JP2002507407A - Heart-derived stem cells - Google Patents

Abstract

(57) Abstract: The present invention relates to a heart capable of producing various cell types including cardiac cells, fibroblasts, smooth muscle cells, skeletal muscle cells, keratinocytes, osteoblasts and chondrocytes during proliferation and differentiation. Derived pluripotent stem cells are provided. The cells can be used in methods of treating patients suffering from cardiac cell necrosis. Stem cells proliferate and differentiate to produce heart cells that replace necrotic tissue. Cells can also be used to screen compounds for activity in promoting the growth and / or differentiation of heart-derived stem cells.

Description

DETAILED DESCRIPTION OF THE INVENTION

TECHNICAL FIELD [0001] The present invention belongs to the technical fields of cell biology, drug supply and medicine.

BACKGROUND OF THE INVENTION [0002] In some tissues, the terminal state of cell differentiation is incompatible with cell division. For example, in outer skin cells the cell nucleus is degraded, in erythrocytes the nucleus is absent, and in myocytes myofibril prevents mitosis and cytokinesis. Develop, regenerate and repair (repai)
r) The ability of such cells and tissues to undergo additional differentiated progeny (addit) during division.
ional differentiated pregeny cells).

[0003] Stem cells do not terminally differentiate and can divide endlessly, giving rise to progeny, which can continue to divide or differentiate. Stem cells can be totipopent, pluripotent or unipotent. Totipotent stem cells (eg, embryonic stem cells) can give rise to all cell types in a mature organism. Pluripotent stem cells can give rise to more than one differentiated cell type. Differentiated pluripotent stem cells can give rise to unidifferentiated cell types. Stem cells are generally characterized by small size, low granularity, low cytoplasm to nuclear ratio, and no expression of osteoponoutin, collagen and alkaline phosphatase.

[0004] The presence of stem cells has been well documented for the epidermis, small intestinal epithelium and the hematopoietic system.
Hematopoietic cells are present in the circulation as well as in the bone marrow. Circulating hematopoietic cells can colonize organs, such as the spleen. Stem cells for bone, cartilage, fat and cells of three types of muscle (smooth, skeletal and cardiac) are common mesenchymal stem cell precursors (common m).
esenchymal stem cell precursors, but also mesenchynal stem cells or additional committed tissue-specific ancestors (committed t
issue specific proglniton) (Owen et al., Ciba Fdn
Symp. 136, 42-46, 1988; Owen et al., J. Cell Sci. 87, 731-738 (1987)).

[0005] Zohar, Blood 90, 3471-3481 (1997) describes the tetal rat perioste
um), and attempts to identify ancestors of osteogenic cells by screening for differentiation expression of markers, protein content and cell cycle location. Huss et al., PNAS 92, 748-752 (1995) report the identification of stromal cell precursors from dog bone marrow cultures. Progenitors have been reported to differentiate in the presence of growth factors and display mature differentiation markers. Robbin
s et al., Trends Cardiovasc. Med. 2, 44-50 (1992) report that mouse embryonic stem cells can differentiate into embryoid bodies that mimic some aspects of cardiac development.

Definitions An isolated cell is a cell that has been at least partially purified from other types of cells with which it is naturally associated. Often, an isolated cell will have at least
25, 50, 75, 90, 95 or 99% are present in a population that is an isolated cell type. At times, the isolated cells give rise to a cell line in which all cells are essentially identical, except for spontaneous mutations that can occur upon expansion of the cell line. At times, the isolated cells undergo spontaneous differentiation, producing a mixed population of mature cell types.

[0007] A series of differentiation markers can be identified and are specific for particular cell types.
Means one or more phenotypic characteristics. Differentiation markers are displayed transiently at various stages of the cell lineage. Pluripotent stem cells that can regenerate without commitment to lineage express a series of differentiation markers that can be lost if commitment to cell lineage is achieved. Progenitor cells express a series of differentiation markers that may or may not continue to be expressed as the cells descend the cell lineage pathway to maturation. Differentiation markers that are exclusively expressed by mature cells usually exhibit such cell products, enzymes for producing the cell products, as well as functional properties such as receptors.

