HK1067661B - Cells of the cardiomyocyte lineage produced from human pluripotent stem cells - Google Patents

Description

Generation of cardiomyocyte lineage cells from human pluripotent stem cells

Technical Field

The present invention relates generally to the field of cell biology of embryonic cells and their differentiation. More specifically, the invention provides controlled differentiation of human pluripotent stem cells into cardiomyocytes and their precursors using specific culture conditions and screening techniques.

Reference to related application

This application claims priority from U.S. pre-patent application 60/305,087 filed on 12/7/2001 and 60/322695 filed on 10/9/2001. This priority document, as well as international patent publication W001/51616, is hereby incorporated by reference in its entirety for all purposes in the united states and other jurisdictions.

Background

Heart disease is one of the most serious diseases of concern in the western world. It is estimated 6100 million americans (almost 1 in every 5 men and women) suffer from 1 or more cardiovascular diseases (national health and nutrition research survey III, 1988-94, centers for disease control and american heart disease association). Widely disseminated diseases include coronary heart disease (1240 million), congenital cardiovascular defects (100 million) and congestive heart failure (4700 million). A major challenge for regenerative medicine research is the development of cellular compositions that can help to restore cardiac function in these diseases.

Most of the research work that has been done to date has employed various types of stem cells developed in rodent models.

Maltsev, Wobus et al (Mechauisms Dev.44: 41, 1993) reported that mouse embryonic stem cells (ES) can differentiate into spontaneously beating cardiomyocytes in vitro by embryonic-like aggregates, and Wobus et al (Ann.N.Y.Acad.Sci, 27: 460, 1995) reported that mouse pluripotent ES cells recapitulate the development of cardiomyocytes from the unformed embryonic cells into a cardiomyocyte (layer) specific cellular phenotype. Embryoid bodies can be inoculated, cultured, isolated and assayed using immunofluorescence and electrophysiological studies. This cell is reported to express heart-specific genes and all major heart-specific ion channels. Wobus et al (J.mol.cell.Cardiol.29: 1525, 1997) reported that retinoic acid accelerates cardiac differentiation of ES cells and increases development of ventricular cardiomyocytes. The cell clones used in this study expressed beta-galactosidase under the control of the MLC-2V promoter after transfection.

Kolosssov et al (J.cell.biol.143: 2045, 1988) reported isolation of mouse ES cell-derived cardiac precursor cells using a vector containing green fluorescent protein under the control of a cardiac alpha-actin promoter. Patch clamp (Patch clamp) and Ca⁺⁺Imaging suggests that expression of L-type calcium channels begins at day 7 of embryoid body development. Narifa et al (Development 122: 3755, 1996) reported that ES cells from GATA-4 deficient mice were able to differentiate into cardiomyocytes. GATA-4 deficient thin mouse is found in endocardium, myocardium and epicardium of chimeric miceAnd (4) cells. These authors suggest that other GATA proteins may compensate for the lack of GATA-4.

U.S. Pat. No. 5,6,015,671 (Field) and Klug et al (J.Clin.invest.98: 216, 1996) report that cardiomyocytes selected from differentiated mouse ES cells using genetic engineering methods form stable intracardiac grafts. Driving glucosaminide phosphotransferase or neo with alpha-cardiac Myosin Heavy Chain (MHC) promoter^rAnd screening with antibiotic G418, selecting these cells from differentiated mouse ES cells, transplanting them into the heart of adult dystrophic mice, and reporting labeled cardiomyocytes found up to 7 weeks after transplantation. International patent publication WO00/78119(Field et al) proposes a method for increasing the proliferative potential of cardiomyocytes by increasing the activity of cyclin D2.

Doeverdans et al (J.mol.cell Cardiol.32: 839, 2000) propose that in floating embryonic-like bodies, the differentiation of cardiomyocytes corresponds to embryonic cardiomyocytes. Cardiomyocytes produced from rodent stem cells have been reported to differentiate into ventricular myocytes and have sodium calcium and potassium flux.

Muller et al (FASEB J.14: 2540, 2000) reported that ventricular-like cardiomyocytes were isolated from mouse ES cells transfected with a ventricle-specific 2.1Kb myosin light chain 2V promoter and green fluorescent protein under the control of the CMV enhancer. Electrophysiological studies suggest the presence of ventricular phenotype but no paced spotted cardiomyocytes. Gryschenko et al (Pflegers Arch 439: 798, 2000) studied the output current in cardiomyocytes produced by mouse ES cells. The dominant repolarization current in early ES-derived cardiomyocytes is the 4-aminopyridine-sensitive transient output current. The authors concluded that these transient output currents play an important role in controlling cardiac electrical activity in early cardiomyocytes.

International patent publication WO 92/13066 (university of Loyola) reports the construction of rat cardiomyocyte cell lines from foetal material genetically modified with the oncogene V-myc or V-ras. U.S. Pat. Nos. 6,099,832 and 6,110,459(Mickle et al, Geugyme) report improved cardiac function in rat models using different combinations of adult cardiomyocytes, pediatric cardiomyocytes, fibroblasts, smooth muscle cells, endothelial cells, or skeletal muscle cells. Us patent 5,919,449(Diacrin) reports the use of porcine cardiomyocytes for the treatment of cardiac insufficiency in xenogeneic individuals, the cells being obtained from porcine embryos pregnant for 20-30 days.

Cardiovasculary tissue engineering was performed by producing neonatal cardiomyocytes from mesodermal stem cells by Makino et al (j.clin.invest.103: 697, 1999) and k.fukuda (Artificial orgems 25: 1978, 2001), generating cardiomyocyte lines from bone marrow stroma and culturing for more than 4 months. Treatment of cells with 5-azadeoxycytidine for 24 hours to induce cell differentiation caused 30% of the cells to form myotube-like structures, acquire cardiomyocyte markers and begin beating.

Most established cardiomyocyte cell lines are obtained from animal tissue, and no cardiomyocyte cell line has been established that is widely approved for human cardiac therapy.

Liechty et al (Nature Med 6: 1282, 2000) reported transplantation of human mesodermal stem cells and demonstrated that site-specific differentiation occurred after intrauterine sheep transplantation. It has been reported that the graft survives long for 13 months after transplantation and develops immunological activity as desired. International patent publication WO 01/22978 proposes a method for improving heart function in heart failure patients, which comprises transplanting autologous bone marrow stromal cells into the myocardium to generate new muscle fibers.

International patent publication WO 99/49105(Zymogenetics) proposes the isolation of human non-adherent pluripotent cardiac stem cells. The cardiomyocytes were suspended for density gradient centrifugation, cultured and tested for their myocardium specific markers. After proliferation and differentiation, the cell line produces progeny cells which are fibroblasts, myocytes, cardiomyocytes, keratinocytes or osteocytes or chondrocytes.

It is not clear whether the cell preparations exemplified in these patents can be mass-produced for marketing as therapeutic complexes for regenerating cardiac function. A more promising source of novacells for treating heart disease is human pluripotent stem cells obtained from embryonic tissue.

Thomson et al (Proc. Natl. Acad. Sci. USA 92: 7844, 1995) first successfully cultured primate embryonic stem cells in the rhesus or marmoset model, which subsequently generated a human embryonic stem (hES) cell line from human blastocysts (Science 828: 114, 1998) Gearhart and colleagues generated a human embryonic germline (hES) cell line from embryonic ovarian tissue (Shamboot et al, Proc. Natl. Acad. Sci. USA 95: 13726, 1998) International patent publication WO 00/70021 sets forth differentiated human embryonic-like cells and methods for their production from hES cells. International patent publication WO 01/53465 outlines the preparation of embryoid body-derived cells from hEG cells.

Both embryonic stem cells and embryonic germ line cells can be propagated in vitro without differentiation, and they retain normal karyotype and the ability to differentiate to produce a variety of adult-type cells. However, it is known that the proliferation and differentiation of human pluripotent stem cells follows a rule that is quite different from that followed by the development of cultured rodent stem cells.

Geron corporation developed novel tissue culture conditions that enable human pluripotent stem cells to proliferate in a substantially feeder-free environment, see Australian patent AU729377 and International patent publication WO 01/51616. The ability to culture stem cells in a feeder cells-free environment provides a system in which the cell composition used can be readily prepared to meet the requirements set forth for human therapy.

In order to exploit this potential of pluripotent stem cells in the treatment of human health and disease, it is now necessary to develop new protocols to develop these cells into cell populations that can treat important tissue types.

Summary of The Invention

The present invention provides a system for efficiently producing primate cells that can differentiate from pluripotent stem cells into cardiomyocyte lineage cells.

One embodiment of the invention is a cell population comprising cells of the cardiomyocyte lineage having the particular properties described herein. For example, they may be:

late cardiomyocytes

Cardiomyocyte precursor cells capable of proliferation in vitro and differentiation in vitro or in vivo into cells with one of the above characteristics

One or more markers capable of expressing the following endogenous genes: cardiac troponin I (cTnI), and Atrial Natriuretic Factor (ANF)

The ability to express three or more additional phenotypic markers as described herein.

Can be produced by primate pluripotent stem (pPS) cells

Has the same genome as an established human embryonic stem (hES) cell line.

Exhibit spontaneous periodic contractile activity.

Exhibit other characteristics of the cardiomyocytes, such as ion channels or corresponding electrophysiological behavior. The cell population of the invention can be enriched to such a degree that about 5%, about 20% or about 60% of the cells have the characteristic. If desired, these cells may also be genetically modified with telomerase reverse transcriptase to extend replication capacity, or to express growth factors, centripetal factors or transcriptional regulatory elements.

Another embodiment of the invention is a method of generating such a cell population, the method comprising differentiating pPS cells or progeny thereof in a suitable growth environment. In an exemplary method, hES cells are cultured under conditions substantially free of feeder cells. It then differentiates into cardiomyocytes or cardiomyocyte precursor cells with one or more of the above-described characteristics. In some cases, the differentiation method may include one or more of the following steps: culturing the pPS cells in suspension culture to form embryoid bodies or cell aggregates, culturing in a growth environment containing one or more centripetal factors, and separating the spontaneously contracting cells from other cells in the cell population or culturing in a growth environment containing one or more cardiomyocyte-enriching factors.

One embodiment of the invention is a method of screening for compounds that affect cardiomyocytes. The method comprises combining the compound with a cell population of the invention and measuring the modulation produced by the compound. This may include examination of cellular toxicity, metabolic changes, or effects on contractile activity.

In yet another embodiment of the invention, the cell population is formulated for administration to a human or animal body for the purpose of treating a myocardial disorder. Another embodiment of the invention is a method of reconstituting or providing contractile activity in cardiac tissue, the method comprising contacting the cardiac tissue with a cell population of the invention, comprising administering to an individual a suitable dosage form of a cell population of the invention to treat a cardiac disease in the individual.

These and other embodiments of the present invention will be apparent from the following description. The compositions, methods, and techniques described herein hold considerable promise for diagnostic, drug screening, and therapeutic applications.

The human embryonic stem cells of the present invention are provided by established cell lines.

