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Definitions

• a differentiation outcome of a population of pluripotent stem cells is less predictable. Placing the cells under conditions which induce specific cell types has been one form of an attempt to regulate the differentiation outcome. These conditions include growing the cells to high or low density, changing media, introducing or removing cytokines, hormones and growth factors, creating an environment which suits differentiation toward a specific cell type, such as providing a suitable substrate. Generally, when a stem cell culture is induced to differentiate, the differentiated population is analysed for particular cell types by expression of genes, markers or phenotypic analysis. In any case, the respective cell types may then be selectively cultured to enrich their percentage population to eventually obtain a single cell type and culture.
• the induction of stem cells to differentiate into muscle cells is useful for muscle transplantation and therapeutic purposes, as well as providing potential human disease models in culture (e.g. for testing pharmaceuticals).
• the induction of cardiomyocyte differentiation in stem cells is especially useful in developing therapeutic methods and products for heart disease and abnormal heart conditions.
• the molecular pathways that lead to specification and terminal differentiation of specific cell types, such as myocytes, from embryonic stem cells during development are not entirely clear.
• a method of inducing differentiation of a stem cell including: culturing a stem cell in the presence of a factor derived from an embryonic cell or extracellular medium of an embryonic cell under conditions that induce differentiation of the stem cell.
• Figure 3 shows co-cultures of stem cells with the mouse visceral endoderm-like cell line END-2.
• P19 EC in normal monolayer culture 3 days after initiation of co-culture with END-2 cells and after 10 days, when beating muscle (B.M.) is evident
• mES cell lineRI in monolayer on its normal 'feeder' cells SNL
• SNL normal 'feeder' cells
• B.M. beating muscle
• GCT27X human EC cell line on mouse embryonic fibroblast (MEF) feeder cells 3 days after initiation of co-culture and after 16 days.
• a method of inducing differentiation of a stem cell including: culturing a stem cell in the presence of an embryonic cell and/or extracellular medium of an embryonic cell, under conditions that induce differentiation of the stem cell.
• the stem cell is induced to differentiate into a myocyte (muscle cell) including cardiomyocytes and skeletal muscle cells, a vascular endothelial cell or a haematopoietic cell.
• a myocyte muscle cell
• the embryonic cell is derived from extraembryonic tissue and more preferably from embryonic endoderm or ectoderm.
• a method of inducing differentiation of a stem cell including: culturing a stem cell in the presence of extracellular medium of an embryonic cell under conditions that induce differentiation of the stem cell.
• the extracellular medium preferably includes cellular factors, such as secreted proteins, that are capable of inducing differentiation of a stem cell. Such secreted proteins will typically bind receptors on a cell surface to trigger intracellular pathways which can initiate differentiation of the cell. Examples of suitable extracellular factors include Ihh and BMP2 as described in Dyer et al 2001, Dev. 128: 1717-1730.
• the embryonic cell is derived from extraembryonic tissue and more preferably is derived from visceral endoderm.
• Endoderm cells are typically adjacent to sites of heart formation in vertebrates. In individuals where endoderm differentiation is defective or absent, the heart develops abnormally.
• the stem cell In order to induce differentiation of the stem cell to a cardiomyocyte it is preferable to introduce the stem cell to an extraembryonic, endoderm cell monolayer in culture.
• the monolayer is produced by proliferation of the embryonic cell derived from embryonic endoderm, more preferably extraembryonic endoderm.
• the embryonic endoderm is preferably extraembryonic, visceral endoderm. More preferably, the cell monolayer is produced by END-2 cells. It is preferred that the embryonic cells are cultured and passaged before allowing them to grow to form a monolayer.
• the monolayer is preferably grown to confluence in a suitable medium, such as DMEM or M16 medium.
• the monolayer may then be treated with certain agents to prevent further division of the cells. For instance, the monolayer can be treated with mitomycin and then the stem cell can be plated on the mitogenically inactive monolayers.
• the present invention preferably provides a myocardial model for testing the ability of stems cells that have differentiated into cardiomyocytes to restore cardiac function.
• a myocardial model for testing the ability of stems cells that have differentiated into cardiomyocytes to restore cardiac function.
• it is important to have a reproducible animal model with a measurable parameter of cardiac function.
• the parameters used should clearly distinguish control and experimental animals (see for example Palmen et al. (2001), Cardiovasc. Res. 50, 516-524) so that the effects of transplantation can be adequately determined.
• PV relationships are a measure of the pumping capacity of the heart and may be used as a read-out of altered cardiac function following transplantation.
• a method of inducing differentiation of a stem cell to a skeletal muscle cell including: culturing a stem cell in the presence of an embryonic ectoderm cell and/or extracellular medium of an embryonic ectoderm cell, under conditions that induce differentiation of the stem cell into a skeletal muscle cell.
• Ectoderm cells can be isolated according to methods described in Roelen et al 1994, Dev. Biol. 166:716-728. Ectoderm cells are known to express oct-4 and have alkaline phosphatase activity and they also have SSEA-1 on their cell surface. Therefore, ectoderm cells can be identified and isolated based on the above characteristics.
• Ectoderm cells may secrete (growth) factors that induce differentiation to skeletal muscle. It is preferred that the ectoderm cells are derived from E7.5, embryonic mouse tissue. More preferably, the ectoderm cells are co-cultured with the stem cells using similar methods as discussed earlier. An ectoderm monolayer is preferably established in culture and preferably dispersed stem cells are introduced to the culture for a period of time sufficient to induce differentiation of the stem cells to skeletal muscle cells.