[0008] The ability of cells to adhere in culture is a marker of progression from an undifferentiated state to a differentiated state. The adherent cells form a monolayer on the plastic support. Cell lineage refers to the differentiated cell type and the ancestral cell type from which the differentiated cell type was obtained. For example, cardiomyocytes and myoblasts are two cell types of the same lineage.

SUMMARY OF THE INVENTION [0009] In one aspect, the invention provides an isolated nonadherent pluripotent candiac-derined hcemm stem cell. During (and after) proliferation and differentiation, such cells are derived from adherent cardiac-derinel stem cells, fibroblasts, smooth muscle cells, skeletal muscle cells, skeletal umscle cell), cardiocyte, keratinocyte, osteoblast
And progeny cells comprising a cell type selected from the group consisting of chondrocytes.

[0010] Some such stem cells generate all cell types selected from the group. The non-adherent heart-derived stem cells of the present invention can be obtained by growing a population of cardiac tissue-derived cells in a liquid medium on a support, discarding cells from the population adhering to the support, and suspending non-adherent heart-derived stem cells. It can be produced by leaving the liquid.

[0011] The present invention further provides isolated non-adherent pluripotent heart-derived stem cells that upon proliferation and differentiation produce cardiac cells and progeny cells including chondrocytes or keratinocytes. Some such stem cells, when proliferating and differentiating, further comprise at least one cell type selected from the group consisting of adherent heart-derived stem subpopulations, fibroblasts, smooth muscle cells and skeletal muscle cells. Produce cells. Such stem cells can be, for example, human or mouse stem cells. Optionally, mouse stem cells are obtained from p53-deficient mice.

The invention further comprises a cell type proliferating and differentiating selected from the group consisting of fibroblasts, smooth muscle cells, skeletal muscle cells, heart cells, chondrocytes, keratinocytes and osteoblasts. Provided are isolated adherent human heart-derived stem cells that produce progeny cells. The invention further provides an isolated adherent heart-derived stem cell that proliferates and differentiates to produce cardiac cells and progeny cells comprising chondrocytes or keratinocytes.

[0013] In another aspect, the present invention provides a method for preparing an isolated non-adherent heart-derived stem cell. Such a method involves centrifuging a suspension of cells from a subject's heart tissue on a density gradient, isolating a band of cells, including myocytes, and leaving adherent heart cells dying or leaving suspension cells. Growing the cells until they are discarded, and culturing the suspended cells until a population of non-adherent heart cells is detectable.

The present invention also provides a method for preparing adherent heart-derived stem cells. Such a method
Start with a non-adherent heart-derived stem cell as described above. Stem cells are expanded until adherent progeny cells appear. From the group consisting of myoblasts, smooth muscle cells, skeletal muscle cells, heart cells, osteoblasts, keratinocytes and chondrocytes, which upon proliferation and differentiation produce progeny cells that include at least one cell type from the group. Adherent cells that lack markers for the selected cells are identified.

The present invention further provides a method for preparing heart cells. Some such methods provide a non-adherent heart-derived stem cell as described above, wherein the cell is grown under conditions in which the cell proliferates and differentiates to produce progeny cells, including adherent cells, and Identifying the adherent cells using differentiation markers characteristic of cardiac cells. Alternatively, such a method may be initiated using a non-adherent heart-derived stem cell as described above. The cells are grown under conditions in which the cells proliferate and differentiate to produce adherent progeny cells. Adherent cells can then be identified using differentiation markers characteristic of cardiac cells.

[0016] In another aspect, the method provides an isolated population of cells comprising smooth muscle cells, skeletal muscle cells, heart cells, fibroblasts, keratinocytes, osteoblasts and chondrocytes. In another aspect, the method provides a method of treating a patient suffering from necrotic heart disease. Such a method implies that an effective dose of such non-adherent or adherent stem cells is administered to the patient, whereby the stem cells proliferate and differentiate to produce cardiac cells, which displace necrotic tissue. .