Drawings

FIG. 1 shows the detection of marker expression in human undifferentiated embryonic stem (hES) cells using immunohistochemistry. The culture is cultured on mouse embryo feeder cells or with extracellular matrixOr laminin, has a phenotypic marker similar to that of hES grown on primary mouse fibroblast feeder layers.

FIG. 2 is a graph of cardiomyocytes obtained from pPS cells (top panel) and a kinetic graph of cardiomyogenesis (bottom panel). Example 2 provides an illustration of the initiation of differentiation of hES cells in suspension culture to form embryoid bodies, transfer of embryoid bodies to gelatin-coated plates after 4 days in suspension culture, spontaneous shrinkage observed in different areas of the culture on day 8 of differentiation, and the cell population which increased in number to over 60% over one week containing contractile cells.

FIG. 3 shows markers detected in cardiomyocytes differentiated from human embryonic stem (hES) cells. The upper panel shows the results of the analysis of the cardiac troponin I (cTnI) marker by immunostaining. cTnI and GATA-4 are found in contractile cells, but not in other wells that do not contain contractile cells. The following figure shows the kinetics of expression of the heavy chain of myosin (. alpha.MHC) during development. Expression of α MHC was significant by day 8, corresponding to the time when contractile cells were very abundant in culture.

Figure 4 shows single cells and cell colonies isolated and positive for tropomyosin, titin, Myosin Heavy Chain (MHC), alpha-actin, binding protein, cardiac troponin i (ctni) and cardiac troponin t (ctnt). Single cells and colonies stained positive for all these markers. Striated features of the myofibrillar structure were observed.

Figure 5 shows the effect of the drug on hES-derived cardiomyocyte contractile activity. The L-type calcium channel inhibitor diltiazem hydrochloride counteracts contractile activity in a dose-dependent manner. The adrenergic receptor agonists isoproterenol, phenylephrine, and clenbuterol, a mucolytic agent, have a chronotropic effect.

FIG. 6 shows the ability of the cytosine analog 5-azadeoxycytosine to act as an inducer of cardiomyocyte differentiation. Embryoid bodies formed by suspension culture of hES cells for 4 days were plated on gelatin-coated plates and 5-azadeoxycytidine was added to the culture broth over a period of 1-4 days, 4-6 days, or 6-8 days. This drug is most effective after the hES cells have well differentiated.

Figure 7 evaluates the potential cardiogenic factors for their ability to increase the ratio of cardiomyocytes in a cell population by adding activin and some growth factors during embryoid body formation (group I), other growth factors (group II) and 5-azadeoxycytidine after seeding on gelatin, and other factors after differentiation (group III), and the effect of the combinations was tested at three concentrations, with low concentrations of growth factors being most effective in combination with 5-azadeoxycytidine.

FIGS. 8(A) and 8(B) show further elaboration of the experimental protocol by individually adjusting each cytokine set. When low levels of factors from groups I and 2 were used, followed by treatment with 5-azadeoxycytosine, the alpha-MHC markers characteristic of cardiomyocytes were produced in the greatest amount. Under these conditions, the expression of the early cardiomyocyte-associated gene GATA-4 was also increased. The effect on α -MHC and GATA-4 was selective compared to the endoderm related gene HNF36, which was increased with any combination of growth factors but not with 5-azadeoxycytidine.

Fig. 9 shows that enrichment was achieved by culturing a cell population containing cardiomyocytes in a culture broth containing creatine, carnitine, and taurine (CCT) for 1-2 weeks. Each line represents the beating zone seen in one well throughout the experiment. CCT media increased the number of beating zones in culture by approximately 4-fold compared to cells cultured with standard differentiation media.

FIG. 10 shows PercoII discontinuity^TMThe effect of separating the cell population resulting from differentiation of hES cells on a gradient, with component I at the upper boundary, component II at the 40.5% level, component III at the lower boundary, and component IV-at the 58.5% level, was that the expression of the cardiomyocyte marker α -myosin heavy chain was highest in the higher density fractions as determined by real-time RT-PCR analysis.

Detailed Description

The invention provides a system for preparing and characterizing primate pluripotent stem cell derived cardiomyocytes and precursor cells thereof.

There are a number of obstacles to overcome in developing a large enriched cell population for obtaining primate pluripotent stem (pPS) cell derived cardiomyocyte cell lines. Some of the obstacles are due to the rather fragile, difficult culture of primate pluripotent stem cells and their abnormal sensitivity and dependence on various factors present in the culture environment. Another obstacle is the need to understand the terminal differentiation requirements of progenitor cardiomyocytes for the presence of visceral, embryonic endodermal and embryonic lineages (Arai et al Dynamucs 210: 344, 1997). In order for pPS cells to differentiate into progenitor heart cells in vitro, all of the activities that occur in the natural ontogeny of such cells in developing embryos must be simulated or replaced.

Despite these obstacles, it has now been found that such cell populations can be obtained from pPS cultures relatively enriched in cells expressing cardiomyocyte characteristics. Figure 4 shows individual cells staining positive for tropomyosin, titin, Myosin Heavy Chain (MHC), alpha-actin, binding protein, cardiac troponin i (ctni) and cardiac troponin t (ctnt), and shows striated features of the myofibrillar structures. These cells have spontaneous periodic contractions in tissue culture. Figure 5 shows that its contractile activity is inhibited by the L-type calcium channel inhibitor belzupine hydrochloride and enhanced in response to the adrenergic receptor agonists isoproterenol and phenylephrine.

It should be understood that the route of preparing human pluripotent stem cell-derived cardiomyocytes differs from many previously reported routes of preparing mouse cardiomyocytes. First, human pPS cells in an undifferentiated stage need different culture systems for proliferation and preparation for differentiation of cardiomyocytes, and mouse embryonic stem cells can be easily proliferated without differentiation by adding Leukemia Inhibitory Factor (LIF) to the culture medium. Whereas LIF by itself is not sufficient to prevent differentiation of human ES cells, human ES cells are routinely propagated on primary embryonic fibroblast feeder layers (Thomson et al, supra). In addition, factors that enable mouse stem cells to produce cardiomyocytes, such as retinoic acid (Wobus et al J.mol.Cardiol.29: 1525, 1997) and DMSO (McBurney et al Nature 299: 165, 1982), are much less effective when used on human stem cells under similar conditions.

The present invention solves the problem of preparing important cells from human pluripotent stem cells by providing a novel system that can highly enrich the cell population of a desired cardiomyocyte lineage. The system lends itself readily to implementation on a commercial scale. Methods useful for increasing cardiomyocyte production include:

1. undifferentiated pPS cells are placed in culture conditions that initiate the differentiation process to form embryoid bodies or are directly differentiated.

2. The pPS cells are cultured in the presence of one or more centripetal factors believed to contribute to their differentiation into cardiomyocyte lineage.

3. Cardiomyocytes are separated from other cells by density centrifugation or other suitable separation methods.

4. A cell population comprising a cardiomyocyte cell line is cultured in the presence of a cardiomyocyte-enriching factor believed to contribute to preferential growth of the desired cell type.

These and other steps described herein can be used alone or in effective combinations, as described in example 9, and only a few of these combined methods provide new cell populations containing greater than 69% of cardiomyocyte cell lines, and the significant homogeneity and functional properties of the cells produced according to the present invention make these cells extremely valuable for the development of new therapeutic agents and as a tool for in vitro studies of cardiac tissue.

Definition of

The techniques and compositions of the present invention relate to pPS derived cardiomyocytes and their precursors, the phenotypic characteristics of which are provided in the sections hereafter. There is no specific feature in the definition of cardiomyocyte precursor cells, but it is known that undifferentiated pPS cells differentiate into mesodermal cells first in the normal course of ontogeny and then into functional (terminal) cardiomyocytes through various precursor cell phases.

Thus for the purposes herein, a cardiomyocyte precursor cell is defined as a cell that is capable (without dedifferentiation or reprogramming) of producing daughter cells (including cardiomyocytes) and that expresses at least one of the following markers (preferably at least 3 or 5 markers): cardiac troponin I (cTnI), cardiac troponin T (cTnT), myofibrillar Myosin Heavy Chain (MHC), GATA-4, NKx2.5, N-cadherin, β 1-adrenoceptor (β 1-AR), ANF, transcription factor MEF-2 family, creatine kinase MB (CK-MB), myosin, or Atrial Natriuretic Factor (ANF).

The techniques described herein above and compositions referred to as "cardiomyocytes" or "cardiomyocyte precursor cells" can be applied equally to cells at any stage of occurrence in a cardiomyocyte individual, without limitation, unless otherwise specified. Such cells may or may not have proliferative capacity or exhibit contractile activity.

Certain cells of the invention exhibit spontaneous, periodic contractile activity, meaning that it is in an appropriate tissue culture environment and appropriate Ca⁺⁺When cultured in concentration and electrolyte equilibrium, it can be observed that the cells shrink laterally along one axis of the cells in a periodic manner and then relax without adding any other components to the culture solution.

The prototype "primate pluripotent stem cells" (pPS cells) are pluripotent cells derived from any type of embryonic tissue (embryonic or pre-embryonic tissue) and are characterized by the ability to produce progeny cells of different types (all three germ layers: endoderm, mesoderm and ectoderm) under appropriate conditions, as identified by standard assays accepted in the art, such as the ability to form teratomas in 8-12 age-matched SCID mice or the ability to form all three germ layers in tissue culture.

The pPS definition includes various types of embryonic-like cells, such as human embryonic stem (hES) cells described by Thomson et al (Science 282: 1145, 1998); embryonic stem cells of other primates such as rhesus monkey stem cells (Thomson et al, Proc. Natl. Acad. Sci. USA 92: 7844, 1995), marmoset monkey stem cells (Thomson et al, biol. reprod.55: 254, 1996) and human embryonic germ line (hEG) cells (Shamblott et al, Proc. Natl. Acad. Sci. USA 95: 13726, 1998). These types of cells can be provided as established cell lines. Other types of pluripotent cells are also included in this term, and any primate origin is included that produces all three germ layer progeny. pPS cells cannot be derived from malignant tumor sources and require (but not always required) that such karyotypes be normal.

pPS cell cultures are described as "undifferentiated" when a high proportion of stem cytoplasm in the cell population, whose derived cells exhibit morphological characteristics of undifferentiated cells, makes them clearly identifiable as differentiated cells of embryonic or adult origin. One skilled in the art would readily recognize undifferentiated pPS cells, which typically appear as a cell colony in a two-dimensional microscopic field of view, with a high nuclear/cytoplasmic ratio and prominent nucleoli. It is known that colonies of undifferentiated cells in a cell population often surround differentiated adjacent cells.

During ontogenesis of a cell, the adjective "differentiated" is a relative term, and a "differentiated cell" is a cell that progresses further downstream than the path of development of the cell. Thus. Pluripotent embryonic-like stem cells can differentiate into germ-line restricted precursor cells (e.g., mesodermal stem cells), which in turn can differentiate into other types of precursor cells further downstream in the developmental pathway (e.g., cardiomyocyte precursor cells), and then develop into terminally differentiated cells, which play a particular role in certain types of tissues and may or may not retain the ability to proliferate further.

