WO2003023022A2

WO2003023022A2 - Method to induce the differentiation of endothelial cells to cardiomyocites

Info

Publication number: WO2003023022A2
Application number: PCT/IT2002/000576
Authority: WO
Inventors: Giulio Cossu; Elisabetta Dejana
Original assignee: Science Park Raf Spa
Priority date: 2001-09-11
Filing date: 2002-09-10
Publication date: 2003-03-20
Also published as: ITRM20010550A1; ITRM20010550A0; WO2003023022A3; AU2002341391A1

Abstract

Method to induce the differentiation of endothelial cells into cardiomyocites, comprising the co-cultivation of said endothelial cells with differentiated cardiomyocites.

Description

METHOD TO INDUCE THE DIFFERENTIATION OF ENDOTHELIAL CELLS INTO CARDIOMYOCITES

The instant invention concerns a method to induce the differentiation of endothelial cells into cardiomyocites.

It was believed that tissues that are not able to regenerate, as cerebral and muscular tissues, did not contain stem cells and were unable to regenerate following injuries. Recently some data show the contrary, with the isolation and growth of neural stem cells, able to give rise to neurons, oligodendrocytes and astrocytes, either from fetal or adult brain (1,2). Moreover, neural stem cells, as well as hematopoietic and mesenchimal cells, are able to generate different tissues as liver, brain, blood, and skeletal muscle, suggesting the presence of one or more fully totipotent stem cell types (3-14). On the other hand environment induced modifications are not limited to stem cells, and involve progenitor cells at different differentiation stages (15-17).

The possibility to utilize stem/progenitor cells for a tissue specific cell therapy opens new perspectives of clinical applications. Cardiac tissue is obviously a very interesting target tissue, given the fact that myocardium lesions are responsible of many deaths in western countries. The cardiomyocite pool is believed to be established soon after birth, when the proliferation stops and each subsequent loss of tissue can not be repaired anymore. The increase of blood vessels is one of the strategies to prevent the cell death (18). Recently it has been reported that hemangioblasts of bone marrow contribute to the formation of new vessels in the post- ischemic myocardium (19) and that c-Kit positive, bone marrow hematopoietic stem cells, differentiate into cardiomyocites, endothelium and smooth muscle upon injection in a ventricular post-ischemic ventricular wall (20). The authors of the invention have surprisingly found that either embryonal endothelial progenitors or endothelial cells isolated from umbilical vein are able to differentiate into cardiomyocites and to express endothelial markers, such as the Von Willebrand factor and the sarcomeric myosin.

Then the authors developed a method to regenerate myocardial tissue by using endothelial cells. The method of the invention is of particular interest in view of the fact that hematopoietic stem cells are not easily propagated in culture, instead of endothelial cells, and particularly cells from umbilical vein that are easily available at high concentrations and as homogeneous population.

It is therefore the object of the instant invention a method to induce the differentiation of endothelial cells into cardiomyocites, comprising the co-cultivation of said endothelial cells with differentiated cardiomyocites. Preferably the differentiation into cardiomyocites is demonstrated by the activation of at least one cardiac specific gene and/or by the devolpment of sarcomere and/or by electric coupling with the differentiated cardiomyocites and/or by in vivo myocardium formation after injection into post-ischemic ventricular wall. Endothelial cells may be derived from embryonic or neonatal tissues or from vessels, preferably umbilical vein.

In the instant invention the term co-cultivation refers to the cultivation of the inducing (cardiomyocite) and the inducible (endothelial) cells in the same culture dish so that either cell-cell contact is established or soluble products are exchanged. In the latter case cell-cell contact is ruled out.

It is a further object of the invention cardiomyocites obtainable from endothelial cells according to the invented method. In the instant invention the term cardiomyocite refers to a cell wherein at least one of the following markers is present: a cardiac specific gene (i.e. the NKX 2.5 gene as disclosed in Lints TJ, et al. Development. 1993 Oct;119(2):419-31), the development of sarcomeres, electric coupling with the differentiated cardiomyocites, in vivo myocardium formation after injection into post-ischemic ventricular wall. It is a further object of the invention a method to obtain immunologically compatible cardiomyocites of a subject including the steps of: a) isolating endothelial cells from the umbilical vein of the subject; b) optionally, storing the same, to keep their viability; c) inducing the differentiation of said endothelial cells into cardiomyocites according to the invented method.