[0017] In some such methods, non-adherent stem cells are administered intravenously. In some methods, fibroblast growth factor is also administered to the patient to stimulate the proliferation and / or differentiation of non-adherent cells. In some methods, stem cell factor is administered to a patient to stimulate non-adherent cell differentiation into heart cells. In some methods, the patient has a congestive heart defect. In some methods, stem cells are obtained from a patient's blood and expanded in vitro before re-administration to the patient.

In another aspect, the present invention provides a method of screening for a potential agent for activity in promoting proliferation and / or differentiation of heart-derived stem cells. Such methods involve growing non-adherent or adherent heart-derived stem cells in the presence of a potential agent and monitoring changes in progeny cell differentiation status with respect to non-adherent or adherent heart-derived stem cells. Implies. In some such methods, the change in differentiation status is the attachment of progeny cells. In some methods, changes in differentiation status are monitored by detecting the appearance of cardiac cells. In some methods, the appearance of adherent heart-derived stem cells is monitored.

DETAILED DESCRIPTION OF THE INVENTION Overview The present invention provides at least two types of pluripotent heart-derived stem cells. One type of cell is non-adherent and the other is adherent. Adherent cells are partially differentiated progeny of non-adherent cells. Both types of stem cells can be expanded and differentiated into various differentiated cell types.

[0020] Heart-derived stem cells can be isolated from humans and other vertebrates. The cells are
It is typically isolated from a fraction of a suspension of heart-derived cells that has been discarded as debris by conventional workers in the art. Muscle cell bands are typically harvested from density gradients and cells are grown in media on a support. Conventional workers have left the cells attached to the support and discarded it from the false view that the medium contained only debris. The present invention retained the medium and discarded the adherent cells. After the medium is increased, a population of non-adherent suspension cells becomes apparent.

Cells have several uses in therapy and drug discovery. In therapeutic applications,
The cells are administered to a patient suffering from cardiac deficiency, eg, tissue necrosis due to myocardial infarction. Administered stem cells clone the patient's heart to produce cardiomyocyte progenitor cells that replace necrotic tissue and / or supplement pre-existing cardiac tissue.

In drug discovery applications, stem cells are used to screen compounds for activity that promotes or inhibits differentiation of stem cells into cardiac cells or other mature differentiated cell types. Compounds that promote the differentiation of stem cells into heart cells, optionally together with the stem cells of the present invention, can be used for the treatment of patients with cardiac deficiency. Other compounds find use in other therapeutic applications where promoting or inhibiting stem cell differentiation is desirable.

Another use for the stem cells of the present invention is in screening for compounds that expand stem cell populations in culture. Some such compounds induce differentiation, others do not. Another therapeutic use for the cells of the present invention is to provide a method of screening for compounds that promote the recruitment of heart-derived stem cells from the heart to the circulatory system.

In vivo assays for assessing cardiac neogenesis include treating newborn and adult animals with the cells of the invention. Animal heart function includes heart rate, blood pressure, L
Measured as V pressure-heart rate product (tdp / dt) and cardiac output to establish left ventricular function. Post-mortem methods for assessing cardiac improvement include the following:
Staining of heart tissue sections to determine increased heart weight, nuclear / cytoplasmic volume, proliferating cell nuclear antigen (PCNA) versus cytoplasmic effect level (Quaini et al., Circulation Res. 75: 1)
050-1063, 1994 and Reiss et al., Proc. Natl. Acad. Sci. 93: 8630-8635, 1
996).

