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  • C12N2502/02—Coculture with; Conditioned medium produced by embryonic cells
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  • 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

Definitions

• the present invention relates to human embryonic stem cells (hES) and their differentiation.
• hES cells can give rise to cardiomyocytes that are a better model for studying human physiology than murine embryonic stem (mES) cell-derived cardiomyocytes or primary cardiomyocytes from adult or fetal mice.
• mES murine embryonic stem
• hES cells can give rise to cardiomyocytes which provide a model of relevance to the study of human cardiac disease.
• hES cells can give rise to normal and mutant cardiomyocytes suitable for testing drugs.
• Cardiomyocytes have potential in restoring heart function after myocardial infarction or in heart failure.
• Human embryonic stem (hES) cells are a potential 5 source of transplantable cardiomyocytes but detailed comparison of hES derived cardiomyocytes with primary human cardiomyocytes is necessary before transplantation into patients becomes feasible.
• VE visceral endoderm
• EC mouse P19 embryonal carcinoma
• mES mouse embryonic stem
• END-2 cells can induce the differentiation of epiblast cells from the mouse embryo to undergo 0 hematopoiesis and vasculogenesis and respecify prospective neuroectodermal cell fate. This effect was largely attributable to Indian hedgehog (Ihh) a factor secreted by END-2 cells and VE of the mouse embryo.
• Ihh Indian hedgehog
• the present work is thus the first describing induction of cardiomyocyte differentiation in hES cells, which do not undergo cardiogenesis spontaneously, even at high local cell densities and is the first direct electrophysiological comparison of hES-derived cardiomyocytes with primary human fetal cardiomyocytes in culture.
• the present invention provides a method for inducing cardiomyocyte differentiation of a human embryonic stem cell (hES ), the method comprising co-culturing the hES cell with a cell excreting at least one cardiomyocyte differentiation inducing factor or with an extracellular medium therefrom, under conditions that induce differentiation.
• hES human embryonic stem cell
• the cell produces a protein excretion profile that is at least substantially as produced by mouse VE-like cells.
• the stem cells suitable for use in the present methods may be derived from a patient's own tissue. This would enhance compatibility of differentiated tissue grafts derived from the stem cells with the patient.
• hES cells can include adult stem cells derived from a person's own tissue.
• Human stem cells may be genetically modified prior to use through introduction of genes that may control their state of differentiation prior to, during or after their exposure to the embryonic cell or extracellular medium from an embryonic cell. They may be genetically modified through introduction of vectors expressing a selectable marker under the control of a stem cell specific promoter such as Oct-4.
• the stem cells may be genetically modified at any stage with a marker so that the marker is carried through to any stage of cultivation.
• the marker may be used to purify the differentiated or undifferentiated stem cell populations at any stage of cultivation.
• Cells providing differentiating factor(s) may be embryonic cells derived from visceral endoderm tissue or visceral endoderm like tissue isolated from an embryo.
• visceral endoderm may be isolated from early postgastrulation embryos, such as mouse embryo (E7.5). Visceral endoderm or visceral endoderm like tissue can be isolated as described in Roelen et al, 1994 Dev. Biol. 166:716-728. Characteristically the visceral endoderm may be identified by expression of alphafetoprotein and cytokeratin ENDO-A).
• the embryonic cell may be an embryonal carcinoma cell, preferably one that has visceral endoderm properties.
• cell producing differentiation factor(s) is a mouse VE-like cell or a cell derived therefrom.
• the cell is an END-2 cell.
• the embryonic stem cell may be derived from a cell line or cells in culture.
• the embryonic cell may be derived from an embryonic cell line, preferably a cell line with characteristics of visceral endoderm, such as the END-2 cell line (Mummery et al, 1985, Dev Biol. 109:402-410).
• the END-2 cell line was established by cloning from a culture of P19 EC cells treated as aggregates in suspension (embryoid bodies) with retinoic acid then replated (Mummery et al, 1985, Dev Biol. 109:402- 410).
• the END-2 cell line has characteristics of visceral endoderm (VE), expressing alpha-fetoprotein (AFP) and the cytoskeletal protein ENDO-A.
• the cell is a liver parenchymal cell.
• the liver parenchymal cell is HepG2.
• the human embryonic stem cell may be derived directly from an embryo or from a culture of embryonic stem cells, (see for example Reubinoff et al, Nature
• the stem cell may be derived from an embryonic cell line or embryonic tissue.
