WO2006021460A1 - Moyens et procedes pour generer des cardiomyocytes et un tissu correspondant ainsi que leur utilisation - Google Patents

Moyens et procedes pour generer des cardiomyocytes et un tissu correspondant ainsi que leur utilisation Download PDF

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WO2006021460A1
WO2006021460A1 PCT/EP2005/009297 EP2005009297W WO2006021460A1 WO 2006021460 A1 WO2006021460 A1 WO 2006021460A1 EP 2005009297 W EP2005009297 W EP 2005009297W WO 2006021460 A1 WO2006021460 A1 WO 2006021460A1
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cells
tissue
cell
gene
differentiation
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Uta C. Hoppe
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Cell Center Cologne Gmbh
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0657—Cardiomyocytes; Heart cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5061—Muscle cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/365—Endothelin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • 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.
  • 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.
  • 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.
  • 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).
  • pacemaker cells and the specific cardiac conduction tissue origin also from cardiac precursor cells (Moorman et al., Circ. Res. 82 (1998), 629-644).
  • 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.
  • 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.
  • ES murine embryonic stem
  • EGFP enhanced green fluorescent protein
  • ANP human atrial natriuretic peptide
  • transgenic ES cell-derived cell types such as cardiomyocytes
  • the techniques of this invention are designed in part to provide cell populations with improved characteristics for human therapy.
  • 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.
  • Fig. 1 Typical pattern of EGFP expression under the transcriptional control of the hANP promoter in a spontaneously beating EB (6+22 d).
  • Fig. 2 Immunohistochemistry corroborated the cardiac nature of ANP-EGFP- expressing EBs.
  • 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 ( ⁇ 395.3 ⁇ 30.7 ms at -150 mV).
  • 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 ( ⁇ 681.1 ⁇ 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.
  • 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).
  • 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).
  • F secondary Alexa Fluor 555 anti-rabbit antibody
  • E untreated EBs
  • 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 ANP-EGFP-expressing ES cells.
  • stem cell can refer to either stem cell or germ cell, for example embryonic stem (ES) and germ (EG) cell, respectively.
  • ES embryonic stem
  • EG germ
  • 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.
  • 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.
  • 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.
  • embryonic germ cell is used to describe cells of the present invention that exhibit an embryonic pluripotent cell phenotype.
  • EG human embryonic germ cell
  • embryonic germ cell 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.
  • 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.
  • ES cells include 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.
  • 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.
  • STO cell refers to embryonic fibroblast mouse cells such as are commercially available and include those deposited as ATCC CRL 1503.
  • EBs embryoid bodies
  • 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.
  • 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.
  • 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” .
  • 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.
  • promoters 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.
  • 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.
  • 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.
  • polypeptide polypeptide
  • peptide protein
  • modified amino acids it may be linear or branched, and it may be interrupted by non-amino acids.
  • 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.
  • ANP atrial natriuretic peptide
  • 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.
  • EGFP green fluorescent protein
  • ABP human atrial natriuretic peptide
  • Atrial natriuretic peptide also known as cardionatrin, atrionatriuretic factor, Pronatriodilatin (PND), atriopeptin and atrial natriuretic factor (ANF)
  • PND Pronatriodilatin
  • ANF atrial natriuretic factor
  • 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.
  • EBs embryoid bodies
  • Suitable regulatory sequences derived from the ANP gene 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).
  • TIGR The Institute for Genomic Research
  • NCBI National Center for Biotechnology Information
  • 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.
  • 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.
  • genomic sequences of the human ANF gene see, e.g., Genbank accession no. X01471 and Greenberg et al., Nature 312 (1984), 656-658.
  • Maki et al. Biochem. Biophys. Res. Cornmun 125 (1984), 797- 802
  • said regulatory sequence is or is derived from the human ANP promoter.
  • 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.
  • 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).
  • 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.
  • 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.
  • exfoliated human deciduous tooth a comparable very accessible tissue
  • neural cells adipocytes
  • odontoblasts see Miura et al., Proc. Natl. Acad. Sci. USA 100 (2003), 5807-5812.
  • those cells were found to be able to induce bone formation, generate dentin, and survive in mouse brain along with expression of neural markers.
  • multilineage potential of homozygous stem cells derived from metaphase II oocytes has been described by Lin et al.