Conveniently umbilical vein may be isolated from frozen collected umbilical cords. It is a further method of the invention a method to identify genes and/or gene products induced during endothelial cell differentiation to cardiomyocites comprising the steps of: a) co-cultivate endothelial cells with differentiated cardiomyocites for different times; b) separate endothelial cells from differentiated cardiomyocites; c) identify and analyze genes selectively expressed from said endothelial cells in respect to same cells that were not exposed to cardiomyocites or alternatively were exposed to other cell types.

Preferably the gene analysis is performed by large scale expression arrays, differential proteomic screening and/or generation of specific monoclonal antibodies.

Preferably other cell types comprise fibroblast cells. The invention will be now described with reference to examples, in relation to the following figures: Fig. 1 In vitro differentiation of endothelial cells into cardiomyocites.

A-C: Double fluorescence of a co-culture of rat neonatal cardiomyocites, stained with anti-MyHC MF20 (red in A), having progenitor endothelial cells transduced with a lentiviral GFP vector (green in B). Arrows show a double stained cell (orange in C). D-E Co-culture of progenitor endothelial cells, isolated from embryos MLC1/3F-nLacZ E9 with rat neonatal cardiomyocites, stained with X-Gal (D) and with anti-cardiac Troponina I (E). Arrows show a binucleated cell expressing β-galactosidase into the nucleus and cardiac Troponin I in the cytoplasm. F-H: Double fluorescence of a co-culture of rat neonatal cardiomyocites, pre-labelled with 6'carboxi- fluorescein (green in G), having endothelial progenitors transduced with an adenoviral vector expressing LacZ and stained with anti-β-galactosidase antibodies (red in F). Arrows show one of numerous endothelial cells wherein fluorescein entered trough open junctions (orange in H). K-M: Double fluorescence of a co-culture of rat neonatal cardiomyocites, stained with anti-MyHC MF20 (red in K) having endothelial cells from human umbilical vein transduced with a lentiviral GFP vector (green in L). The double arrow shows a double labelled cell (orange in M), whereas the arrow shows a HUVEC that did not trans-differentiate. N: EM micrographs showing two proximal cells, both containing sarcomeres (arrows), where only one is also labelled with Bluo-Gal (arrow head). J: Magnification of a cell showing the Bluo-Gal staining (arrow head) among sarcomeres (arrow).

Figure 2: In vivo cardiac differentiation of endothelial cells. Double fluorescence of sections from mouse myocardium (A-C) and mouse post- ischemic myocardium (D-F), 2 weeks further injection of endothelial progenitors labelled with a GFP lentiviral vector (green in B e E). Sections were labelled with anti-MyHC MF20 (red in A e D). The nuclear staining (Hoechst) is shown in C e F. Few double labelled cells may be evidentiated in the healthy myocardium (arrow in A-C). On the contrary numerous double labelled cells are present in the post ischemic myocardium, (arrow in D-F) at the injection zone; arrow heads show normal myocardium and asterisk an ischemic area, with no injected cells.

Figure 3: In vivo cardiac differentiation of endothelial and non endothelial cells. Endothelial progenitors from embryonic aorta (EEC) and from ES (44b), endothelial cells from adult myocardium (5HV) and lung (1G11) and from human umbilical vein (HUVEC), mouse fibroblasts (3T3) and neural stem cells (NSC) were infected with the GFP expression vector and co-cultivated with rat neonatal cardiomyocites as described in Methods. The cardiac differentiation in these co-cultures was monitored by counting double labelled cells (GFP+/MyHC+) in thirty randomly selected fields and expressing the number as percentage the total of GFP+ cells. Data are the average of at least 3 separate experiments, having a SE of around 10% of the mean. In a separated group of experiments same cells were exposed to BMP4 and FGF2 as described in Methods and after 5 days stained with anti-MyHC. No positive cell was detected in two separated experiments. Finally, in one experiment, same molecules were added to a co-culture of the same cell lines with cardiomyocites. Figure 4: In vitro cardiac trans-differentiation of endothelial cells.

Double fluorescence of a co-culture of rat neonatal cardiomyocites with HUVEC, stained with polyclonal anti-MyHC antibodies (green in A), with a monoclonal anti-Von Willebrand antibody (red in C). The nuclear staining (Hoechst) is shown in B. The arrow shows granules containing Von Willebrand in the cytoplasm of a differentiated cardiomyocite (orange in D). Figure 5: RT- PCR for human Nkx-2.5 at different times of co- culturing rat neonatal cardiomyocites with HUVEC, with cell-cell contact.