• the embryonic stem cells may be cells which have been cultured and maintained in an undifferentiated state.
• the hES cells may be hES cells which do not undergo cardiogenesis spontaneously.
• the present invention provides a differentiated cardiomyocyte produced from an hES cell that does not undergo cardiogenesis spontaneously.
• the invention also provides a cardiomyocyte produced by a method according to the first aspect of the invention.
• the differentiated cardiomyocyte may express cardiac specific sarcomeric proteins and display chronotropic responses and ion channel expression and function typical of cardiomyocytes.
• the differentiated cardiomyocyte resembles a human fetal ventricular cell in culture.
• the differentiated cardiomyocyte resembles a human fetal atrial cell in culture. In another preferred form the differentiated cardiomyocyte resembles a human fetal pacemaker cell in culture.
• the present invention provides a plurality of differentiated cardiomyocytes of the invention wherein the differentiated cardiomyocytes are coupled.
• the coupling may be functional or physical.
• the coupling is through gap junctions.
• the coupling is through adherens junctions.
• the coupling is electrical.
• the present invention provides a colony of differentiated cardiomyocytes produced by dissociating beating areas from differentiated cardiomyocytes of the invention.
• the dissociated cells are replated. Preferably they adopt a two dimensional morphology.
• the present invention provides a model for the study of human cardiomyocytes in culture, comprising differentiated cardiomyocytes of the invention. This model is useful in the development of cardiomyocyte transplantation therapies.
• the present invention provides an in vitro system for testing cardiovascular drugs comprising a differentiated cardiomyocyte of the invention.
• a differentiated cardiomyocyte of the invention provides a mutated differentiated cardiomyocyte of the invention prepared from a mutant hES cell. It will be recognized that methods for introducing mutations into cells are well known in the art.
• the present invention provides a method of studying cardiomyocyte differentiation and function (electrophysiology) comprising use a mutated differentiated cardiomyocyte of the seventh aspect.
• the present invention provides an in vitro system for testing cardiovascular drugs comprising a mutated differentiated cardiomyocyte of the seventh aspect.
• the present invention provides an in vitro method for testing cardiovascular drugs comprising using a mutated differentiated cardiomyocyte of the seventh aspect as the test cell.
• the present invention describes the genes and proteins present in cardiomyocytes derived from hES.
• Ion channels play an important role in cardiomyocyte function. If we know which channels are expressed we can make hES cells lacking specific ion channels, and study the effect on cardiac differentiation and function (using electrophysiology). Furthermore, drugs specific for a cardiac ion channel can be tested on cardiomyocyte function (looking at indicators such as action potential, beating frequency, and morphological appearance). Expression of cardiac specific ion channels was determined in undifferentiated hES cells and in differentiating cells 8 and 15-days after initiation of co-culture with END-2 cells ( Figure 3). As shown by others previously (12), areas of beating hES-derived cardiomyocytes express ANF.
• RNA for the delayed rectifier potassium channel KvLQTI was found in undifferentiated cells, but transcripts disappeared during early differentiation and reappeared at later stages.
• the cells of the invention may be formulated with suitable carriers.
• 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.
• Another aspect of the invention is a method of treating or preventing a cardiac disease or condition, the method including introducing an isolated differentiated cardiomyocyte cell of the invention and /or a cell capable of differentiating into a cardiomyocyte cell when treated in accordance with the method of the first aspect of the invention into cardiac tissue of a subject.
• the isolated cardiomyocyte cell is preferably transplanted into damaged cardiac tissue of a subject. More preferably, the method results in the restoration of cardiac function in a subject.
• a method of repairing cardiac tissue including introducing an isolated cardiomyocyte cell of the invention and /or a cell capable of differentiating into a cardiomyocyte cell when treated in accordance with the method of the first aspect of the invention into damaged cardiac tissue of a subject.
• the subject is suffering from a cardiac disease or condition.
• the isolated cardiomyocyte cell is preferably transplanted into damaged cardiac tissue of a subject. More preferably, the method results in the restoration of cardiac function in a subject.
• the present invention preferably also provides a myocardial model for testing the ability of stem cells that have differentiated into cardiomyocytes to restore cardiac function.
• the present invention further provides a cell composition including a differentiated cell of the present invention, and a carrier.