  • HSC rare hematopoietic stem cell(s)
  • 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.
  • ES embryonic stem
  • syngenic or autologous cells and recipients are preferably used in the corresponding embodiments of the invention.
  • 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.
  • cells may be genetically manipulated to suppress relevant transplantation antigens, see also infra, immunosuppressive agents may be used.
  • 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.
  • transgenic non-human animals in particular mammals
  • compositions and methods for making transgenic swines to be used as xenograft donors are described in US patent 5.523,226.
  • international application WO97/12035 describes methods of producing transgenic animals for xenotransplantation.
  • 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.
  • the cells may be isolated from a subject.
  • 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.
  • 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
  • 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.
  • 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).
  • 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.
  • medium component(s) that promote(s) growth of undifferentiated cells, or act(s) as an inhibitor of differentiation.
  • 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.
  • 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.
  • 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.
  • said regulatory sequence is derived from the ANF gene as described above.
  • 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.
  • 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.
  • FuGENE(TM) 6 Transfection Reagent a blend of lipids in non-liposomal form and other compounds in 80 % ethanol, obtainable from Roche Diagnostics Corporation.
  • FuGENE(TM) 6 Transfection Reagent a blend of lipids in non-liposomal form and other compounds in 80 % ethanol, obtainable from Roche Diagnostics Corporation.
  • 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.
  • nucleoside and aminoglycoside- antibiotic-resistance genes for, e.g. puromycin (puromycin-N-acetyltransferase), streptomycin, bleomycin, neomycin, gentamycin or hygromycin.
  • resistance genes are dehydrofolate-reductase,
  • 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.
  • said regulatory sequence of the reporter gene is substantially the same as said regulatory sequence of the marker gene, but not necessarily.
  • the reporter can be of any kind as long as it is non-damaging for the cell and confers an observable or measurable phenotype.
  • the green fluorescent protein (GFP) from the jellyfish Aequorea victoria (described in international applications WO95/07463, WO96/27675 and WO95/121191) and its derivates ;i Blue GFP" (Heim et al., Curr. Biol. 6 (1996), 178-182 and Redshift GFP" (Muldoon et al, Biotechniques 22 (1997), 162-167) can be used.
  • the enhanced green fluorescent protein (EGFP) is particularly preferred.
  • EYFP and ECFP enhanced yellow and cyan fluorescent proteins
  • DsRed, HcRed red fluorescent proteins
  • 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.
  • other detectable proteins, particularly epitopes of those proteins can also be used.
  • 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.
  • stably transfected ES cells contain a further selectable marker gene, which confers e.g. a resistance against an antibiotic, e.g. neomycin.
  • a further selectable marker gene confers e.g. a resistance against an antibiotic, e.g. neomycin.
  • 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.
  • 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.
  • repressor elements e.g. a doxicyclin-inducible repressor element.
  • 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.
  • 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.
  • 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.
  • ET-I endothelin-1
  • endothelin-1 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.
  • Endothelin-1 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.
  • ECE-I endothelin- converting enzyme- 1
  • ETA endothelin- A receptor-mediated signaling pathway
  • endothelin-1 refers to 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.
  • ET-I agonists may also be referred to as ET-I receptor agonists.
  • 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.
  • endothelins mediate their actions via only two receptor types that have been cloned and classified as the ET(A) and ET(B) receptors.
  • 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.
  • ET-I is a dual agonist for both endothelin type A and B receptors.
  • 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.
  • ET-I stimulates or induces ES cell-derived cardiocytes towards pacemaker cells in an endothelin receptor-dependent manner without affecting electrophysiological properties.
  • ET-I acts directly or indirectly as an endothelin receptor agonist for both ET(A) and ET(B) receptors.
  • 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.
  • selective agonists for endothelin B receptors such as sarafotoxin 6c (Granstr ⁇ m 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.
  • ET(B) receptor agonists that may be employed in accordance with the present invention are described for example in international application WO2004/037235.
  • 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.
  • 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,
  • 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.
  • 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.
  • 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.
  • pacemaker-like cardiomyocytes 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).
  • 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.
  • 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.
  • 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
  • EBs embryoid bodies
  • 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).
  • spinner flasks 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.
  • 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.