Fig 6: RT PCR for human NKX 2.5 showing that co-culture without cell-cell contact (B) is effective in inducing Nkx 2.5 as efficiently as the co- culture where cells are mixed (A). Materials and Methods

Cell Culture Primary endothelial embryonic cells (EEC) were isolated from explants of mouse dorsal aorta as previously described (21). For clonal expansion, cells were plated at a concentration of 10⁴/60 mm dish on a feeder layer of mitomycin C (2μg/ml) treated STO cells. Clonal isolates were collected and tested by RT-PCR for the expression of VE- Cadherin, CD34, Myf-5 and MyoD as described (21). More than 90% of the selected clones were positive for endothelial and negative for myogenic markers. Clones were used between the second and third passage.

The 44B endothelial cell line (22), derived from murine ES cells, H5V endothelial cells (23), derived from mouse heart, and 1G11 endothelial cells (24), isolated from adult mouse lung were grown as described. Human Umbilical Vein Endothelial Cells (HUVEC) were isolated and grown as described (25). STO and 3T3 mouse fibroblasts were grown in DMEM supplemented with 10% FCS. Primary neonatal rat cardiomyocites were isolated as described

(26) and plated at 2 x10⁵ cells/ml, treated with Mitomycin C (2 μg/ml to prevent overgrowth of contaminating fibroblasts) and used for the co- culture experiments.

Endothelial cells (either genetically labeled, or previously infected with lentiviral or adenoviral vectors (see below)) were added to the cardiomyocites at a 1:4 ratio, in DMEM containing 10% fetal calf serum.

After 2 days, the cultures were shifted to DMEM containing 5% horse serum and at various periods thereafter processed for analysis.

When indicated endothelial cells were treated with different concentrations of BMP4 (either 1 or 10 nM) and FGF2 (either 1 or 10 nM) for different periods and thereafter stained for the expression of MyHC (Myosin heavy chains).

For the coupling experiment, rat cardiomyocytes were labeled with

10nm 6'-carboxifluorescein for 10 min at 37°C in serum free medium. The cultures were extensively washed with serum containing medium and then endothelial cells, labeled with an adenoviral vector expressing LacZ, were added. After 4 days cultures were fixed and stained with an anti-β galactosidase antibody.

Vectors An adenoviral vector encoding the LacZ reporter gene was used as previously described (27). The third generation lentiviral vector pRRLsin.PPT-PGK.GFPpre expressing Green Fluorescent Protein (GFP) was used as described (28).

In vivo analysis For in vivo assay, 2 x 10^ GFP-labeled endothelial cells or 3T3 fibroblasts were injected into the myocardial layer of adult NOD-SCID mice in a total volume of 2 μl, using a 29 G needle adapted to a Hamilton syringe. After 2 weeks the heart was removed and cryostat sections were stained with MF20 antibody. Heterotopic cardiac transplants in mice were performed as described by Corry et al. (29). The recipient NOD-SCID mice were anesthetized with Ketamine (35 mg/kg)ip +Xylazine (5 mg/kg ) ip. The aorta and vena cava were separated between the renal vessels and the bifurcation of the iliacs. The donor heart was harvested from a NOD-SCID mouse and the left descending coronary artery was cauterized to induce ischemic damage. Immediately afterwards, GFP labeled, aorta-derived cells were injected into the heart parenchyma near the lesion site or into an uninjured heart (as a control) with a microsyringe fitted with a 29G needle. The ascending aorta of the donor heart was then connected via an end-to-side anastomosis with the recipient aorta. A similar anastomosis was also created between the recipient vena cava and the superior vena cava of the donor heart. The total ischemic time averaged 15 minutes. Heterotopic cardiac survival was monitored by direct palpation of the heartbeat through the abdominal wall. Cytochemistry β-galactosidase activity was detected at the light microscopy level as previously described (30). For detection of β- galactosidase activity at the E.M. level, cells were fixed with glutaraldehyde (2.5%) and paraformaldehyde (1%) in 0.1 M PBS at pH7.4 for 1.5h. After extensive rinsing in PBS, the cells were stained in histochemical Bluo-Gal solution at 30° overnight. The Bluo-Gal reaction contains a mixture of 1mg/l 4CI-5Br-3 indolyl β-galactopyranoside (Bluo- Gal), 10mM Kferricyanide, 10mM Kferrocyanide, 2mM MgCl2 in PBS. The cells were then post-fixed in 1 % Osθ4 in 0.2M collidine buffer (pH7.4) for 1.5h and stained with 2% uranyl acetate for 45'. After rinsing, dehydratation in alcohol and propylene-oxide, the samples were embedded in Epoxy resin. Ultrathin sections (80nm) were examined and photographed with a Zeiss EM109 transmission electron microscope. For immunocytochemistry, the following antibodies were used: a mono (MF20) and a polyclonal anti-myosin heavy chain (30), anti-cardiac troponin I monoclonal (31), donated by S. Schiaffino, anti-V. Willebrand from Pharmyngen. Immuno-cytochemistry on tissue sections and cultured cells was carried out as described (30). (RT)-PCR Analysis RT-PCR were performed as described (32). Oligos used for amplification of the following genes were: VE-cad (227 bp): 5' GGA TGC AGA GGC TCA CAG AG 3'; 3' CTG GCG GTT CAC GTT GGA CT 5'; Flk-1 (270 bp): 5' TCT GTG GTT CTG CGT GGA GA 3';