The heart-derived mesenchymal stem cells of the present invention may be used for the treatment of heart diseases, ie myocardial infarction, coronary artery disease, congestive heart failure, hypertrophic cardiomyopathy, myocarditis, congenital heart disease and disorders associated with dilated cardiomyopathy. Can be used for therapy. The cells of the invention improve cardiac function by inducing cardiomyocyte neogenesis and / or hyperplasia, by inducing coronary collateral collateral formation, or by inducing remodeling of the necrotic myocardial region. Useful for

The cells of the present invention may be used to develop coronary collateral circulation, promote angiogenesis or wound healing after angioplasty or endarterectomy, to regenerate blood in the eye, Useful for complications associated with poor circulation, such as leg ulcers, for beating after coronary reperfusion using pharmacological methods, and for other indications where angiogenesis is beneficial It is.

An ischemic event is a disruption of blood flow to an organ, causing necrosis or infarction of a non-perfused area. Ischemia-reperfusion is the disruption of blood flow to an organ such as the heart or brain, and subsequent restoration of blood flow (often sudden). While restoration of blood flow is essential for preserving functional tissue, reperfusion itself is known to be harmful.

Indeed, reperfusion in the ischemic area jeopardizes endothelium-dependent vasorelaxation, causing vasospasm and, in the heart, coronary vasodilation not observed in ischemic events without reperfusion. There is evidence of exposure (Cuevas et al., Growth Factors
15: 29-40, 1997). Both ischemia and reperfusion are important concerns for tissue necrosis, such as myocardial infarction or stroke. The cells of the invention have therapeutic value for reducing damage to tissues caused by ischemia or ischemia-reperfusion events, especially in the heart or brain.

Other therapeutic uses for the present invention include skeletal myogenesis and / or hyperplasia, cartilage regeneration, bone formation, tendon regeneration, neurogenesis, induction of neogenesis in pancreas and kidney, and / or systemic and pulmonary Treatment of hypertension. Various specialized cell functions, for example, stimulate the differentiation of heart-derived mesenchymal stem cells (MSCs) along the appropriate cell pathway into osteoblasts, chondrocytes, skeletal muscle cells or cells of the fibroblast lineage It can be obtained by: The pathway of differentiation is determined by exposure to growth factors that promote the formation of specific differentiated cells. Some combinations of growth factors and differentiated cell types are disclosed below, and others are known in the art.

Heart-derived MSC-induced coronary collateral development is measured in rabbits, dogs or pigs using a model of chronic coronary artery occlusion (Landu et al., Amer. Heart J. 29:92).
4-931, 1995; Sellke et al., Surgery 120 (2): 182-188, 1996 and Lazarous
et al., 1996, ibid.). Cardiac-derived MSC-induced benefits for the treatment of stroke are tested in vivo in rats by utilizing bilateral carotid occlusion and measuring histological changes as well as maze performance (Gage et al., Neurobiol . Aging 9: 6
45-655, 1988). Cardiac-derived MSC-induced efficacy in hypertension is tested in vivo using idiopathic hypertensive rats (SHR) for systemic hypertension (Marche et al.,
Clin. Exp. Pharmacol. Physiol. Suppl. 1: S114-116, 1995).

II. Cells of the Invention Non-adherent pluripotent heart-derived stem cells These cells are highly refractory to light, have a small size (typically less than 10μ, often less than 5μ), are round, and exhibit slow growth. Features. The cells are further characterized by their ability to grow (ie, self-renew) in a medium containing a carbon source, a nitrogen source, insulin and transferrin. Stem cell factor (commercially available from Amgen), acidic or basic fibroblast growth factor, zFGF-
5. Addition of leukemia inhibitor (commercially available) or increased serum concentration stimulates proliferation.

[0032] The cells are further characterized by differentiating into several cell types. These cell types include cells in various states of differentiation that are more advanced than non-adherent heart-derived stem cells. These cell types include adherent heart-derived stem cells (see below), fibroblasts, myoblasts, smooth muscle cells, skeletal muscle cells, heart cells, chondroblasts, keratinocytes and chondrocytes. Presumably, lipoblasts, adipocytes, osteoblasts and osteocytes are also present in progeny cells. Tendon cells may also be present. The relationship of non-adherent heart-derived stem cells to differentiated progeny cell lines and types is shown in FIG.