The term "feeder cells" or "feeder" is used to describe a cell type that, when incubated with another type of cell, provides an environment for the growth of a second type of cell. A pPS cell population is said to be "substantially free" of feeder cells if no fresh feeder cells are added to the pPS cells after isolation to support their growth, but the pPS cells have been grown for at least one round. It will be appreciated that if a new feeder cells-free culture is performed using a previous feeder cells-containing culture as the source of pPS, some feeder cells will survive the passage, and that a culture that is substantially feeder-free when less than 5% of the viable feeder cells are present. More preferred are compositions containing less than 1%, 0.2%, 0.05% or 0.01% feeder cells (expressed as a percentage of the total number of cells in culture). When a cell line in the same culture is differentiated into multiple cell types, these different cell types are considered to act as feeder cells within the meaning of the present definition with respect to each other, even though they may interact in a supportive manner.

A "growth environment" is an environment in which a cell of interest can proliferate, differentiate, or mature in vitro. The characteristics of this environment include the medium in which the cells are cultured, the presence of growth factors or differentiation inducing factors, and the supporting structure (e.g., a matrix on a solid surface).

A cell is said to be genetically modified when a polynucleotide is transferred into the cell by suitable manual manipulation or when the cell is a progeny of a cell that has been previously engineered to inherit the polynucleotide. The polynucleotide will often contain a transcribable sequence encoding a protein such that the cell expresses elevated levels of the protein, the genetic modification being heritable if the progeny of the modified cell have the same (genetic) change.

The term "antibody" as used herein refers to both polyclonal and monoclonal antibodies. The term is deliberately intended to encompass not only whole immunoglobulin molecules, but also fragments and derivatives of immunoglobulin molecules (e.g., single chain FV structures, diabodies, and fusion structures), which may be prepared by techniques known in the art and which retain the desired antibody binding specificity

General technique

To further elaborate on the general techniques used in practicing the present invention, physicians may refer to standard textbooks and review cell biology, tissue culture, embryology, and cardiac physiology.

With respect to tissue culture and embryonic stem cells, the reader may wish to refer to "teratomas and embryonic stem cells: methods of implementation (Teratoccarinomas and implantable stem cells: A practical propaach) "(E.J. Robertson, eds., LPL Press Ltd, 1987); "Guide to technologies in Mouse Development" (p.m. wasserman et al eds., Academic Press, 1993); "in vitro Differentiation of Embryonic Stem cells (Embryonic Stem Cell Differentiation in vitro)" (M.V.Wiles, meth.Enzymol.225: 900, 1993); "characteristics and applications of embryonic stem cells: application Prospects in human biology and Gene Therapy (Properties and uses: applications to apply to humcn. biology and Gene Therapy) "(P.D. Rathjen et al, record. Fortil. Dev.10: 31, 1998). With respect to cardiac Cell culture, standard references include "Heart cells in culture" (The Heart Cell in culture) "(a. pinson eds., CRC Press 1987)," Isolated Adult Cardiomyocytes (islet Adult cardiocytes) "(volumes I and II, pipe & Isenberg eds., CRC Press, 1989)," cardiac development (Heart development) "(harvest & Rosenthal, Academic Press 1998)," I leave my Heart in San Francisco "(i.e. Left my Heart) (t.bennet, sorards, 1990) and" go with Wnt "(go The Wnt) (m.mitch, Scribner 1996).

General methods of molecular and cell biology can be found in the following standard textbooks: "Molecular Cloning, A Laboratory Manual" third edition (Sambrook et al, harbor Laboratory Press 2001); "Short Protocols in molecular biology" 4 th edition (edited by Ausubel et al, John Wiley & Sons 1999); "protein methods" (Bollag et al, John Wiley & Sons 1996); "non-viral Vectors for Gene Therapy (non viral Vectors for Gene Therapy)" (Wagner et al eds., Academic Press 1999); "Viral Vectors (Viral Vectors)" (Kaplitt and Loewy eds., Academic Press, 1995); "handbook of immunization Methods (Manual)" (i.e. lefkovits eds., Academic Press, 1997) and "cell and tissue culture: methods of testing in biotechnology (Cell and Tissue culture: Laboratory Procedures in Biotechnology) "(Doyle & Griffiths, John Wiley & Sons 1998). The genetically manipulated reagents, cloning vectors, and kits described herein are available from commercial suppliers such as BioRad, Stratagene, Invitrogen, Sigma-Aldrich, and Clon Tech.

Stem cell source

The invention may be practiced with various types of pluripotent stem cells, particularly those derived from embryonic tissue that have the ability to produce progeny from all three germ layers described above.

Examples of cell lines used as existing or established are embryonic stem cells and embryonic germ line cells of primates (including humans) primary embryonic tissue.

Embryonic stem cells

Embryonic stem cells have been isolated from blastocysts of members of the primate (Thomson et al, Proc. Natl. Acad. Sci. USA 92: 7844, 1995).

Propagation of pPS cells in the undifferentiated stage

pPS cells can be propagated in continuous culture using proliferation-promoting but differentiation-promoting culture conditions, and an exemplary serum-containing ES medium is 80% DMEM (e.g., knockout DMEM, Gibco). 20% fetal bovine serum (FBS, Hyclone) or serum replacement (WO 98/30679), 1% non-essential amino acids, 1mML glutamine and 0.1mM beta-Thiosyl ethanol. 4-8ng/ml human bFGF (WO 99/20741Geron Corp) can be added before application

ES cells are routinely cultured on a layer of feeder cells, usually embryonic-like or embryonic tissue-derived fibroblasts. Embryos from CF1 mice at day 13 of gestation were collected and transferred to 2ml trypsin/EDTA. Finely chopped, incubated at 37 ℃ for 5 minutes, debris was allowed to settle by the addition of 1% FBS, and cells were propagated in 90% DMEH, 10% FBS and 2mM glutamine. To prepare the feeder cell layer, cells were irradiated to inhibit proliferation, but allowed to synthesize factors (about 4000 rady gamma rays) that support the ES cells. The plates were coated overnight with 0.5% gelatin, and 37.5 million irradiated mEF cells were seeded per well, which may take 5 hours to 4 days after seeding. Fresh hES medium was used immediately prior to pPS cell inoculation.

Geran corporation developed a new tissue culture environment capable of continuously proliferating pluripotent stem cells in a substantially feeder-free environment, see Australian patent AU729377 and International patent publication WO 01/51616. Can be produced in a conditioned medium by feeder cells or supplemented with growth factors such as FGF and SCFExtracellular matrix in seed cultureOr culturing the stem cells on laminin. The ability to culture stem cells in a feeder cells-free environment provides a system for readily preparing cell compositions that comply with the human therapeutic code requirements, and for the purposes of this application, any application in the united states that claims priority from international patent publication WO01/51616 is incorporated herein by reference in its entirety.

The feeder cells-free culture environment comprises a suitable culture medium, in particular, for exampleOr an extracellular matrix such as laminin. pPS cell inoculation amount is more than 15.000 cells/cm²(optimally 90.000-17.000/cm)²). Enzymatic digestion is usually carried out until the cells are completely dispersed (with collagenase IV for about 5-20 minutes). Then, a patch of approximately 10-2000 cells is seeded directly onto the matrix without redispersion, and feeder cells-free culture is supported with nutrient medium (typically conditioned medium freshly generated for culturing irradiated primary mouse embryonic fibroblasts, telomerase-treated mouse fibroblasts, or pPS cell-derived fibroblast-like cells), either in serum-free medium such as KO DMEM supplemented with 20% serum replacement and 4ng/ml FGF, at about 5-6X 10⁴/cm²And (4) inoculating a feeder cell conditioning culture solution at a density. The 24-hour conditioned medium was filtered through a 0.2 μ M filter and 8ng/ml bFGF was added to support pPS cell culture for 1-2 days.

Microscopically, ES cells exhibit high nuclear/cytoplasmic ratios, prominent nucleoli and dense colonies formed by poorly identifiable cell junctions. Primate ES cells express one or more stage-specific embryonic antigens (SSEA)3 and 4, and a marker detectable with antibodies designated anti-Tra-1-60 and Tra-1-81 (Thomson et al, Science 282: 1145, 1998). Undifferentiated hES cells also typically express Oct-4 and TERT (detectable by RT-PCR) and alkaline phosphatase activity (detectable by enzyme assay). In vitro differentiation of hES cells often results in the loss (if any) of these markers and increased SSEA-1 expression.

Procedure for preparing cardiomyocytes

Cells of the invention can be obtained by culturing or differentiating stem cells in a specific growth environment that can enrich for cells having the desired phenotype (either by growing the desired cells or by inhibiting killing of other cell types). These methods are applicable to many types of stem cells, particularly the primate pluripotent stem (pPS) cells described in the section above.

Differentiation is usually initiated by the formation of embryoid bodies or aggregates, for example by overgrowth of donor pPS cell cultures or suspension culture of pPS cells in culture flasks containing a matrix with low adhesive energy (to form EBs). pPS cells were harvested by simple digestion with collagenase, broken into small pieces, seeded in non-adherent cell culture plates, fed with aggregates every few days, and harvested after an appropriate time (usually 4-8 days). The harvested aggregates are seeded on a solid substrate and cultured for a period of time such that the cells in the aggregates adopt a cardiomyocyte phenotype. Generally, the total time of differentiation is at least 8 days, possibly at least 10 or 12 days.

Alternatively, cells may be cultured according to a differentiation protocol to initiate the differentiation process. Conditions that induce differentiation of hES cells into heterogeneous cell populations include addition of Retinoic Acid (RA) or dimethyl sulfoxide (DMSO) to the culture or withdrawal of cells from the usual extracellular matrix in which they are cultured. See U.S. patent application 60/312,740 and international patent publication WO 01/51616. However, care was taken because in some cases these preparations reduced the cardiomyocyte ratio obtained (example 6).

In some cases, it may be beneficial to add one or more "centripetal factors" to the culture. These simple factors may alone or in combination enhance proliferation or survival of cardiomyocyte cell types, or inhibit growth of other cell types. This effect may be due to direct action on the cardiomyocytes themselves or due to action on another type of cell which in turn favours cardiomyogenesis. For example, factors that induce the formation of ectodermal or endodermal equivalent cells, or factors that cause these cells to produce their own myocardial-enhancing elements, are included within the definition of centripetal factor.

Factors believed to induce differentiation of pPS cells into mesodermal cells or promote further differentiation into cardiomyocyte lineage cells include the following:

nucleoside analogues capable of affecting DNA methylation and altering cardiomyocyte-associated gene expression.

TGF- β ligands (TGF-. beta.1, TGF-. beta.2, TGF-. beta.3, and other members of the TGF-. beta.superfamily described below) that bind to the TGF-. beta.receptor, activate type I and type II serine kinases, and cause Smad effector phosphorylation.

Morphogens such as activin A and activin B (members of the TGF-. beta.superfamily)

Insulin-like growth factors (e.g. IGF11)

Bone morphogenetic proteins (members of the TGF-. beta.superfamily, exemplified by BMP-2 and BMP-4)

Fibroblast growth factors (exemplified by bFGF, FGF-4 and FGF-8) and other ligands that activate cytosolic and mitogen-activated protein kinases (MAPKs).