3' GTA TCA TTT CCA ACC ACC CT 5'; CD34 (300 bp): 5' TTG ACT TCT GCA ACC ACG GA 3'; 3' TAG ATG GCA GGC TGG ACT TC 5'; Myf-5 (132 bp): 5' GAG CTG CTG AGG GAA CAG GTG GAG A 3' 3' GTT CTT TCG GGA CCA GAC AGG GCT G 5';

MyoD (144 bp): 5' CAC TAC AGT GGC GAC TCA GAC GCG 3'; 3' CCT GGA CTC GCG CAC CGC CTC ACT 5'. Results

Endothelial cells isolated from dorsal embryonic aorta are able to differentiate into cardiomyocites

Clonal isolates from embryonic aorta (21) were expanded on a feeder layer of mitomycin C-treated STO fibroblasts. Indeed, under these conditions, these cells express endothelial markers such as Flk-1 , VE- Cadherin and CD34 but not myogenic markers such as Myf5 or MyoD. During their growth the cells were infected with a lentiviral vector TRRLSIN 3 (28) expressing Green Fluorescent Protein (GFP). More than 90% of the population expressed GFP further to a single cycle of infection.

Fluorescent cells were then co-cultivated with a four fold excess of rat neonatal cardiomyocites, for 5 days. After this period, cultures were fixed and stained with MF20 antibody that recognizes sarcomeric myosin heavy chains. Fig. 1 (a-c) shows that, while the majority of GFP fluorescent cells did not express sarcomeric myosin, several cells (approximately 10% of the fluorescent population), were double labeled. Since the lentiviral vector is replication incompetent, the double-labeled cells represent aorta derived, endothelial progenitors that have differentiated into striated muscle. Almost invariably, these double labeled cells were adjacent or inter-mixed with cardiomyocites, suggesting that cell-cell contact is needed for the activation of myogenesis in these progenitors; indeed medium conditioned from cardiomyocite cultures did not induce myogenesis in the aorta derived clonal isolates. In the following we show that soluble factors are involved indeed bu they are likely to be short-lived. To perform ultrastructural studies EEC cells were infected with an adenoviral vector expressing the β-galactosidase reporter gene, then mixed to rat neonatal cardiomyocites.

EM analysis of co-cultures, revealed well developed sarcomeres and junctions in many clustered cells, several of which were labeled with β-Gal. Fig. 1 (N) shows an example of two adjacent cardiomyocites only one of which shows electron dense β-Gal precipitates in the cytoplasm. A magnification of the same field (Fig. 1H) reveals the presence of well developed sarcomeres.

Since MF20 antibody recognizes sarcomeric myosin in both skeletal and cardiac muscle, we analyzed the co-cultures for the expression of cardiac-specific markers. Furthermore, since expansion in culture may alter the potency of freshly isolated progenitors, we repeated the experiments by isolating and pooling individual clones grown on plastic dish (each clone does not exceed 100-200 cells under these conditions) and co-culturing about 1000 progenitor cells with 4000 cardiomyocites in micro-spot cultures. Clones had been derived from MLC1/3F-nLacZ embryos, expressing the β-gal reporter gene only in the nuclei of striated muscle (33). The co-cultures were stained with X-Gal and reacted with a monoclonal antibody directed against cardiac specific troponin I, one of the very few sarcomeric proteins expressed in cardiac but not in skeletal muscle. Fig. 1 (D,E) shows a binucleated cell expressing a nuclear β- galactosidase and cardiac troponin I in the cytoplasm. Since expression of the reporter gene is cell autonomous, the cell indicated must derive from the embryonic aorta and, because it co-expresses a cardiac marker, it has differentiated into a cardiomyocite.