[0033] Heart-derived stem cells can be stimulated to differentiate by growth in FBS-supplemented medium. Differentiation can be stimulated by treatment with a growth factor, eg, a stem cell growth factor, and by contact suppression. Higher concentrations of horse serum, while stimulating proliferation, have a tendency to inhibit differentiation. The type of growth factor used to induce differentiation can bias the differentiation into a selected lineage. Retinoic acid, TGF-β, bone morphogenetic protein (BMP), ascorbic acid and β-glycerophosphate induce osteoblastic acidity.

Indomethacin, IBMX (3-isobutyl-1-methylxanthine), insulin and triiodityrosine (T3) induce adipocyte production. aFGF, bFGF,
Vitamin D3, TNF-β and retinoic acid induce muscle cell production. zFGF-5 induces expansion of cardiac cell ancestry, but may also be effective later in the pathway from adherent to cardiac cells.

(2) Adherent Pluripotent Heart-Derived Stem Cells Adherent pluripotent heart-derived stem cells result from the proliferation and partial differentiation of non-adherent stem cells described in the preceding section. The cells are characterized by amorphous and lack the differentiation markers tested to date. Differentiation markers examined include alkaline phosphatase expression, a marker for pericytes, osteoblast precursors and chondrocyte precursors, and alpha-actin expression. The cells can be induced to proliferate and / or differentiate into the same cell type as the non-adherent stem cells. The growth rate and the lineage from which differentiation can be induced can be controlled by medium supplementation with the growth factors described above.

III. Production of Non-adherent Heart-Derived Stem Cells The starting material is a heart or heart biopsy from a human or animal subject. The subject can be an embryo, neonate, infant or adult past the mesodermal stage. If immortalized cells are desired, the starting material is obtained from the heart of a transgenic animal lacking one or both copies of the tumor suppressor gene. Examples of tumor suppressor genes include p5
3, p21 and the retinoblastoma gene. Transgenic mice with homozygous mutations in p53 are commercially available from Taconic Farms or Jackson Labs.

[0037] Methods for treating whole tissues to prepare cell suspensions are described, for example, in Methods in Molec.
ular Biology: Animal Cell Culture, 5 (Pollard et al. eds., Humana Press,
NJ, 1990), which is incorporated herein by reference. Typically, heart tissue is digested with collagenase, trypsin, other proteases and / or DNase to release cells (Polinger, Exp.
Cell Res. 63: 78-82 (1970); Owens et al., J. Natl. Cancer Instit., 53: 2.
61-269, 1974; Milo et al., In Vitro 16: 20-30, 1980; Lasfargues, "Human M
ammary Tumors ", in Kruse et al. (eds) Tissue Culture Methods and Applica
tions (Acacemic Press, NY, 1973); Paul, Cell and Tissue Culture, Church
ill Livingston, Edinburgh, 1975).

Next, the intact cells are separated from the debris by low-speed centrifugation. Fragments collect in the supernatant. The cell pellet is resuspended and the cells are sorted. Separation is based on density gradient,
For example, it can be performed on a Focl or sucrose gradient. The cells form four bands as shown in FIG. 2 and the myocyte band is the third from the top. The muscle cell band is removed and the cells are cultured in complete medium. Such a medium contains at least a carbon source, a nitrogen source, essential amino acids, vitamins and minerals, and serum. The medium can be further supplemented with growth factors.

The cells are grown on a plastic support at about 37 ° C. in an atmosphere of 95% O ₂ and 5% CO ₂ . Initially, most cells are adherent muscle cells. However, within 5-30 days, most of the adherent myocytes (eg, at least 75 or 90%) die and small, non-adherent cells appear in suspension at increasing concentrations. The period is shorter for the p53-deficient cells and longer for normal cells. Small non-adherent cells are pluripotent heart-derived stem cells.