Platelet-derived growth factor (PDGF. beta. as an example)

Natriuretic factors, such as Atrial Natriuretic Factor (ANF), Brain Natriuretic Peptide (BNP)

Products of related factors such as insulin, Leukemia Inhibitory Factor (LIF) Epithelial Growth Factor (EGF) TGF alpha and Oripto genes.

Specific antibodies having agonist activity to the same receptor

Alternatively, the cells may be incubated with cells that secrete factors that promote differentiation of cardiomyocytes (e.g., various types of endothelial cells).

As described in example 6, nucleoside analogs that affect DNA methylation (and thus gene expression) can be effectively used to increase the proportion of cells of the cardiomyocyte lineage that appear as a result of primary differentiation, e.g., it has been found that the addition of 5-azadeoxycytidine in culture increases the frequency of contractile cells in the population and myocardial aMHC expression. In some cases, enrichment by this step alone can increase the contractile cells of the cell population by about 1% to more than 3%.

The evaluation of the centripetal preparation is further described in example 7. Particularly effective combinations of centripetal formulations include the use of morphogens such as activin A and various growth factors, such as growth factors of the TGF- β and IGF families added early in the investment. Other centripetal agents, such as one or more of fibroblast growth factor, bone morphogenic protein and platelet derived growth factor, may optionally be added.

In elaborating upon the present invention, it was discovered that deletion of insulin-like growth factor 11(IGF11) and related molecules at the end of differentiation in vitro actually increased the expression levels of cardiac muscle genes. IGF11 was found to reduce fibroblast GSD3 β levels in unrelated studies (Scalia et al, J.cell. Bilchem.82: 610, 2001). IGF11 may potentiate the action of Wnt proteins present in the culture or secreted by the cells, which normally stabilize the cytoplasmic molecule β -catenin (which contains part of the transcription factor TCF) and cause its nuclear translocation. This alters the transcriptional activity of multiple genes. In the absence of Wnt, the kinase GSK3 β phosphorylates β -catenin, destabilizing it and keeping it cytosolic.

Since Wnt activators significantly limit cardiomyocyte differentiation like IL-11, it is believed that culture with Wnt antagonists can improve the program or rate of differentiation of hES cells into cardiomyocytes. Wnt signaling can be inhibited by injection of synthetic mRNA encoding DKK-1 or Crescent (secreted proteins that bind and inactivate Wnt) (Schneider et al, Gene Dev.15: 304, 2001), or infection with a retrovirus encoding DKK-1 (Marvin et al, Genes Dev 15: 316, 2001). Or increasing the activity of the kinase GSK3 β, e.g. incubation of cells with IL-6 or glucocorticoids could inhibit the Wnt pathway.

Of course, the use of centripetal factors in the differentiation protocol of the present invention does not always require an understanding of the manner in which they function. The combination of means and amounts of these compounds effective to enrich cardiomyocyte production can be determined empirically by adding such factors to a culture environment and then determining whether the whole compound increases the number of cardiomyocyte lineage cells in the cell population based on the phenotypic markers set forth below.

It has been found that pPS derived cardiomyocytes can be separated into single cell suspensions for reseeding and amplification, enrichment, cloning and phenotypic characterization, respectively. Example 2 illustrates the preparation of isolated individual cardiomyocytes using collagenase B solution. Collagenase II or collagenase mixtures such as Blendzyme IV (Roche) are also suitable for this preparation. After dissociation, the cells were seeded on chambered slides and cultured in differentiation medium. The single cardiac cell that was re-cultured survived and continued to beat.

The pPS-derived cell suspension may be further enriched for cells having the desired characteristics, for example, by mechanical separation or cell division. It has been found that density separation using appropriate techniques enriches the contractile cells by a percentage of about 20-fold. Examples 4 and 9 illustrate that isopycnic enriched cardiomyocyte cell populations can be obtained containing at least about 5%, about 20%, about 60%, and possibly more than 90% of cardiomyocyte lineage cell populations. Many of the research and therapeutic applications described herein benefit from high rates of cardiomyocytes obtained, but generally do not require complete homogeneity.

After initiation of differentiation (and before or after the isolation step, if isolation methods are employed) the cells can be cultured in an environment containing a cardiomyocyte-enriched preparation to increase the percentage of cardiomyocyte lineage cells. Factors in culture or on surface matrices can promote the growth of a desired cell type by promoting cardiomyocyte lineage cell proliferation, or by inhibiting cell growth (or causing apoptosis) in other tissue types. Some of the centripetal factors listed above are suitable for this purpose, as are certain compounds or analogs thereof known to be beneficial to cardiomyocytes in vivo, including compounds capable of forming high energy phosphate bonds (e.g., creatine), acyl carrier molecules (e.g., carnitine), and cardiomyocyte calcium channel modulators (e.g., taurine).

Characterization of cardiomyocyte lineage cells

Cells obtained using the techniques of the present invention can be characterized by a number characteristic of a standard phenotype. pPS cell line-derived cardiomyocytes and precursor cells often have the morphological characteristics of cardiomyocytes of other origins. They may be spindle, circular, triangular or polygonal and have striated features of immunostaining-detectable myofibrillar structures (example 3) that can form myotube-like structures that show typical myofibrillar nodes and anterior chamber granules when examined by electron microscopy.

pPS-derived cardiomyocytes and their precursors typically have at least one of the following cardiomyocyte-specific markers:

cardiac troponin i (ctni), a subunit of the troponin complex, provides a calcium-sensitive molecular switch that regulates striated muscle contraction.

Cardiac troponin T (cTnT)

Anterior chamber natriuretic factor (ANF), a hormone expressed in developing cardiac and embryonic cardiomyocytes but down-regulated at maturation, is considered a good marker for cardiomyocytes because it is expressed in a highly specific manner in cardiomyocytes but not on skeletal myocytes.

The cells also typically express at least one of the following markers:

myosin Heavy Chain (MHC) of myofibrillar fibers

Titin, tropomyosin, alpha-actin and binding proteins

GATA-4, a transcription factor highly expressed in cardiac mesoderm and persistently present in development, regulates many cardiac genes and plays an important role in cardiogenesis.

Nkx2.5, a transcription factor expressed in early mouse embryonic development cardiac mesoderm. Persisting in the developing heart.

MEF-2A, MEF-2B, MEF-2C, MEF-2D, a transcription factor persistently present in developing hearts expressed by cardiac mesoderm

N-cadherins, which mediate adhesion between cardiomyocytes

Gap junction protein, which forms gap junctions between cardiomyocytes

Beta 1-adrenoceptor (beta 1-AR)

Creatine kinase MB (CK-MB) and myosin, both of which are other markers that may be positive on cardiomyocytes and their precursors following post-myocardial infarction at elevated serum levels include α -cardiac actin, early growth response-1 and cyclin D2.

Any suitable immunological technique may be used to detect these tissue-specific markers, such as flow immunocytochemistry or affinity adsorption of cell surface markers, immunocytochemistry of intracellular or cell surface markers (e.g., fixed cells or tissue sections), immunostaining of cell extracts, and enzyme-linked immunoassays of cell extracts or products secreted into culture fluid. A cell expresses an antigen that is detectable by an antibody if a significant amount of the antibody binds to the antigen in a standard immunocytochemistry or flow cytometry assay. The label may optionally be amplified after the cells are fixed and optionally with a labeled secondary antibody, or other conjugate (e.g., biotin-avidin conjugate).

Expression of tissue-specific gene products can also be detected at the mRNA level using northern blot analysis, dot blot analysis, or reverse transcriptase-induced polymerase chain reaction (RT-PCR) using sequence-specific primers using standard amplification methods. See U.S. Pat. No. 5,843,780 for a detailed understanding of these general techniques. Sequence information for the other markers listed herein is available from public databases such as Gen Bank (URL www.ucbi.nlm.mih.gov: 80/entrez). If an assay is performed on a cell sample according to standard procedures, the production of a clearly distinguishable hybridization or amplification product in a typical controlled assay, the expression of mRNA levels is said to be detectable in one of the assays described herein. A tissue-specific marker is considered positive if the expression of the marker is detected at a protein or mRNA level that is at least 2-fold, preferably 10 or more than 50-fold, higher than that of a control cell, such as an undifferentiated pPS cell or other cell type of unrelated type.

Once markers of the desired phenotype are detected on the cell surface, they can be used in immunoscreening to further enrich the cell population with techniques such as immunopanning or antibody-mediated fluorescence-activated cell sorting.

In appropriate circumstances, pPS derived cardiomyocytes often exhibit spontaneous periodic contractile activity, meaning that when cultured in an appropriate tissue culture environment with appropriate Ca + + concentrations and electrolyte balance, one can observe contraction of these cells across one axis of the cell, followed by relaxation, without the need to add any other components to the culture medium. Contraction is periodic, meaning that it repeats regularly or irregularly at a frequency of between about 6-20 times, often between about 20-90 times per minute (FIG. 5), and individual cells may exhibit spontaneous periodic contractile activity by themselves, or in concert with neighboring cells in cell aggregates or cultured cell masses in tissues.

The contractile activity of the cells can be characterized according to the effect of culture conditions on the nature and frequency of contraction. Compounds that reduce Ca + + concentration or interfere with transmembrane transport of Ca + + often affect contractile activity. For example, diltiazem hydrochloride, an L-type calcium channel blocker, can inhibit contractile activity in a dose-dependent manner (fig. 5). On the other hand, adrenergic receptor agonists such as isoproterenol and phenylephrine have positive chronotropic effects. Further characterizing the functional properties of the cell may include identifying Na⁺、k⁺And Ca⁺⁺A channel. The electrophysiology of myocardial cell action potentials can be studied by patch clamp analysis, see igelmount et al, Pflugers Arch 437: 699, 1999; wobus et al, ann.n.y.acad.sci.27: 752, 1995 and Doevendans et al, j.mol.cell cardio 32: 839, 2000.

Functional profiling studies provide a means to characterize cells and their precursors in vitro, but may not be required for certain applications described herein. For example, a mixed population of cells enriched for certain marker cells as described above may not have all of the cells with functional and electrophysiological properties. They can be used to obtain the functional properties required to support cardiac function in vivo if they are transplanted into damaged cardiac tissue, or they are considered to be of therapeutic value.

The cell populations and isolated cells of the invention, as derived from an established pPS cell line, can be characterized as having the same genome as the cell line from which they are derived. This means that the chromosomal DNA identity between the pPS cells and the cardiomyocytes (if the cardiomyocytes were obtained from an undifferentiated cell line by a normal mitotic process) is over 90%. Cells recombinantly introduced with a transgene (e.g., TERT) or knocked-out of an endogenous gene can still be considered to have the same genome as the cell line from which it was derived, since it retains all of the genetic elements that were not manipulated. The two cell populations may be shown to have the same genome using standard techniques, such as DNA fingerprinting. Alternatively, the relationship can be established by observing records preserved during cell division. Characterization of cardiomyocyte lineage cells derived from a parental cell population is important in several respects, in particular, an undifferentiated cell population may be used to generate additional cells containing a shared genome, another set of cardiomyocytes, or other types of cells that may be used in therapy, such as a histocompatibility type of cells that may be used to pre-tolerize a patient to cardiac allograft.