Functional evidence of cardiac differentiation is represented by electrical coupling of cardiac cells, through the establishment of active gap junctions. To test this possibility, rat neonatal cardiomyocites were pre- labeled with 6 carboxy-fluorescein, a membrane soluble dye, that is estherified inside cells and cannot therefore cross again the plasma membrane (34). Fluorescein labeled cardiomyocites were then co-cultured with endothelial progenitors (EEC) that had been previously labeled with an adenoviral vector expressing the β-gal reporter gene (27). Fig. 1 (F,H) shows that in these cultures several cells express both fluorescein and β- galactosidase, indicating that LacZ labeled endothelial progenitors have been electrically coupled to fluorescein positive cardiomyocites. The specificity of this coupling has been verified by exposing sister dishes to TPA (10 ^"~8M) that is known to block gap junctions: in this case no double labeled cells could be detected.

Endothelial cells contribute to the formation of mvocardiomvocites in a post-ischemic myocardium To test whether endothelial progenitors are capable of differentiating into cardiomyocites also in an in vivo environment, 2 x 10⁵ GFP-labeled endothelial progenitors were injected into the left ventricle of adult SCID mice. After one or two weeks, the mice were sacrificed and criostat sectioned. Each section was stained with MF20. Fig. 2 (a,b,c) shows that fluorescent injected cells were detected in a relatively coherent mass along the track of the needle, usually located under the epicardium. Very few double labeled cells could be detected, indicating that transition from endothelium to cardiomyocites is extremely rare in healthy myocardium. In contrast Fig. 2 (d,e,f) shows that many double labeled cells could be detected in the infarcted myocardium, indicating that these endothelial cells had differentiated into myosin positive cardiomyocites in vivo.

To show that cardiac differentiation of endothelial cells would result in significant tissue repair of post-ischemic myocardium, 2x10⁵ GFP- labeled endothelial progenitors were injected in the left ventricle of adult NOD SCID adult mice were the left anterior descending coronary had been cauterized. Further to cell injection, the hearth was transplanted in the abdomen of a recipient NOD SCID mouse, connecting the ascending aorta of the donor heart with the recipient aorta, and the superior vena cava with the recipient vena cava. After two weeks the transplanted heart was removed and cryostat sections were stained with anti-myosin antibody. As shown in fig, 2D-E, in the area of injection the majority of GFP labeled cells had differentiated into myosin positive cells, with the typical shape of cardiomyocites, indicating that the new myocardium had been produced by injected endothelial cells. In transplanted control hearts (that had not been infarcted) very few of the injected cells expressed myosin heavy chains like in control hearts in situ.

Different endothelial cell types show differential potential conversion into cardiomyocites

To investigate how widespread is this potency among other endothelial progenitor cells, as well as in other types of stem cells, we infected with the GFP-expressing lentiviral vector the 44b endothelial cell line (22), derived from murine ES, the H5V endothelial cells (23), from adult mouse heart, and the 1G11 endothelial cells (24), also isolated from adult mouse lung. Furthermore we infected neural stem cells isolated from the same MLC1/3F/nLacZ mouse strain, that had been previously shown to differentiate efficiently into skeletal muscle (10). As a negative control we infected 3T3 mouse normal fibroblasts. Finally, to test whether this capacity was also present in human cells, we similarly infected early passage human umbilical vein endothelial cells (HUVEC). After infection all cell lines were co-cultured with rat neonatal cardiomyocites and the number of cells expressing GFP (β-galactosidase in the case of neural stem cells) was counted in thirty randomly selected microscopic fields and expressed as percentage of the test cell population. Fig. 3 shows that the potency to generate cardiomyocites was well expressed also in the 44b cells, derived from embryonic stem cells and in the human UVEC cells (Fig. 1 K,L). It was very much reduced in G11 and in neural stem cells and it was practically absent in H5V and in 3T3 fibroblast cells. Together these data demonstrate that clonal isolates of endothelial progenitor cells derived from embryonic vessels or established early passage endothelial cell lines are capable of differentiating into functional cardiomyocites when in the presence of differentiated cardiomyocites. The fact that HUVEC are relatively well differentiated endothelial cells and yet maintain the capacity, indicates that they are sufficiently plastic to undergo this kind of trans-differentiation. Trans-differentiation is normally verified by transient co-expression of markers of two different cells in the same cytoplasm. HUVEC cells were then stained, at different co-culture days, with rat cardiomyocites, with a von Willebrand factor recognizing antibody and with an anti sarcomeric myosin antibody. Starting from the third co-culture day, a significant number of cells strongly react with the anti-von Willebrand ab and start to allineate sarcomeres in the same cytoplasm (Fig. 4 A-D). During the last co-culture days double labelled cells were identified with a minor extent and occasionally fully differentiated cardiomyocites showed a strong von Willebrand factor reactivity in a small perinuclear area, suggesting that the von Willebrand factor was shut off and residue protein was doing to be degraded.