• vascular endothelial cells have been differentiated from a stem cell co-cultured under conditions described above.
• the differentiated cell is a cardiomyocyte, skeletal muscle cell, vascular endothelial cell or a haematopoietic cell.
• the present invention also provides differentiated cells produced according to the methods of the invention that may be used for transplantation, cell therapy or gene therapy.
• the invention provides a differentiated cell produced according to the methods of the invention that may be used for therapeutic purposes, such as in methods of restoring cardiac function in a subject suffering from a heart disease or condition.
• the differentiated cells may be used as a source for isolation or identification of novel gene products including but not limited to growth factors, differentiation factors or factors controlling tissue regeneration, or they may be used for the generation of antibodies against novel epitopes.
• the differentiated cells produced according to the methods of the present invention may be clonally expanded.
• a specific differentiated cell type can be selectively cultivated from a mixture of other cell types and subsequently propagated.
• Specific differentiated cell types that are clonally expanded can be useful for various applications such as the production of sufficient cells for transplantation therapy, for the production of sufficient RNA for gene discovery studies etc.
• the differentiated cells may be used to establish cell lines according to conventional methods.
• the differentiated cells produced according to the methods of the present invention may be genetically modified. For instance, a genetic construct may be inserted to a differentiated cell at any stage of cultivation. The genetically modified cell may be used after transplantation to carry and express genes in target organs in the course of gene therapy.
• the cell composition of the present invention can be used in methods of repairing or treating diseases or conditions, such as cardiac disease or where tissue damage has occurred.
• the treatment may include, but is not limited to, the administration of cells or cell compositions (either as partly or fully differentiated) into patients. These cells or cell compositions would result in reversal of the condition via the restoration of function as previously disclosed above through the use of animal models.
• the hES cells were cultured in DMEM with 20% FCS, 0.1 mM ⁇ -mrcaptoethanol, 1 % non-essential amino acids, 2mM glutamine plus antibiotics (pen/strep) on mitomycin (10 ⁇ g/ml) treated embryonic feeder cells. HES were subcultured by treating with dispase and mechanical slicing of individual colonies into 6-10 pieces followed by transfer of the pieces to new feeder cells.
• Visceral endoderm cells were isolated from the three germ layers of gastrulating mouse embryos at E7.5 (where E0.5 is noon on the day of the vaginal plug, as described previously using dispase (Roelen et al, 1994, Dev. Biol. 166:716- 728).
• the separated germ layers were plated on to poly-L-lysine coated culture dishes in M16 medium and allowed to attach overnight. The next day, M16 was replaced by hES complete medium, and on day 3 after germ cell isolation, pieces of undifferentiated cell "transfers" were plated on to the attached endodermal and ectodermal cell from the mouse embryo. Cultures were then grown for 2 to 3 weeks and medium refreshed every 5-6 days.
• Example 4 Co-Culture of Visceral Endoderm Cells and hES and Differentiated Cardiomyocytes
• END-2 cells P19 EC, hEC and hES cells were cultured as described previously (Mummery et al. 1985, 1991 ; vanden Eijnden-van Raaij et al. 1991 ; Slager et al. 1993;Reubinof et al. 2000).
• the hES2 cell line from ESI (Reubinof et al. 2000) was used in all experiments.
• mitogenically inactive END- 2 cell cultures treated for 1 h with mitomycin C (10 ⁇ g ml "1 ), as described in Example 1 , replaced mouse embryonic fibroblasts (MEFs) as feeders for hEC, mES and hES.
• the first step involved a 15-min incubation with 4.0 U mL -1 protease type XXIV (Sigma, St Louis, MO, USA) at 37 °C. Tissues were then transferred to a solution consisting of coUagenase 1.0 mg mL ⁇ 1 and hyaluronidase 0.5 mg mL " 1 , followed by three further incubations with coUagenase (1.0 mg mL -1 ) for 20 min each at 37 °C. Tissue extracts were combined and the calcium concentration restored to 1.79 mmol L ⁇ 1 .
• the human EC cell line GCT27X is a feeder-dependent, pluripotent EC cell line, with characteristics similar to human ES cells (Pera et al. (1989), Differentiation 42, 10-23). In co-culture with END-2 cells, formation of large aggregates was observed (Fig. 3d). However even after 3 weeks, there was no evidence of beating muscle.
• VE-like cells induce/promote differentiation of pluripotent cells to cardiomyocytes.
• These cells include pluripotent mouse EC cells, mouse ES as well as human ES cells, which are demonstrated for the first time to respond to inductive cues derived from cells similar to those normally adjacent to the region of heart development in the embryo.
• a 1.4 French conductance-micromanometer (Millar Instruments, Houston, TX, USA) was delivered to the left ventricle through the carotid artery. Pressure and conductance measurements were recorded using Sigma SA electronic equipment (CDLeycom, Zoetermeer, the Netherlands) and stored for offline analysis. A typical pressure volume (PV) loop recorded in a normal heart is presented in Fig. 7(a). From the PV-loops many haemodynamic parameters can be deduced including the end-systolic PV relationship (ESPVR) and preload recruitable stroke work (PRSW).
• ESPVR end-systolic PV relationship
• PRSW preload recruitable stroke work

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