• Influencing differentiation is taken to mean causing a stem cell to develop into a specific differentiated cell type as a result of a direct or intentional influence on the stem cell.
• Influencing factors can include cellular parameters such as ion influx, a pH change and/or extracellular factors, such as secreted proteins, such as but not limited to growth factors and cytokines that regulate and trigger differentiation. It may include culturing the cell to confluence and may be influenced by cell density.
• the hES cell and the cell providing the differentiating factor(s) are co-cultured in vitro.
• This typically involves introducing the stem cell to an embryonic cell monolayer produced by proliferation of the embryonic cell in culture.
• the embryonic cell monolayer is grown to substantial confluence and the stem cell is allowed to grow in the presence of extracellular medium of the embryonic cells for a period of time sufficient to induce differentiation of the stem cell to a specific cell type.
• the stem cell may be allowed to grow in culture containing the extracellular medium of the embryonic cell(s), but not in the presence of the embryonic cell(s).
• the embryonic cells and stem cells may be separated from each other by a filter or an acellular matrix such as agar.
• the stem cell In general for differentiation of stem cells the stem cell can be plated on a monolayer of embryonic cells and allowed to grow in culture to induce differentiation of the stem cell.
• Conditions for obtaining differentiated embryonic stem cells are typically those which are non-permissive for stem cell renewal, but do not kill stem cells or drive them to differentiate exclusively into extraembryonic lineages. A gradual withdrawal from optimal conditions for stem cell growth favours differentiation of the stem cell to specific cell types. Suitable culture conditions may include the addition of DMSO, retinoic acid, FGFs or BMPs in co-culture which could increase differentiation rate and/or efficiency.
• the cell density of the embryonic cell layer typically affects its stability and performance.
• the embryonic cells are typically confluent.
• the embryonic cells are grown to confluence and are then exposed to an agent which prevents further division of the cells, such as mitomycin C.
• the embryonic monolayer layer is typically established 2 days prior to addition of the stem cell(s).
• the stem cells are typically dispersed and then introduced to a monolayer of embryonic cells.
• the stem cells and embryonic cells are co-cultured for a period of two to three weeks until a substantial portion of the stem cells have differentiated.
• extracellular medium as used herein is taken to mean conditioned medium produced from growing an embryonic cell as herein described in a medium for a period of time so that extracellular factors, such as secreted proteins, produced by the embryonic cell are present in the conditioned medium.
• the medium can include components that encourage the growth of the cells, for example basal medium such as Dulbecco's minimum essential medium, Ham's F12, or foetal calf serum.
• the cardiomyocytes of the invention are preferably beating. Cardiomyocytes, can be fixed and stained with ⁇ -actinin antibodies to confirm muscle phenotype. ⁇ -troponin, ⁇ -tropomysin and ⁇ -MHC antibodies also give characteristic muscle staining. Preferably, the cardiomyocytes are fixed according to methods known to those skilled in the art. More preferably, the cardiomyocytes are fixed with paraformaldehyde, preferably with about 2% to about 4% paraformaldehyde. Ion channel characteristics and action potentials of muscle cells can be determined by patch clamp, electrophysiology and RT-PCR.
• Stem cells from which cardiomyocytes are to be derived can be genetically modified to bear mutations in, for example, ion channels (this causes sudden death in humans). Cardiomyocytes derived from these modified stem cells will thus be abnormal and yield a culture model for cardiac ailments associated with defective ion channels. This would be useful for basic research and for testing pharmaceuticals. Likewise, models in culture for other genetically based cardiac diseases could be created. Cardiomyocytes of the present invention can also be used for transplantation and restoration of heart function.
• ischaemic heart disease is the leading cause of morbidity and mortality in the western world.
• Cardiac ischaemia caused by oxygen deprivation and subsequent oxygen reperfusion initiates irreversible cell damage, eventually leading to widespread cell death and loss of function.
• Strategies to regenerate damaged cardiac tissue by cardiomyocyte transplantation may prevent or limit post- infarction cardiac failure.
• the methods of inducing stem cells to differentiate into cardiomyocytes, as hereinbefore described would be useful for treating such heart diseases.
• Cardiomyocytes of the invention may also be used in a myocardial infarction model for testing the ability to restore cardiac function.
• 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.
• 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 host animal such as but not limited to, an immunodeficient mouse may be used as a 'universal acceptor' of cardiomyocytes from various sources.
• the cardiomyocytes are produced by the method of the present invention.