IV. Differentiation Markers of Different Cell Types Differentiated cell progeny of stem cells are recognized in part by the presence of differentiation markers. There are three types of muscle cells: heart cells (ie, cardiomyocytes), striated muscle, and smooth muscle cells. 3
Types of cells are derived from a common precursor called myoblasts. Cardiac myosin isozyme expression and creatine kinase isozyme expression in a cardiac-specific pattern
When identified together in the same cell or clonal population of cells, they are markers for cardiomyocytes.

Heart cells can also be recognized by light microscopy by their branch appearance and the ability to form gap junctions. Such cells can be recognized by creating a potential through the fusogenic cells and detecting the movement of the signal through the cells. Muscle α-
Actin mRNA and smooth muscle cell actin are differentiation markers for muscle cells. Myosin isozyme expression and muscle-specific pattern creatine kinase isozyme expression, when identified in a cell or clonal population, are markers for skeletal muscle cells.

[0042] Osteoblasts secrete bone matrix material. ALP, osteocalcin expression, PTH-induced cAMP expression and bone mineralization capacity, identified together in cells or clonal populations of cells, are markers of differentiation for osteoblasts. Cartilage secretes type II cartilage. Alcian blue staining, which detects the production of aggrecan and type IIB collagen, chondroitin sulfate, identified in cells or clonal populations of cells, is a marker for chondrocytes. Keratinocytes secrete keratin and can be recognized using commercially available dyes. Adipocytes produce lipids and can be recognized by Oil Red O staining.

V. Growth Factor Basic FGF (also known as FGF-2) is associated with endothelial cells, vascular smooth muscle cells, fibroblasts, and, generally, of mesodermal or neuroectodermal origin. Cells are mitogenic in vitro for cells such as cardiomyocytes and skeletal muscle cells (Gospod
arowicz et al., J. Cell. Biol. 70: 395-405, 1976; Gospodarowica et al., J
Cell. Biol. 89: 568-578, 1981 and Kardami, J. Mol. Cell. Biochem. 92,
124-134, 1990).

Non-proliferative activities associated with acidic and / or basic FGFs include: increased endothelial release of tissue plasminogen activator, stimulation of extracellular matrix synthesis, chemotaxis to endothelial cells Sex, induction of fetal contractile gene expression in cardiomyocytes (Parker et al., J. Clin. Invest. 85, 507-514, 1990), and enhanced pituitary hormone responsiveness (Baird et al., J. Cellular Physiol. 5, 101-106, 1987).

[0045] zFGF-5 is a type of fibroblast growth factor that is expressed at high levels in human fetal and adult heart tissue. This growth factor is also expressed at reduced levels in fetal lung, skeletal muscle, smooth muscle tissue such as the small intestine, colon and trachea. In the fetal and adult heart
High levels of expression of zFGF-5, and its effects in cells derived from heart tissue,
Have particular efficacy in stimulating the proliferation and / or differentiation of heart-derived stem cells into cardiac cells.

The isolation of zFGF-5 and its properties were described in commonly owned PCT US97 / 186, filed Oct. 16, 1997.
35, the contents of which are incorporated herein by reference. The nucleotide sequence encoding zFGF-5 is set forth in SEQ ID NO: 1, and its deduced amino acid sequence is set forth in SEQ ID NO: 2. The sequence of zFGF-5 shows some sequence similarity with FGF-8.

VI. Cell Immortalization Cells that can be cultured continuously and do not die after a limited number of cells have been produced are called immortalized. Cells that only survive 20-80 population doublings are considered finite (Wiley-Liss, NY, 1994), and cells that survive more than 80, preferably at least 100, are considered immortalized.

Some, but not all, immortalized cells are tumorigenic. As mentioned above,
By using a transgenic animal deficient in a tumor suppressor gene as a donor for heart tissue, cells can be immortalized. Cells from other sources can be immortalized by other means. These measures include transformation and expression by a gene whose product plays a role in cellular aging, or overexpression or mutation of one or more oncogenes that abolish the action of the aging gene. .