For therapeutic applications, it is often desirable that the differentiated cell population of the invention be substantially free of undifferentiated pPS cells. One method of eliminating undifferentiated stem cells from the cell population is to transfect them with a vector. The responsive gene in the vector is under the control of a promoter which causes it to be preferentially expressed in undifferentiated cells. Suitable promoters include the TERT promoter and the OCT-4 promoter. The effector gene may directly lyse the cell (e.g., a gene encoding a toxin or apoptotic mediator). Or the effector gene can confer sensitivity to the toxic effects of a foreign agent such as an antibody or prodrug. An example is the herpes simplex virus thymidine kinase (tk) gene, which causes cells expressing it to become sensitized to ganciclovir. Suitable pTERT-tk constructs are provided in WO 98/14593(Morin et al).

Since it has been demonstrated that cardiomyocytes and their precursors can be generated from pPS cells, the reader can determine whether the differentiation protocol described herein is suitable for his or her purpose. For example, the reader can readily test the suitability of such culture conditions by culturing pPS cells or cells derived therefrom under test conditions comparable to those described herein, and other types of control cells (e.g., primary human cardiomyocytes, hepatocytes, or fibroblasts), and then comparing the phenotype of the above-described markers for the cells obtained. Adjusting culture and cell separation conditions to alter specific components is a routine optimization of the normally expected performance of such culture methods without departing from the concept of the present invention.

Genetic modification of differentiated cells

Where it may be desirable for such cells to have replicative capacity and the ability to provide a reservoir for cardiomyocytes and their precursors for use in certain drug screening and therapeutic applications, the cells of the invention may optionally be telomerised to enhance their replicative capacity before and after their progression to restricted developmental lineage cells or to terminally undifferentiated cells, telomerised pPS cells may enter the above-mentioned differentiation pathways, or alternatively the cells may be terminally treated directly to differentiate.

Teleomorphized cells are transfected or transduced with an appropriate vector; homologous recombination, or other suitable technique, genetically modifies them to express the telomerase catalytic component (TERT), which is usually under the control of a heterologous promoter, which increases telomerase expression over that under the control of an endogenous promoter. Particularly suitable is the catalytic component of human telomerase (hTETR), provided in international patent publication WO 98/14592. Ethnic homologues thereof such as mouse TERT (WO 99/27113) may also be used in certain applications. Transfection and expression of telomerase in human cells is described in Bodnar et al, Science 279: 349, 1998 and Jiang and nat gene.21: 111, 1999. In another example, the hTERT clone (WO 98/14592) was used as the source of hTERT coding sequence, spliced into the ECOR1 site of the PBBS 212 vector under the control of MPSV promoter, or into the EcoRI site of the commercial pBABE retroviral vector under the control of LTR promoter.

Differentiated or undifferentiated pPS cells genetically modified with the vector are cultured in a supernatant containing 8 to 16 hours of culture, and then replaced with a growth medium for 1 to 2 days. Cells that have been genetically modified are screened for puromycin, G418 or blasticidin using a drug screening agent and re-cultured. Their hTERT expression was then assessed by RT-PCR telomerase activity (TRAP assay), immunocytochemical staining or replicative capacity of hTERT. The following kits are available from commercial markets for research:XL telomerase detection kit (Cat. No. s 7707; Intergen co Purchase NY) and Telotaggg telomerase PCRELISSAplus (Cat. No. 2,013, 89; Roche Diagnostics, Indianapolis IN). TERT expression can also be assessed at the mRNA level using FT-PCR. A commercial reagent for research purposes is the LigheCycler Telo TAGA hTERT quantitation kit (Cat. No. 3,102,344; Roche Diagnostics). The constantly replicating colonies can be enriched by further culturing under conditions that support proliferation and cells with the desired phenotype can be cloned by limiting dilution.

In certain embodiments of the invention, the pPS cells are differentiated into cardiomyocyte precursor cells and then genetically modified to express TERT, and in other embodiments of the invention, the pPS cells are genetically modified to express TERT and then differentiated into cardiomyocyte precursor cells or terminally differentiated cells. Successful modification to increase TERT expression can be determined by the TRAP assay, or whether the replication capacity of the cell is increased.

Depending on the cell used, other methods of rendering the cell immortal are also acceptable, such as transformation of the cell with DNA encoding myc SV40 large T antigen or MOT-2 (U.S. Pat. No. 5,869,243, International patent applications WO 97/32972 and WO 01/23555). Transfection with oncogenes or oncogenic viruses is less appropriate when the cells are used for therapeutic purposes. Of particular interest for use in the present invention are terminally granulated cells which have the advantage that they proliferate and maintain their karyotype during, for example, drug screening and during therapies given to patients to differentiate cells to enhance cardiac function.

The cells of the invention may also be genetically modified to enhance their ability to participate in tissue repair, or to deliver therapeutic genes to the site of administration. A vector is designed with the known coding sequence of the desired gene operably linked to a promoter that is specifically or specifically active in differentiated cell types. Cells of particular interest are those genetically modified to express one or more of a variety of growth factors, centripetal factors such as atrial natriuretic factor, Cripto, and cardiac transcriptional regulators such as GATA-4, Nkx2.5, and MEF 2-C. Production of these factors at the site of administration has the beneficial effect of promoting the adoption of a functional phenotype, increasing the number of cells administered, or enhancing the proliferation or activity of host cells adjacent to the site of treatment.

Use of cardiomyocytes and their precursors

The present invention provides methods for the generation of large numbers of cardiomyocyte lineage cells that can be used for a number of important research, development and commercial purposes.

The cells of the invention can be used to prepare a cDNA library that is relatively free of contaminating cDNAs that are preferentially expressed in other cells. For example, cardiomyocytes are harvested by centrifugation at 1000rpm for 5 minutes, and mRNA from the settled cell mass is prepared by standard methods (Sambrook et al, supra) and reverse transcribed to cDNA, which removes cDNA from undifferentiated pPS cells, other progenitor cells, or cardiomyocytes or end-stage cells derived from other developmental pathways.

The differentiated cell of the present invention may be also used in preparing specific antibody for cardiac muscle cell and its precursor cell mark, and injecting the cell of the present invention in immunogen form into vertebrate to prepare polyclonal antibody. Monoclonal antibody production can be found in standard references such as U.S. Pat. nos. 4,491,632, 4,472,500 and 4,444,887 and methods of enzymology 73B: 3(1981). Libraries of immunocompetent cells or viral particles may also be contacted with a target antigen and selected positive clones cultured to produce specific antibody molecules. See Marks et al, New eng.j.med.335: 730, 1996 and McGuiincess et al, Nature Biotechnol.14: 1449, 1996. Another approach is to reassemble the random DNA fragment into the antibody coding region as described in European patent application 1,094,108A.

By using specific cells of the invention for positive selection and cells with more widely distributed antigens (e.g., embryonic cell progeny with other phenotypes) or adult cardiomyocytes for negative selection, the desired properties (antibodies) can be obtained, which in turn can be used to identify or rescue cardiac cells of a desired phenotype in a mixed population of cells for the purpose of co-staining or isolating terminally differentiated cardiomyocytes and other lineage cell precursors in immunodiagnosis of tissue samples.

The cells of the invention are also interesting for identifying the expression patterns of transcripts and newly synthesized proteins characteristic of cardiac myocytes, which may help to direct differentiation pathways or promote interactions between cells. Expression patterns of differentiated cells are obtained and compared to patterns of control cell lines such as undifferentiated pPS cells, other types of committed precursor cells (e.g., pPS cells differentiated to other lines), or terminally differentiated cells.

For a review of gene expression analysis using microarrays, see Frifg et al science.288: 316, 2000; com, Miceoarmy Biochip Technology, L Shi, www.Gene-chips. The exemplary method can be carried out with an array generator and TM scanning. First, cDNA fragments encoding the marker sequences to be analyzed are amplified and spotted directly onto a glass slide to prepare a microarray. To compare mRNA preparations of two cells of interest, one preparation was converted to Cy 3-labeled cDNA, while the other was converted to Cy 5-labeled cDNA. Both cDNA preparations were hybridized to microarray slides simultaneously and then washed to remove non-specific binding. The slide is scanned at a wavelength appropriate for each marker and the resulting fluorescence is quantified and expressed as the relative abundance of mRNA for each marker on the array.

Drug screening

The cardiomyocytes of the present invention can be used to screen for factors (e.g., solvents, small molecule drugs, peptides, oligonucleotides) or environmental conditions (e.g., culture conditions or manipulations) that can affect the characteristics of the cell and its various progeny.

In certain applications, pPS cells (differentiated or undifferentiated) are screened for factors that promote their maturation into late cardiomyocyte precursor cells or terminally undifferentiated cells or that promote proliferation and maintenance of such cells in long-term culture. For example, candidate maturation or growth factors are added to cells in different wells and tested by measuring the resulting phenotypic changes according to criteria for further culturing and application of such cells.

Other screening applications of the invention relate to testing the effect of a drug compound on the maintenance or repair of myocardial tissue, as contemplated compounds have pharmacological effects on such cells or as contemplated compounds may have undesirable side effects on such tissue. The precursor cells or the final undifferentiated cells of the present invention can be used for screening.

The reader is referred to the standard textbook "In Vitro Methods for drug studies (In Vitro Methods In pharmaceutical Research)", Academic Press, 1997 and U.S. Pat. No. 5,030,015. Evaluation of a candidate pharmaceutical composition typically involves mixing the differentiated cells of the invention with a candidate compound, alone or in further combination with other drugs. The researcher can determine changes in the morphology, marker phenotype or functional activity of such cells due to the compound (as compared to untreated cells or cells treated with an inert compound) and then correlate the effect of the compound with the change seen.

Cytotoxicity can be determined by first assaying for effects on cell viability, survival morphology, and expression of certain markers and receptors. The effect of a drug on chromosomal DNA can be determined by assaying the effect of said drugDNA synthesis or repair [ 2 ]³H]Thymidine or BrdU incorporation, particularly at unplanned times in the cell cycle, or levels above that required for cell replication). Adverse effects may also include rare sister chromosome exchange rates (as measured by metaphase spread). The reader is referred to the A.Vickers (In Vitro Methods In pharmaceutical Research), Academic Press, 1997, page 375-410) for further review.

The effect on cell function can be assessed by observing the phenotype or viability of cardiomyocytes using standard assays, such as marker expression, receptor binding, contractile activity or electrophysiology, in cell culture or in vivo. The effect of the candidate drug on contractile activity may also be tested, e.g., whether contractile program and frequency are increased or decreased. Observing this effect the concentration of titratable compounds to determine the effective dose median value (ED)₅₀)。

Therapeutic applications

The invention also provides the use of cardiomyocytes and their precursors to enhance the maintenance and repair of myocardial tissue that is essential due to congenital defects in cardiac metabolic function, disease-induced or severe creation.