• the myocardial model of the present invention is preferably designed to assess the extent of cardiac repair following transplant of cardiomyocytes or suitable progenitors into a suitable host animal. More preferably, the host animal is an immunodeficient animal created as a model of cardiac muscle degeneration following infarct that is used as a universal acceptor of the differentiated cardiomyocytes.
• This animal can be any species including but not limited to murine, ovine, bovine, canine, porcine and any non-human primates.
• Parameters used to measure cardiac repair in these animals may include, but are not limited to, electrophysiological characteristic of heart tissue or various heart function. For instance, contractile function may be assessed in terms of volume and pressure changes in a heart. Preferably, ventricular contractile function is assessed. Methods of assessing heart function and cardiac tissue characteristics would involve techniques also known to those skilled in the field.
• the present invention further provides a cell composition including a differentiated cell of the present invention, and a carrier.
• the carrier may be any physiologically acceptable carrier that maintains the cells. It may be PBS or other minimum essential medium known to those skilled in the field.
• the cell composition of the present invention can be used for biological analysis or medical purposes, such as transplantation.
• 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.
• FIG. 1 Cardiomyocyte markers in hES/END2 co-cultures compared with primary human fetal and adult cardiomyocytes.
• A-G,0 hES-derived cardiomyocytes.
• H-J,P Human fetal ventricular cardiomyocytes. ti, . Human fetal atrial cardiomyocytes.
• M Adult human ventricular cardiomyocytes.
• N Adult human atrial cardiomyocytes.
• Cells were stained with Hoechst (A,C,M,N), anti- ⁇ actinin (green) (B,E,F,H,M,N), anti-MLC-2a (red) (N), anti-MLC-2v (red) (M) and anti-tropomyosin (green) (D,G,I, J,L).
• FIG. 1 Vital staining of ryanodine receptors in hES derived cardiomyocytes (O) and human fetal ventricular cardiomyocytes (P).
• Figure 3 Expression of cardiomyocyte marker and ion channel mRNA in cocultures of hES and END-2 cells by RT-PCR. RT-PCR on hES cells co-cultured for 8d with END-2 cells (8d+END-2), hES beating muscle (BM), adult human heart and directly on RNA (-RT).
• Figure 4 Action potentials and chronotropic responses.
• A Action potentials in hES derived beating cardiomyocytes and isolated human fetal ventricular and atrial cells.
• B Effect of Verapamil on action potentials in hES-derived and primary human fetal cardiomyocytes (hfetal).
• C Chronotropic responses of hES and human fetal cardiomyocytes to different stimuli; mean beat frequency ⁇ S.E.M.
• Figure 5. Calcium transients and L-type calcium channels.
• A The first image of an image stack (100 images, total time 30 seconds) of a group of 7 cells. The lines indicate line scans in time through the image stack.
• B Intensity plot of the horizontal line through the image stack in time (time running from top to bottom).
• C C.
• Intensity plot of the vertical line from the image (time running from left to right).
• D Image stamps of the first 36 images, with time.
• E Calcium transients in a single cell from the upper right corner of the image in A. The dots are the average value of the same region of interest in one cell in each slice in the image stack.
• F and G Confocal images of ⁇ -actinin (green) and ⁇ 1C (red) positive cells in hES (F) and human fetal ventricular cardiomyocytes (G).
• FIG. Junctional communication in hES-derived and human fetal cardiomyocytes.
• A B. Human ventricular fetal cardiomyocytes.
• C D. hES-derived cells. Double-staining of phalloidin (red) and anti-pan-cadherin (green) (A, C) or anti- Cx43 (green) (B, D).
• E Injection of Lucifer yellow into a single cell (arrows) within a group of beating hESderived cardiomyocytes results in dye spreading to multiple cells (arrow heads, left bottom; phase contrast, right bottom) within minutes as determined by 2D projected Zseries (upper panel from left to right).
• hES cells were co-cultured with visceral-endoderm (VE) like cells from the mouse. This initiated differentiation to beating muscle.
• VE visceral-endoderm
• Sarcomeric marker proteins, chronotropic responses and ion channel expression and function were typical of cardiomyocytes. Electrophysiology demonstrated that most cells resembled human fetal ventricular cells, with atrial-like responses in a minority population.
• Real-time intracellular calcium measurements, lucifer yellow injection and connexin 43 expression demonstrated that fetal and hES derived cardiomyocytes are coupled by gap junctions in culture.