  • 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.
  • the stem cells are substantially differentiated into atrial cardiomyocytes or precursors thereof.
  • said pacemaker cells are identified by their morphology and/or electrophysiological properties; see also the Examples.
  • 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:
  • 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.
  • MLC-2v myosin light chain-2v
  • CMV(enh) 0.5 kb enhancer element of the cytomegalovirus
  • 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.
  • 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.
  • said cells are preferably embryonic cardiac cells.
  • 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.
  • 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.
  • 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.
  • 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.
  • said stem cell is stem cell as described and produced in Example 1.
  • 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.
  • 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%.
  • 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
  • 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.
  • 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 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.
  • 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..
  • 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:
  • 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;
  • 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.
  • 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).
  • MEA microelectrode array
  • 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.
  • MEA microelectrode arrays
  • 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.
  • 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.
  • factors such as solvents, small molecules, drugs, peptides, polynucleotides, and the like
  • environmental conditions such as culture conditions or manipulation
  • 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.
  • compounds are screened initially for potential toxicity (Castell et al., pp.
  • 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.
  • 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:
  • 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;
  • 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.
  • EST ES cell test
  • preferred compound formulations for testing do not include additional components such as preservatives, that have a significant effect on the overall formulation.
  • preferred formulations consist essentially of a biologically active compound and a physiologically acceptable carrier, e.g. water, ethanol, DMSO, etc.
  • a physiologically acceptable carrier e.g. water, ethanol, DMSO, etc.
  • the formulation may consist essentially of the compound itself.
  • a plurality of assays may be run in parallel with different compound concentrations to obtain a differential response to the various concentrations.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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 ⁇ l 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 10 3 to about 10 5 , 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.
  • ASO allele-specific oligonucleotide
  • 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.
  • 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.
  • cells cultured or modified using the materials and methods provided by the present invention are mounted to support surfaces to screen for bioactive substances.
  • 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.
  • measuring devices such as a computer
  • 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.
  • high throughput 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.
  • 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.
  • 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.
  • the cells can be introduced into a microfiuidics device, such as those provided by Caliper (Newton, MA, USA).
  • the method of the present invention comprises taking 2, 3, 4, 5, 7, 10 or more measurements, optionally at different positions within the container.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • the isolated compound or corresponding drug supports wound healing and/or healing of damaged cardiac tissue.
  • 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.
  • the method may further comprise mixing the substance isolated or modified with a pharmaceutically acceptable carrier.
  • the drug or a pro-drug thereof can be synthesized in a therapeutically effective amount.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • ES cells were electroporated into 2.5 x 10 7 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 (ESGROTM, Chemicon International Inc., Temecula, CA) and selected for 10 days using G418 (250 ⁇ g/ml).
  • ESGROTM leukemia inhibitory factor 1000 units/ml
  • G418 250 ⁇ g/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 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 ⁇ -mercaptoethanol.
  • 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
  • the recombinant peptide containing the ⁇ 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.
  • 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.
  • EGFP fluorescence was exclusively detected in these beating areas, indicating the cardiac specificity of the human ANP promoter.
  • not all beating areas within the same EB displayed a prominent fluorescence, suggesting a spatial- restricted expression of the ANP promoter.
  • 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 0 C for 2 h.
  • the mouse monoclonal antibody recognizing ⁇ -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 0 C.
  • 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).
  • 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
  • R-Phycoerythrin conjugated anti-goat IgG Sigma
  • 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).
  • the micropipette electrode solution was composed of (mM): K-glutamate 130, KCl 15, NaCl 5, MgCl 2 1, HEPES 10, and Mg-ATP 5; pH was adjusted to 7.3 with KOH. Borosilicate microelectrodes had tip resistances of 2-4 M ⁇ 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.
  • 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).
  • ANP-EGFP-expressing 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
  • ET- 1 endothelin- 1
  • 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.
  • the concentration-response relation for ET-I revealed an EC 50 of 1.1 x 10. '9 M (Fig. 4A).
  • Total protein (20 ⁇ g) 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, Kunststoff, Germany).
  • minK and MiRPl protein levels were estimated in ET-I treated EGFP -positive cells compared with controls.
  • no effect of ET-I or ET-I with additional BQ123 or BQ788 on minK or MiRPl protein amounts was obtained (Fig. 4C).