To determine whether a cell composition is suitable for therapeutic administration, the cells can first be tested in a suitable animal model at a level to assess the ability of the cells to survive and maintain their phenotype in vivo. After a period of re-growth of the cell composition administered to an immunodeficient animal (e.g., a nude mouse or an immunodeficient animal that has been chemically or by irradiation), tissues are harvested and the presence of the pPS-derived cells is assessed.

This can be done by administering cells expressing a detectable marker (e.g., green fluorescent protein or β -meter lactylate), pre-labeled (e.g., with Brdo or [ beta ], [ beta ] -meter³H]) Cells, and then detecting constitutive cellular markers (e.g., with human-specific antibodies). The presence and phenotype of the cells administered can be assessed by immunohistochemistry or ELISA using human specific antibodies, or by means of a nucleic acid sequence that results in a human polynucleotide(according to published sequence data) specific amplification of primers and hybridization conditions for RT-PCR to evaluate.

This suitability may also be determined by evaluating procedures for restoring the heart as a result of treatment with pPS-derived cardiomyocytes. A number of animal models have been used for such testing. For example, pre-chilled aluminum rods may be placed in contact with the anterior wall surface of the left ventricle to freeze the heart (Marry et al, J.Clin. invest.98.2209, 1996, Reinecke et al, Circulation 100: 193, 1999; U.S. Pat. No. 6,099,832). For larger animals, a 30-50mm copper disk probe cooled in liquid nitrogen can be placed on the anterior wall of the left ventricle for approximately 20 minutes to cause frostbite (Chiu et al, Ann Thorac. Surg.60: 12, 1995). Ligation of the left-hand main crown animal can cause obstruction (Li et al, J.Clin invest.100: 1991, 1997). The damaged part is treated by the cell preparation of the invention, and whether the damaged area of the heart tissue has the cells or not is examined by a histological method. The cardiac function can be monitored by measuring parameters such as the left ventricular end diastolic pressure, the generated pressure, the pressure rising speed and the pressure falling speed.

Upon sufficient detection, the differentiated cells of the invention may be used for tissue reconstruction or regeneration in a patient or other subject in need of such treatment. These cells are administered by grafting or otherwise migrating into the desired tissue site and reconstructing or regenerating the functionally deficient area, and special devices are available to administer these cells directly to the desired site within the heart chamber, pericardium or myocardium to reconstruct cardiac function.

The indications for such treatment include various acute and chronic heart diseases, such as coronary heart disease, cardiomyopathy, congenital cardiovascular defects, and congestive heart failure. The treatment effect can be monitored according to clinically accepted standards, such as scar tissue area reduction; scar tissue regrows out of the blood vessel; angina pectoris is reduced in frequency and severity; the generated pressure, the systolic pressure and the diastolic pressure are improved; delta pressure/delta time, patient movement and quality of life.

The cardiomyocytes of the present invention can be provided in the form of a pharmaceutical composition comprising an isotonic vehicle prepared under substantially sterile conditions for human consumption. The general principles of pharmaceutical formulation the reader is referred to "cell therapy: stem Cell Transplantation, gene Therapy and Cellular Immunotherapy (Cell Therapy: Stem Cell Transplantation, Gene Therapy and Cellular Immunotherapy) "G.Morstyn & W.Sheridan eds, Cambridge university Press, 1996 and" Hematopoietic Stem Cell Therapy "E.D.ball, JLIster & P.Law, Churchill Livingstone, 2000. The choice of cellular excipients and accompanying elements for the composition will depend on the route of administration and the device used. The composition may also contain or be accompanied by one or more other ingredients that promote cardiomyocyte transplantation and functional activity. Suitable components include matrix proteins or helper cell types, particularly endothelial cells, which are capable of supporting or promoting adhesion of cardiomyocytes.

The composition is optionally packaged in a suitable container with instructions for use, such as reconstituting cardiomyocyte function to ameliorate certain abnormalities of the myocardium.

The following examples are provided as further non-limiting illustrations of specific embodiments of the invention.

Examples

Example 1: feeder cell-free proliferation of embryonic stem cells

Established undifferentiated human embryonic stem (hES) cells are maintained in a substantially feeder cells-free culture environment.

Feeder cells-free cultures were maintained using conditioned medium prepared from primary mouse embryo fibroblasts isolated by standard methods (WO 01/51616). With no Ca⁺⁺/Mg⁺⁺The fibroblasts in the T150 flasks were harvested after one PBS wash and incubated in 1.5-2ml trypsin/EDTA (Gibco) for 5 minutes. After the fibroblasts were detached from the flask, they were collected in mEF medium (DMEM + 10% FBS), irradiated with 4000 rads, counted and counted at 5.5 ten thousand cells/cm²Inoculating to mEF culture solution6-well plates 52.5 ten thousand cells per well).

At least 4 hours later, the medium was replaced with SR containing ES medium (80% knock-out DMEM, Gibo BRL Rockville MD) 20% knock-out serum replacement (Gibco), 1% nonessential amino acids (Gibco), 1mML glutamine (Gibco), 0.1mMB mercaptoethanol (Sigma St Louis MO) supplemented with 4ng/ml recombinant human basic fibroblast growth factor (bFGF, Gibco). There were 0.3-0.4 ml of culture per square centimeter of plate surface area, and 4ng/ml human bFGF was added to this conditioned medium prior to addition to the hES culture.

Coating of cultured hES cell plates(Becton-Dickinson. Bedford MA), the stock solution was diluted with cold KODMEM 1: 30 and dispensed at 0.75-1.0 ml per well of 9.6 square centimeters, incubated at room temperature for 1-4 hours or coated overnight at 4 ℃.

The hES culture was passaged at 200U/ml collagenase IV at 37 ℃ for about 5-10 minutes. The cells were scraped and then gently dissociated into small pieces in conditioned medium for seedingThe culture was confluent about one week after inoculation on the coated plates for passaging. Cultures were maintained under these conditions for more than 180 days, with an ES-like morphology being constantly displayed. The samples were incubated with primary antibodies against SSEA-4 (1: 20), Tra-1-60 (1: 40) and Tra-1-81 (1: 80) diluted in knockout DMEM for 30 minutes at 37 ℃ for immunocytochemistry examination, the cells were washed with hot knockout DMEM and fixed with 2% paraformaldehyde for 15 minutes, then washed with PBS, the cells were incubated with PBS containing 5% goat serum for 30 minutes at room temperature, and then incubated with FITC-conjugated goat anti-mouse IgG (1: 125, Sigma) for 30 minutes. Cells were washed, stained with DAPI and mounted.

Cells were also examined for alkaline phosphatase (a marker for undifferentiated ES cells) expression. Cells were incubated on chambered slides and fixed with 4% paraformaldehyde for 5 minutes. This check was then performed by washing with PBS. The cells were then incubated with alkaline phosphatase substrate (Vector Labe-rates.inc. burlingame.ca) for 1 hour at room temperature in the dark. The slides were rinsed with 100% ethanol for 2-5 minutes and then mounted.

FIG. 1 shows marker expression on hES cells detected by the histochemical procedure. The hES colonies expressed SSEA-4, Tra-1-60, Tra-1-81 and alkaline phosphatase as seen on the feeder layer, but not as expressed by the differentiated cells between colonies.

Expression of undifferentiated cell markers was detected by reverse transcriptase PCR amplification. For the relative quantification of the respective gene products by radioactivity, Quantum RNA was used according to the manufacturer's instructions^TMAlternate18S internal standard primers (ambion. Briefly, the linear range of amplification for a particular primer pair was examined and then amplified using an Alternate18S primer: co-amplification with an appropriate mixture of competitors (competitors) yielded PCR products with overlapping linear ranges. Subjecting Ampli Tag to^TM(Roche) before addition to the PCR reaction, the enzyme was reacted with TagStart^TMAntibodies (ProMega) were incubated together according to the manufacturer's instructions. Radioactive PCR reactions were analyzed on 5% non-denaturing polyacrylamide gels, dried and exposed to a phosphorus imaging screen (Molecular Dynamisc) for 12 hours, scanned with Molecular dynamics Strom860 and ImageQuant^TMThe software quantitatively determined the band intensity. Results are expressed as the ratio of radioactivity incorporated in the hERT or OCT-4 bands, normalized to the radioactivity incorporated in the 18S band. The primer sequence for this experiment can be found in international patent publication WO 01/51616.

Transcription factor OCT-4 is normally expressed in undifferentiated hES cell and is down-regulated during differentiation and maintained in conditioned mediumThe cells expressed hERT or OCT-4. Telomerase activity was measured by the TRAP assay (Kim et al, Science 266: 2011, 1977; Weinrich et al, Nature Genetics 17: 498, 1997). Cells maintained in a feeder cells-free culture environment showed telomerase activity after 180 days of culture.

Pluripotency of undifferentiated cells in feeder cells-free culture, as measured by formation of embryoid bodies by suspension culture for 4 days followed by culture on poly-ornithine coated plates for 7 days, immunocytochemistry showed staining patterns consistent with neuronal and cardiomyocyte lineage cells and positive staining for cellular alpha-fetoprotein, a marker of endoderm. Undifferentiated cells were also tested for their ability to produce telomerase by intramuscular injection of SCID mice. The resulting tumors were excised 78-84 days later and all three germ layer-derived cell types were identified by histological analysis.

Example 2: differentiation of hES cells into cardiomyocytes

The hES cell line: h1, H7, H9 and H9.2(H9 derived clonal lines) were maintained on feeder cells and then cultured under feeder cell-free conditions as described in example 1. Incubating at 37 deg.C with 200U/ml collagenase IV for 5-10 minutes per week, and then 9-17 ten thousand cells/cm at a ratio of 1: 3-1: 6²Is inoculated inCulture passages were performed on coated plates and maintained in conditioned medium produced by primary mouse embryonic fibroblasts.

Figure 2 (top panel) shows the time schedule for differentiating hES cells into cardiomyocytes. Culturing the suspended hES cells to initiate differentiation into embryoid bodies, dissociating the hES cells into small pieces by incubation with 1mg/ml collagenase IV at 37 ℃ for 5-10 minutes, and then culturing in a differentiation medium to form aggregates. The differentiation medium contained 80% knock-out DMEM (KO-DMEM) (Gibco, BRL, Rockville MD)1 mML-glutamine, 0.1% mM beta-thioglycol and 1% stock solution of non-essential amino acids (Bibco BRL Rockvi l.MD) supplemented with 20% fetal bovine serum.

After 4 days of suspension culture, embryoid bodies were transferred onto gelatin-coated plates or chambered slides, EBs were attached to the surface after inoculation, proliferated and differentiated into heterogeneous cell populations, and spontaneously contracted cells were observed in each region of the culture on day 8 of differentiation.

FIG. 2 (lower panel) shows that the rate of seeding with embryoid bodies containing beating cells increases as the cells differentiate. Contractile cells were visible in the culture up to 32 days.