• Antibody staining and inhibition of electrical responses by Verapamil demonstrated the presence of functional ⁇ 1c calcium ion channels.
• END-2 cells and hES2 cells were cultured as described previously (1 ,15,16).
• mitogenically inactive END-2 cell cultures treated for 3hr with mitomycin C (mit.C; 10 ⁇ g/ml) (1), replaced mouse embryonic fibroblasts (MEFs) as feeders for hES cells.
• MEFs mouse embryonic fibroblasts
• Co-cultures were then grown for up to 6 weeks and scored for the presence of areas of beating muscle from 5 days onwards.
• HepG2 cells a carcinoma cell line resembling liver parenchymal cells (17), were cultured in DMEM plus 10% fetal calf serum (FCS) and passaged twice weekly. Co-cultures were initiated as for END-2 cells.
• beating aggregates were dissociated using collagenase and replated on gelatine-coated coverslips.
• Data were recorded from cells at 33 °C in spontaneously beating areas using an Axopatch 200B amplifier (Axon Instruments Inc., Foster City, CA, U.S.A.). Cell attached patches were made in the whole cell voltage-clamp mode. The pipette offset, series resistance and transient cancellation were compensated; subsequent action potentials were recorded by switching to the current-clamp mode of the 200B amplifier. Output signals were digitized at 4 kHz using a Pentium III equipped with an AD/DAC LAB PC+ acquisition board (National Instruments, Austin, TX, U.S.A.). Patch pipettes with a resistance between 1 and 3 M ⁇ were used.
• Bath medium was 140 mM NaCl, 5mM KCL, 2mM CaCl2, 10 mM HEPES, adjusted to pH 7.45 with NaOH.
• Pipette composition 145 mM KCI, 5 mM NaCl, 2 mM CaCl2, 4 mM EGTA, 2 mM MgCb, 10 mM HEPES, adjusted to pH 7.30 with KOH.
• Verapamil was used at 5 ⁇ M, as indicated.
• Cardiomyocyte differentiation of human ES cells Cardiomyocyte differentiation of human ES cells.
• HES on HepG2 cells did form areas of beating muscle as in Figures 1 C and D, usually attached to HepG2 cell colonies.
• hES-derived cardiomyocytes beat 35-90 times per minute (Table 2). Cardiomyocyte colonies could be frozen and sometimes resumed beating upon thawing.
• immunofluorescent staining for sarcomeric proteins Figure 2A-G
• BIDOPY- ryanodine as a vital stain for ryanodine receptors in the sarcoplasmic reticulum
• Figure 3 analyzed the expression of ion channels by RT-PCR
• hES- derived cardiomyocytes also stained with myosin light chain-2a, MLC-2v (data not shown) and tropomyosin (Figure 2G) although again the sarcomeres were less evident than in human fetal and adult cardiomyocytes ( Figure 21, J).
• Carbachol addition decreased the beating rate of hES derived cardiomyocytes and human fetal ventricular cells while an increase in response to phenylephrine and isoprenaline was observed in both cell types. Similar effects were reported in mES derived cardiomyocytes 21 and mouse fetal cells (22).
• L-type calcium channels comprise the predominant route for calcium entry into cardiac myocytes and are key components in excitation contraction coupling.
• the dominant cardiac specific isoform is ⁇ 1C (23).
• ⁇ 1C antibody (24) we observed positive cardiomyocytes in both differentiated hES cultures
• Kehat et al (1 1 ) recently reported similar findings in independently derived hES cardiomyocytes.
• cellular Ca 2+ entry is regulated by the sympathetic nervous system.
• L-type Ca 2+ channel currents are markedly increased by beta-adrenergic (beta-A) agonists, which contribute to changes in rate and contractile activity of the heart.
• beta-A beta-adrenergic
• hES-derived and early human fetal cardiomyocytes show some features of early mouse cardiomyocytes, their calcium channel modulation resembles that in the adult mouse.
• hES cells may thus represent an excellent system for studying changes in calcium channel function during early human development which appears to differ significantly from that in mice.
• the appropriate calcium handling makes the cells more suitable for transplantation.
• Kikuchi Y Agathon A, Alexander J et al. casanova encodes a novel Sox-related protein necessary and sufficient for early endoderm formation in zebrafish. Genes Dev 2001 ;15:1493-1505.

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