  • MiRPl minK was barely detectable in treated and untreated cells, indicating a weak expression of this protein in ES cell-derived atrial cardiomyocytes.
  • 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.
  • ET-I a vascular cytokine
  • 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.
  • I f is the characteristic ionic current of primary and secondary adult pacemaker cells (DiFrancesco, Annu. Rev. Physiol. 55 (1993), 455-472).
  • 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.
  • connexin 40 and connexin 45 increased the expression of connexin 40 and connexin 45.
  • 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).
  • 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).
  • 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.

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Abstract

L'invention concerne des cellules cardiaques dérivées de cellules souches ainsi qu'un tissu correspondant. Elle concerne notamment des systèmes destinés à la génération de novo de cardiomyocytes fonctionnant à la manière d'un stimulateur cardiaque ainsi que l'utilisation de ces systèmes dans la transplantation ou dans le développement de médicaments.
PCT/EP2005/009297 2004-08-27 2005-08-29 Moyens et procedes pour generer des cardiomyocytes et un tissu correspondant ainsi que leur utilisation WO2006021460A1 (fr)

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Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
CHEN SONGCANG ET AL: "Suppression of ANP gene transcription by liganded vitamin D receptor: Involvement of specific receptor domains", HYPERTENSION (DALLAS), vol. 31, no. 6, June 1998 (1998-06-01), pages 1338 - 1342, XP002357707, ISSN: 0194-911X *
CHEN YIU-FAI ET AL: "Hypoxia stimulates atrial natriuretic peptide gene expression in cultured atrial cardiocytes", HYPERTENSION (DALLAS), vol. 29, no. 1 PART 1, 1997, pages 75 - 82, XP002357706, ISSN: 0194-911X *
CHIEN K R ET AL: "REGULATION OF CARDIAC GENE EXPRESSION DURING MYOCARDIAL GROWTH AND HYPERTROPHY MOLECULAR STUDIES OF AN ADAPTIVE PHYSIOLOGIC RESPONSE", FASEB JOURNAL, vol. 5, no. 15, 1991, pages 3037 - 3046, XP002357708, ISSN: 0892-6638 *
GASSANOV NATIG ET AL: "Endothelin induces differentiation of ANP-EGFP expressing embryonic stem cells towards a pacemaker phenotype.", THE FASEB JOURNAL : OFFICIAL PUBLICATION OF THE FEDERATION OF AMERICAN SOCIETIES FOR EXPERIMENTAL BIOLOGY. NOV 2004, vol. 18, no. 14, November 2004 (2004-11-01), pages 1710 - 1712, XP002357488, ISSN: 1530-6860 *
KOLOSSOV E ET AL: "FUNCTIONAL CHARACTERISTICS OF ES CELL-DERIVED CARDIAC PRESURSOR CELLS IDENTIFIED BY TISSUE-SPECIFIC EXPRESSION OF THE GREEN FLUORESCENT PROTEIN", THE JOURNAL OF CELL BIOLOGY, ROCKEFELLER UNIVERSITY PRESS, US, vol. 143, no. 7, 28 December 1998 (1998-12-28), pages 2045 - 2056, XP002938941, ISSN: 0021-9525 *
MUELLER M ET AL: "Selection of ventricular-like cardiomyocytes from ES cells in vitro", FASEB JOURNAL, FED. OF AMERICAN SOC. FOR EXPERIMENTAL BIOLOGY, BETHESDA, MD, US, vol. 14, no. 15, December 2000 (2000-12-01), pages 2540 - 2548, XP002180458, ISSN: 0892-6638 *
SACHINIDIS A ET AL: "Cardiac specific differentiation of mouse embryonic stem cells", CARDIOVASCULAR RESEARCH, vol. 58, no. 2, 1 May 2003 (2003-05-01), pages 278 - 291, XP002313488, ISSN: 0008-6363 *
SPIELMANN HORST ET AL: "The use of transgenic embryonic stem (ES) cells and molecular markers of differentiation for improving the embryonic stem cell test (EST)", CONGENITAL ANOMALIES, vol. 40, no. Supplement, December 2000 (2000-12-01), pages S8 - S18, XP008029541, ISSN: 0914-3505 *

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