art without departing from the spirit of the invention embodied in the claims that follow.

Claims

Claims

1. Method of providing pacemaker-like cardiomyocytes comprising culturing stem cells under conditions allowing differentiation of said cells into cardiomyocytes, wherein said stem cells comprise a recombinant nucleic acid molecule comprising a selectable marker and/or reporter gene operably linked to a regulatory sequence, which is derived from the gene encoding atrial natriuretic peptide (ANP) or from a gene which exhibits substantially the same expression pattern as the ANP gene.

2. The method of claim 1, wherein said regulatory sequence is or is derived from the human ANP promoter.

3. The method of claim 1 or 2, wherein said stem cells are embryonic stem (ES) cells, embryonic germ (EG) cells or adult stem cells.

4. The method of any one of claims 1 to 3, wherein said cardiomyocytes comprise or substantially consist of atrial and/or pacemaker cells.

5. The method of any one of claims 1 to 4, wherein said selectable marker confers resistance to puromycin, bleomycin, hygromycin, methothrexate, or neomycin.

6. The method of any one of claims 1 to 5, wherein said reporter is selected from different color versions of enhanced green fluorescent protein (EGFP).

7. The method of any one of claims 1 to 6, wherein said marker gene and said reporter gene are contained one the same recombinant nucleic acid molecule.

8. The method of any of claims 1 to 7, wherein said pacemaker cells are identified by determining reporter gene activity.

9. The method of any one of claims 1 to 8, further comprising exposing said cell to endothelin-1 or to a compound which has substantially the same biological activity as endothelin-1.

10. A vector or a composition of vectors comprising a recombinant nucleic acid molecule as defined in any one of claims 1 to 7.

11. A cell or a plurality of cells comprising the vector or the composition of vectors of claim 10 or which are cells as defined in any one of claims 1 to 9.

12. A stem cell as defined in any of claims 1 to 9 comprising stably integrated into its genome a recombinant nucleic acid molecule comprising a fluorescent reporter gene, preferably an EFGP gene, operably linked to a regulatory sequence which is derived from the ANP gene.

13. The cells of claim 11 or 12, which are a cell aggregate.

14. Cells or tissue obtainable by the method of any one of claims 1 to 9 or comprising cells of any one of claims 11 to 13.

15. An organ comprising cells of any one of claims 11 to 14 or a tissue of claim 14.

16. An implant or transplant comprising cells of any one of claims 11 to 14, a tissue of claim 14, or an organ of claim 15.

17. A composition of matter comprising recombinant nucleic acid molecules as defined in any one of claims 1 to 7, a vector of claim 10, cells of any one of claims 11 to 14 or a tissue of claim 14.

18. Use of the method of any one of claims 1 to 9, recombinant nucleic acid molecules as defined in any one of claims 1 to 7, a vector of claim 10, cells of any one of claims 11 to 14 or a tissue of claim 14 for analyzing early steps of tissue formation during embryonic development or the influence of factors and compounds on this process.

19. A method of treatment of damaged cardiac tissue in a subject comprising implanting or transplanting to the subject in need thereof cells of any one of claims 11 to 14, a tissue of claim 14 or an organ of claim 15.

20. A method of improving cardiac tissue repair and/organ function in a mammal comprising the steps of:

(a) introducing a cellular inoculum comprising a culture of cells of any one of claims 11 to 14, in which differentiation has been initiated, or tissue of claim 14 to at least a portion of the previously damaged area of the tissue; and

(b) allowing said introduced cellular inoculum to engraft in situ as viable cells or tissue situated within the previously damaged area of the tissue, wherein the engraftment results in improved cardiac tissue and/or organ function in said mammal.

21. A method for improving cardiac function in a mammal after a myocardial infarct, said method comprising the steps of:

(a) culturing undifferentiated mammalian embryonic stem (ES) cells comprising a resistance gene and/or a reporter gene under the control of a regulatory sequence as defined in claim 1 or 2 in vitro in a culture medium, optionally containing the selective agent for the resistance gene under conditions allowing differentiation of said ES cells into cardiomyocytes, in particular pacemaker cells or precursors thereof,

(b) isolating said differentiated cardiomyocytes or precursors thereof and/or eliminating non-differentiated cells, optionally along with cells differentiating towards irrelevant cell types from said cardiomyocytes in the course of differentiation;

(c) subsequently transplanting said cardiomyocytes or precursors thereof to at least a portion of the previously infarcted area of the heart tissue; and (d) allowing said introduced cellular inoculum to engraft in situ as viable ceils situated within the previously infarcted area of the heart tissue, wherein the engraftment results in improved cardiac function in said mammal.

22. A method for obtaining and/or profiling a test substance capable of influencing cardiomyocyte development, comprising the steps:

(a) contacting a test sample comprising cells of any one of claims 11 to 14, a tissue of claim 14 or an organ of claim 15 with a test substance; and

(b) determining a phenotypic response in said test sample compared to a control sample, wherein a change in the phenotypic response in said test sample compared to the control sample is an indication that said test substance has an effect on cell development and/or tissue structure formation.

23. The method of claim 22, wherein said test sample is contacted with said test substance prior to, during or after said cell or tissue passed through the method of any one of claims 1 to 9.

24. The method of claim 22 or 23, wherein said contacting step further includes contacting said test sample with at least one second test substance in the presence of said first test substance.

25. The method of any one of claims 22 to 24, wherein preferably in a first screen said test substance is comprised in and subjected as a collection of test substances.

26. The method of claim 25, wherein said collection of test substances has a diversity of about 10³ to about 10⁵.

27. The method of claim 26, wherein the diversity of said collection of test substances is successively reduced.

28. The method of any one of claims 22 to 27, wherein the phenotypic response comprises electrophysiological properties during the ongoing differentiation process.

29. The method of any one of claims 1 to 9 or 22 to 28, wherein said one or more cells are genetically engineered to (over)express or inhibit the expression of a target gene.

30. The method of any one of claims 1 to 9 or 22 to 29, wherein a compound known to activate or inhibit differentiation process and/or tissue structure formation is added to the culture medium.

31. A method of manufacturing a drug comprising the steps of any one of claims 22 to 30.

32. A method of manufacturing an agent which supports wound healing and/or healing of damaged cardiac tissue comprising the steps of any one of claims 22 to 31.

33. The method of claim 31 or 32, further comprising modifying said substance to alter, eliminate and/or derivatize a portion thereof suspected causing toxicity, increasing bioavailability, solubility and/or half-life.

34. The method of any one of claims 31 to 33, further comprising mixing the substance isolated or modified with a pharmaceutically acceptable carrier.

35. A kit or composition useful for conducting a method of any one of claims 1 to 9 or 22 to 34, containing the vector or the composition of vectors of claim 10, a multi- or pluripotent cell, a differentiation-promoting compound and optionally a culture medium, recombinant nucleic acid molecules, and/or standard compounds.

36. Use of cells of any one of claims 11 to 14, a tissue of claim 14 or an organ of claim 15 as biological pacemaker or precursor thereof.

37. Use of a regulatory sequence as defined in claim 1 or 2, a recombinant nucleic acid molecule as defined in any one of claims 1 to 7 or a vector or the composition of vectors of claim 10 for the staining, identification and/or (pre)selection of in vitro differentiated atrial and/or pacemaker cells.