On days 11-14 of differentiation, the beating cardiomyocytes were mechanically isolated from the growing EBs, collected in 15ml tubes containing low calcium medium or PBS, and washed. Different reagents including trypsin, EDTA, collagenase IV or B were tested for their ability to produce a single cell suspension of viable cardiomyocytes. The cells were incubated with collagenase B solution for 60-120 min at 37 deg.C (depending on collagenase activity) to obtain single viable contractile cardiomyocytes, resuspended in KB medium (85mM KCl, 30mM K)₂HPO₄5mM MgSO₄1mM EGTA, 5mM creatine, 20mM glucose, 2mM Na₂ATP, 5mM pyruvate and 20mM taurine, buffered to ph7.2) (Maltsev et al, circ.res, 75: 233, 1994). The cells were cultured in this culture medium at 37 ℃ for 15 to 30 minutes, dissociated and then seeded on a glass slide with a chamber to perform differentiation culture. Single cardiomyocytes survived and continuously jumped after subculture.

All hES cell lines, including H1, H7, H9, H9.1, and H9.2, were tested for their ability to produce beating cardiomyocytes even after 50 passages of maintenance (approximately 260 diploid population).

Example 3: characterization of cardiomyocytes

The hES-derived cells prepared in example 2 were analyzed for the presence of phenotypic markers characteristic of cardiomyocytes.

Immunostaining of EB growth cultures or dissociated cardiomyocytes was performed as follows: the differentiation cultures were fixed with methanol/acetone (3: 1) -20 ℃ for 20 min, the cells were washed 2 times with PBS, blocked with 5% normal sheep serum (NGS) PBS overnight, and then incubated with primary antibody (diluted 1: 20-1: 800 in primary antibody dilution buffer (Biomeda Corp., FoterCity CA)) at room temperature for 30-60Minute, rewashing cells, staining with DAP1, and staining with Vectashi led^TM(Vector classifications Inc., Burl ingeme CA). In Nikonlabphot^TMOptical microscopy was performed on (equipped with epi-fluorescence and SPOT CCD anti-freeze camera).

On day 15, each contractile center in H9.2 cell differentiation cultures was photographed, the contractile regions were recorded, and the cultures were fixed and stained for cardiac troponin (cTnI) to match the optical micrographs to determine the percentage of contractile regions stained positive for cTnI. The contractile region stained positive for 100% cTnI, while little staining was seen in the non-beating cells.

The immuno-printing of cTnI expression was performed as follows: undifferentiated and differentiated cells were lysed with lysis buffer, separated in 10% SDS-PAGE, and transferred to nitrocellulose membrane (Schleichef)&Schull), room temperature with a solution containing 0.05% Tween^TMThe membrane was blocked with 5% skim milk in 20PBS (PBST) for 1 hour. This membrane was then incubated with horse anti mouse IgG (H + L) antibody conjugated to horseradish peroxidase (vector laboratories Inc., Burlingame CA) diluted 1: 8000 with PBST-conjugated 1% skim milk for 1.5 hours at room temperature. SuperSignal for antibody binding^TMWest Pilo chemical system (Pierce, Rockford. TN). As a control, β -actin on the same membrane was probed as follows: ECL assay followed by PBS washing of the membrane and Vector exposure^TMthe-SG plates were taken for about 5 minutes (Vector Laboratories Inc) and then examined with anti-beta-actin (Sigma) monoclonal antibodies.

FIG. 3 (top panel) shows the results of the immunostaining assay. The wells containing the contractile cells (lanes 2 and 3) had a band of approximately 31kDa (molecular weight corresponding to human cTnI), but the wells containing undifferentiated hES cells (lane 1) or no contractile cells (lane 4) had no band. All lanes present β -actin (standard for protein recovery) staining.

Real-time reverse transcription PCR was performed using a LightCycler, and for relative quantitation of α MHC, RNA samples and primers were mixed with RT-PCR reaction mixtures (LightCycler RNA amplification Rit-hybridization Probes, Roche Molecular Biochemicals) according to kit instructions. The reaction conditions were as follows: reverse transcription at 55 ℃ for 10 min, denaturation at 95 ℃ for 30 sec, amplification for 45 cycles: 95 ℃ for 10 seconds, 60 ℃ for 15 seconds, 72 ℃ for 13 seconds. The reactions were analyzed using the LightCycler3 program and the relative MHC levels were expressed as the ratio of MHC and 28S for each sample in triplicate.

Fig. 3 (lower panel) shows the results that the α MHC level was significantly increased after 7 days of differentiation, but could not be detected in undifferentiated hES cells or early differentiated cells. Expression levels thereafter increased continuously, consistent with the appearance of beating cells. Decreased hTERT expression was found during differentiation.

hES-derived cardiomyocytes were isolated as single cells with collagenase β as described in the examples, and the isolated cardiomyocytes were examined for expression of the myofibrillar Myosin Heavy Chain (MHC), titin, tropomyosin, α -activin, binding protein, cTnI, and cardiac troponin t (ctnt).

FIG. 4 shows the results, single cells and colonies all staining positive for these markers, single cardiomyocytes stained in spindle, round and triangular or polygonal shapes. The striated nature of the myofibrillar structures is seen, consistent with the contractile apparatus necessary for muscle function.

GATA-4 is a highly expressed transcription factor in the embryonic layer of the heart, and a strong GATA-4 immune response was observed in the nuclei of all cTnI positive cells. Immunostaining revealed that GATA-4 was strongly expressed in differentiated hES cells containing contractile cells (FIG. 1, lanes 2 and 3), but not in differentiated cultures without contractile cells (FIG. 1, lane 4). A weak signal was also detected in undifferentiated cells (lane 1), possibly due to spontaneous differentiation into visceral endoderm, which also expressed GATA-4, or due to low levels of GATA-4 expressed by undifferentiated cells.

Immunocytochemistry detected MEF2 cardiac transcription factor in the nuclei of all cTnI-positive cells. Semi-quantitative RT-PCR of the cardiac transcription factor NKx2.5 (Xu et al, Dev Biol, 196: 237, 1998) showed high expression in cultures containing beating cardiomyocytes, but not detectable in undifferentiated cells. The adhesion marker N-cadherin and gap junction marker connexin 43 were detectable in cardiomyocytes identified as either cTnI or MHC expression, but not in peripheral non-cardiomyocytes. Furthermore, we stained partially dissociated cells with anti- β 1-adrenoceptor (. beta.1-AR) and cTnI antibodies. Specific staining of surface markers indicates that these cells can be further enriched by sorting techniques based on these markers.

Creatine kinase MB (CK-MB) and myosin can also be detected by immuno-technical staining of hES derived cardiomyocytes co-stained with MHC. CK-MB is thought to be responsible for high-energy storage and is only present in muscle cell lines. Myosin is a cytosolic oxygen binding protein responsible for oxygen storage and diffusion in muscle cells, and both CK-MB and myosin can be used to diagnose acute myocardial infarction. A strong immune response to the β 1 adrenergic receptor (. beta.1-AR) was observed on the cTnI positive cell membrane.

Atrial Natriuretic Factor (ANF) is up-regulated during differentiation of hES cells and can be detected by semi-quantitative RT-PCR. Double staining of 18% of cTnI positive cells with Ki-67, a protein present in actively dividing cells but absent in quiescent G0 cells, indicated that the cells still were proliferative.

Taken together, these data indicate that hES-derived cardiomyocytes have an appropriate gene expression pattern consistent with the early (embryonic) cardiomyocyte phenotype.

EXAMPLE 4 enrichment of cardiomyocytes by Density centrifugation

Percoll in discontinuous gradient^TM(a dense separation medium containing colloidal PVP coated silica) to further enrich cardiomyocytes. Cardiomyocytes were generated by inducing differentiation of suspended hES cells for 4 days and then on clear detachment risk coated plates for 15 days. The cells were dissociated with collagenase B37 ℃ for 2 hours, and the washed cells were resuspended in differentiation medium. Standing for 5 min, and pulverizingThe cell suspension was added to 40.5% Percoll^TM(Pharmacia about 1.05g/ml) on a layer of 58.5% Percoll^TM(about 1.075 g/ml). The cells were then centrifuged at 1500g for 30 minutes. After centrifugation Percoll was collected^TMThe above cells (fraction 1) and two layers of Percoll^TMA layer of cells in the interface (component 2). The collected cells were washed and resuspended in differentiation medium 10 cells per well⁴Individual cells were seeded on chambered slides.

After one week, cells were fixed and stained for Myosin Heavy Chain (MHC) expression (example 3) and the percentage of MHC positive cells was determined by counting cells in 30 images per group of triplicate wells, expressed as mean ± standard deviation of triplicate wells. Beating cells were seen in both fractions, but fraction 2 contained more. The results are shown in Table 1. The enrichment in fraction 2 was at least 20-fold higher than that of the starting cell population.

Example 5: pharmacological reactions

The function of hES-derived cells was determined by determining whether cardiomyocytes responded appropriately to the chronotropic effect of cardioactive drugs.

Study of pharmacological responses

EB cells were seeded on gelatin-coated 24-well plates to differentiate as described in example 2. The pharmacological response was examined using contractile cardiomyocytes at day 15 of differentiation. Spontaneous beating frequency was determined by counting the rate of contraction of the beating area in culture medium maintained during the tillering phase in an inverted microscope chamber heated at 37 ℃. The cells and test compounds were then incubated in an incubator for 20-30 minutes and the rate of contraction observed. The dose-dependent effect was determined by gradually adding increasing concentrations of each substance. Data are presented as mean beat rate ± standard deviation measured over 10-20 beat regions.

To demonstrate that these cells are able to express functional L-type calcium channels that play a key role in myocardial contraction, we examined the effect of the L-type calcium channel blocker diltiazem hydrochloride on the beating of hES-derived cardiomyocytes. Differentiated cells were incubated with different concentrations of this drug and the number of beats per minute was calculated. The cells were washed with the culture medium, maintained in the differentiation medium for 24 hours, and the time to recover the contractile force was observed.

FIG. 5(A) shows that diltiazem hydrochloride inhibits pulsatile velocity in a concentration-dependent manner. When using 10^-5When cells were treated with diltiazem hydrochloride, 100% of the pulse zone stopped the contraction. Contraction was returned to normal levels 24-48 hours after removal of the drug. Fisher's PLSD test determined that P < 0.05, P < 0.005, and P < 0.0005 was statistically significant. This observation indicates that hES-derived cardiomyocytes have functional L calcium channels. In a separate experiment, clenbuterol was found to increase the beating rate of 72 days cells from 72 beats per minute to 98 beats per minute (1-10nM, P < 0.005).

FIGS. 5B and 5C show positive chronotropic effects induced by isoproterenol (a beta-adrenoceptor agonist) and phenylephrine (an alpha-adrenoceptor agonist). Figures 5D and 5E show that phosphodiesterase inhibition IBMX and clenbuterol, a β 2-adrenoceptor agonist, have similar effects. Thus hES-derived cells can respond to cardioactive drugs in a manner consistent with that of cardiomyocyte lineage cells.