HUVEC cells were detached from the dish and added to primary cutiures of rat neonatal cardiomyocytes as described above. At different timepoints, RNA was extracted from the co-cultures, retrotranscribed and used for RT-PCR with oligo specific for the 3' untranslated region of the human Nkx2.5 gene (Ace. No. U34962): hNkx2,5 forw S'-CTCCCAACATGACCCTGAGT-S' hnkx2,5 rev 5'-GAGCTCAGTCCCAGTTCCAA-3'.

Negative control were represented by rat primary fibroblasts.

Figure 5 shows that after 6 hours of co-culture, human Nkx 2.5, the earliest marker of cardiomyocyte differentiation, is activated in human endothelial cells. This result demonstrates that even though terminal differentiation (monitored by sarcomere assembly in the cytoplasm) occurs after 3 days of co-culture, activation of cardiac genes in human endothelial cells is very rapid, marking the time when high throughput screening techniques may identify early response genes to cardiomyocyte-induced transdifferentiation. It also rules out the possibility that cell fusion may be responsible for the observed phenomenon.

Endothelial-cardiomvocite cell-cell contact is not necessary to induce differentiation

HUVEC were plated in the center 25 mm, of a 90 mm dishes whereas rat neonatal cardiomyocites were plated in a peripheral ring of 25mm. An intermediate ring devoid of cells and medium was left until all cells had adhered to the dish. At this time medium was added to cover the all dish so that soluble factors could be exchanged but no cell-cell interaction could occur. After 6 hours RNA was extracted from HUVEC cells and retro-transcribed. RT-PCR revealed expression of Nkx2.5 thus demonstrating that soluble factors are sufficient to activate cardiogenesis in HUVEC cells (Fig 6 ). The activation by soluble factors is as efficient as that induced by endothelial-cardiomyocites cell-cell contact (Fig 6DB). These factors are labile, because stored conditioned medium cannot elicit the same effect.

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Claims

1. Method to induce the differentiation of endothelial cells into cardiomyocites, comprising the co-cultivation of said endothelial cells with differentiated cardiomyocytes.

2. Method according to claim 1 wherein the differentiation into cardiomyocites is demonstrated by the activation of at least one cardiac specific gene and/or by the development of sarcomere and/or by electric coupling with the differentiated cardiomyocites and/or by in vivo myocardium formation after injection into post-ischemic ventricular wall.

3. Method according to claim 1 wherein endothelial cells may be derived from embryonic or neonatal tissues or from vessels, preferably umbilical vein.

4. Cardiomyocites obtainable from endothelial cells according to the method of previous claims.

5. Method to obtain immunologically compatible cardiomyocites of a subject including the steps of: a) isolating endothelial cells from the umbilical vein of the subject; b) optionally, storing the sames to keep their viability; c) inducing the differentiation of said endothelial cells into cardiomyocites according to the invented method.

6. Method to identify genes and/or gene products induced during endothelial cell differentiation to cardiomyocites comprising the steps of: a) co-cultivate endothelial cells with differentiated cardiomyocites for different times; b) separate endothelial cells from differentiated cardiomyocites; c) identify and analyze genes selectively expressed from said endothelial cells in respect to same cells that were not exposed to cardiomyocites or alternatively were exposed to other cell types.

7. Method according to claim 6 wherein the gene analysis is performed by large scale expression arrays, differential proteomic screening and/or generation of specific monoclonal antibodies.

8. Method according to claim 7 wherein other cell types comprise fibroblast cells.