Example 6: centripetal factor as differentiation inducer

Differentiation for 1-4 days, 4-6 days, or 6-8 days with 5-azadeoxycytidine (a cytosine analog that affects DNA methylation, thereby activating gene expression)Becoming embryoid-like alpha MHC. Tagman^TM7700 the sequencing system used the RT-PCR assay of example 3 with the same primers for 40 cycles of amplification 9515 min at 60 ℃ for 1 min. By using Tagman^TMRibosomal RNA control reagent kit (Applied Biosystems) sugar-increased 18S ribosomal RNA was used as a control. With AB1Prism^TM7700 the sequencing system analyzes the reaction.

Figure 6 shows the results of using 5-azadeoxycytidine as a differentiation inducer (mean ± S. determination of the ratio of α MHC to 18S RNA in triplicate). The data show that on days 6-8, 1-10. mu.M 5-azadeoxycytosine significantly increased myocardial alpha MHC expression with an increased rate of beating zones in the culture.

Other agents formulated to induce cardiomyocyte differentiation were examined, including methine (DMSO) and all Retinoic Acid (RA) formulas. Treatment of embryonic-like bodies for days 0-4 with 0.5% DMSO produced less pulsatile areas than untreated cultures, cultures treated with 0.8% or 1% DMSO lacked pulsatile cells, and 1.5% DMSO was actually toxic to cells. DMSO treatment also caused a significant decrease in alpha MHC expression compared to no treatment.

At 10^-9And 10^-5Dose between M Retinoic acid was added to differentiated hES cultures, RA was cytotoxic to cells at days 0-4, while beating cells did not increase on days 4-8, 8-15, or 4-15 compared to untreated cultures.

Thus 5-azadeoxycytidine is a potent cardiomyocyte differentiation inducer, increasing the cardiomyocyte ratio in a population of cells. In contrast, DMSO and retinoic acid inhibit cardiomyocyte differentiation, even though these compounds are capable of producing cardiomyocytes from embryonic cancers or embryonic stem cells (Wobus et al, J.mol.cell.Cardiol.29: 1525, 1997; MeBurney et al, Nature 299: 165, 1982)

Cardiomyocyte differentiation was also achieved in the direct differentiation protocol. Dissociated H7 line undifferentiated hES cells were seeded directly onto gelatin coating without going through the embryoid body phase. The seeded cells were cultured in differentiation medium (80% KO-DMEM, 1 mML-glutamine, 0.1mM β -mercaptoethanol, 1% amino acids and 20% fetal bovine serum). Contractile cardiomyocytes were found in cultures treated with 10 μ M5-azadeoxycytidine on day 14, and in all cultures on days 10-12 or 12-14 and later.

Example 7: effective combination of centripetal factors

This example is a study of the combined effect of added growth factors and 5-azadeoxycytidine on the differentiation of human ES cells into cardiomyocytes.

The human ES cell line, designated H1, typically produces fewer pulsatile cardiomyocytes following standard embryoid body procedures than the H4 and H9 lines. To increase cardiomyocyte production, a series of growth factors and 5-azadeoxycytidine were added to the differentiating H1 cultures.

The principle is as follows: the first group of factors were screened because they can provide endoderm function in the initial typing. Group 2 was screened for ability to provide endoderm function in combination with group 1 factors during subsequent development. The third group of factors was screened because they are survival factors for prolonged culture of cardiomyocytes. Typical working concentrations are defined as "medium" levels, 4-fold lower and 4-fold higher as "low" and "high" levels. The concentrations are shown below

TABLE 2 exemplary centripetal factors

FIG. 7 (top panel) shows a schedule using these factors. H1 cells from passage 48 were collagenase treated and mechanically scraped with a 5ml pipette, and H1 cells were removed from the plate for the production of embryoid bodies. One well 10 cm square of cells was transferred to one 10 cm square well in a low adhesion plate and cultured in 4ml of DMEM with 20% FBS (with or without other factors) for 4 days. Each embryoid body suspension was aliquoted 4 days later into 2 wells of gelatin-coated adherent 6-well tissue culture plates. Adherent embryoid bodies and their growth were cultured in 4ml of DMEM with 20% FBS (with or without other factors) for 11 days. After observing the number of beating zones in each well with an optical microscope, RNA from each well was harvested for quantitative PCR analysis.

The first set of factors was added on day 0 (the day that undifferentiated cells were engrafted in suspension culture to produce embryoid bodies) until day 8 (4 days after embryoid body seeding into gelatin coated wells). Group 2 factors were added on day 4 (time of inoculation) until day 8. Group 3 factors were added on day 8 until the end of the experiment (day 15). A portion of the culture was exposed to 5-azadeoxycytidine for 48 hours (days 6-8), 6.8.11 and 13 days, and the culture was fed with fresh medium with or without addition of factors.

It was found that no pulsatile zone was observed in the control cultures (maintained in the absence of added growth factor/5-azadeoxycytidine) or those maintained with added growth factor but lacking 5-azadeoxycytidine, but pulsatile zones were observed in all wells with the addition of the growth factor and 5-azadeoxycytidine mixture.

FIG. 7 (lower panel) shows quantitative PCR analysis (Tagman)^TM) The expression of the cardiac gene α myosin heavy chain (α MHC) corresponds to the level of normal cardiac RNA. The expression levels were much higher in cells exposed to Growth Factor (GF) plus 5-azadeoxycytidine, with the lowest concentration tested being sufficient to obtain higher alpha MHC expression (30-fold higher than control).

Subsequent experiments detail these results. H1 cells (passage 38) were cultured as described above, except that: a) the expression level of the marker relative to undifferentiated cells was determined in a real-time PCR assay using only the lowest concentration of growth factor used in the previous experiment and b) deleting the factor group 3 treatment in one set of samples.

FIG. 8 shows that deletion of group 3 factor treatment from the protocol results in a further 3-fold increase in RNA expression of α MHC, and an increase in expression of the early cardiomyocyte-associated gene GATA-4 was also detected. In contrast, no endoderm-related gene HNF3b was specifically induced under these conditions. The effect on α MHC and GATA-4 was selective compared to the endoderm related gene HNV3 b. While the expression of HNF3b was increased with any combination of growth factors but without 5-azadeoxycytosine.

These results indicate that group 1 and group 2 growth factors increase the proportion of beating cells characteristic of cardiomyocytes.

Example 8: culturing in culture medium containing enrichment factor

hES cells of the H9 line were differentiated by forming embryoid bodies in suspension for 5 days and cultured in differentiation mediumThe coated plates were further differentiated for 12 days. The cells were dissociated with PBS containing 200U/ml collagenase II (Worthington), 0.2% trypsin (Invine Scientific) and 0.02% glucose, and seeded in differentiation mediumCoated on plates and cultured for 14 days.

Then the cells were transferred to a cell containing 10^-7M insulin (Sigma), 0.2% bovine albumin (Sigma), 5mM creatine (Sigma), 2mM carnitine and 5mM taurine (Sigma) in "CCT" medium (supplemented with Gibco medium 199). See Volz et al, j.mol.cell cardio.23: 161, 1991 and Li et al, j.tiss. cult.meth.15: 147, 1993. For comparison, control cultures were maintained in standard differentiation medium containing 20% FBS.

Figure 9 shows the number of beating zones after transfer into CCT broth (separate lines indicate observations made after separation of wells during the study). Cells in CCT medium showed an increase in the number of beating zones after 7-14 days of growth, indicating that creatine, carnitine and taurine, alone or in combination, increased the proportion of cardiomyocyte lineage cells in culture.

Example 9: four-phase centrifugal separation method

hES cells from the H7 line produced cardiomyocytes by forming embryoid bodies on day 4 and then proliferating on gelatin coated plates (without 5-azadeoxycytidine and growth factors). Dissociated cells with collagenase B are resuspended in differentiation medium and the cell suspension is then plated onto a discontinuous gradient of Percoll^TMAt the top, 1500g were centrifuged for 30 minutes. 4 fractions were collected: I. an upper interface layer; II.40.5% of a layer; III, a lower interface layer; iv.58.5% layer. Cells were washed and resuspended in differentiation medium. Immunostained cells were seeded on glass slides with chambers, 10000 cells per well, cultured for 2 or 7 days, and then fixed and stained.

The results are shown in Table 3. The percentage of MHC positive cells in 30 images of each triplicate well was calculated and expressed as mean ± standard deviation of 3 wells of cells.

Beating cells were observed in all fractions, but fractions III and IV contained the highest percentage of beating cells.

FIG. 10 shows the results of a similar procedure using hES cells of line H1. Percoll was used 22 days after differentiation^TMThe cells are isolated. Real-time RT-PCR analysis detected myocardial MHC levels much higher than cells before isolation. The data show that with 18S RNA as the standard, components III and IV have the highest level of MHC expression in terms of total transcription ratio.

Table 4 shows the cell phenotype of the indirect immunocytochemistry assay.

The cardiomyocyte population isolated by density gradient centrifugation was identified by cTnI and MHC staining. Myogenin, alpha fetoprotein, or beta tubulin III did not stain indicating a lack of skeletal muscle, endodermal cells, and neurons. The absence of SSEA-4 and Tra-1-81 staining confirmed the absence of undifferentiated hES cells.

alpha-Smooth Muscle Actin (SMA) has been reported to be present in embryonic-like and embryonic cardiomyocytes, but not in mature cardiomyocytes (Leor et al, Circulation 97: 1332, 1996; Etzion et al, mol.cell.Cardiol.33: 1321, 2001). Virtually all cTnI-positive and a subset of cTnI-negative cells obtained by this cardiomyocyte differentiation protocol were SMA positive, suggesting that they may be in the early stage and capable of proliferation.

When the cells of fractions III and IV were cultured for an additional 2 days, 43. + -. 4% of sMHC positive cells expressed BrdU, indicating that they were in the S phase of the cell cycle. In other experiments, a subset of cTnI-positive cells were found to express Ki 67. These results indicate that approximately 20% or 40% of the cardiomyocytes in this cell population are undergoing active proliferation.

Those skilled in the art can effect modifications to the compositions and methods provided herein without departing from the spirit of the invention as set forth in the following claims.

Claims (4)

1. A method of producing a cellular composition comprising human cardiomyocytes or cardiomyocyte precursor cells, the method comprising:

a) culturing human embryonic stem cells from an established cell line in a substantially feeder cells-free environment comprising an extracellular matrix and a fibroblast conditioned medium;

b) forming the human embryonic stem cells of step a) into embryoid bodies;

c) differentiating the cultured cells into cardiomyocytes or cardiomyocyte precursor cells using a centripetal factor, said centripetal factor being 5-azadeoxycytidine.

2. The method of claim 1, further comprising culturing the cells in a culture medium comprising creatine, carnitine, and taurine for at least one week.

3. A method of enriching for a plurality of cardiomyocytes derived from human embryonic stem cells derived from an established cell line, the method comprising:

a) passing human embryonic stem cells from an established cell line through the method of claim 1 to produce a cell composition comprising human cardiomyocytes;

b) centrifugation with density gradient increased the percentage of cardiomyocytes.

4. The method of claim 3, wherein the density gradient centrifugation is performed with a Percoll gradient.