[0049] Expression of the gene that produces a positive signal for cell proliferation is determined by the SV40 large T antigen (Linder
et al., Exp. Cell Res. 191, 1-7 (1990)), polyoma large T antigen (Ogris
et al., Oncogene 8, 1277-1283, 1993), adenovirus ElA (Braithwaite et
al., J. Virol. 45, 192-199, 1983), myc oncogene (Khoobyarian et al., Vi.
rus Res. 30, 113-128, 1993) and the E7 gene of type 16 papillomavirus (McDo
ugall, Curr. Top. Microbiol. Immunol. 186: 101-119, 1994).

VII. Treatment Regimens Heart disease is the leading cause of fat in the United States, accounting for up to 30% of total fat. In the United States, more than 5 million people have been diagnosed with coronary artery disease. Coronary artery disease includes congestive heart failure, hypertrophic cardiomyopathy, cardiomyopathy, viral psoriasis or myocardial infarction (
MI). Myocardial infarction accounts for 750,000 hospitalizations annually in the United States. In MI patients, ischemia stimulates fibroblast proliferation and promotes the development of larger than normal fibrous tissue, displacing necrotic muscle tissue. Risk factors for MI include diabetes mellitus, hypertension, trunk obesity, smoking, high levels of low density lipoproteins in plasma or genetic predisposition.

Some proliferation of heart cells apparently occurs in normal aging humans and animals (Olivetti et al., J. Am. Coll. Cardiol. 24 (1), 140-9 (1994) and Anv
ersa et al., Circ. Res. 67, 871-885, 1990), but this is inappropriate for repairing damage due to coronary artery disease. Thus, the necrosis that occurs in MI and other coronary artery diseases is essentially irreversible.

[0052] Such conditions are treated by administration of the heart-derived stem cells as described above. Adherent or non-adherent cells can be used. The cells can be administered intravenously, intracoronarily or intraventricularly. A catheter may be used for the latter two routes of administration, but this is more conventional for adherent cells. The cells are administered at a therapeutically effective dose. Such doses generate a significant number of new cardiac cells in the heart and / or at least partially displace necrotic heart tissue and / or produce clinically significant changes in cardiac function Is enough.

[0053] A clinically significant improvement in cardiac performance was due to a decrease in left ventricular ejection fraction before and after administration of cells.
Can be determined by measuring and increasing at least 5% of the total ejection fraction, preferably
Or more than 10%. Standard method when measuring by ejected blood / pulsation
In addition, it can be used to determine the ejection fraction. Dosage is about 100 ~ 10⁷, 1000-10 ⁶ Or 10^Four~Ten^FiveCan vary from cell to cell.

A cell is a pharmaceutically acceptable, typically sterile, non-toxic carrier or diluent defined as a vehicle commonly used to formulate pharmaceutical compositions for animal or human administration. Can also be administered as a pharmaceutical composition, which can include depending on the desired formulation. The diluent is chosen so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate buffered saline, Ringer's solution, dextrose solution and Hanks' solution. In addition, the pharmaceutical compositions or formulations may also include other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers and the like.

The administration of the stem cells can be done simultaneously, or before, the administration of the growth factor (s) that stimulates the proliferation and / or differentiation of the stem cells to the heart cells. Growth factors can be administered intravenously or intraventricularly. When the growth factor produces a significant number of new cardiac cells in the heart and / or at least partially replaces necrotic heart tissue and / or causes a clinically significant change in cardiac function Can be administered at a dose sufficient to cooperate with the stem cells administered.

[0056] Suitable growth factors include zFGF-5 and stem cell growth factor. In some methods, cardiac-derived stem cells are administered in combination with an anti-inflammatory drug that blocks, reverses, or partially ameliorates inflammation associated with coronary artery disease. Suitable anti-inflammatory drugs include Mac-1 and antibodies to L-, E- and P-selection (Springer, Nature 346, 425-433 (1990); Osborn, Cell 62, 3 (1990). ; Hy
nes, Cell 69, 11 (1992)). Heart-derived stem cells can be further administered with diluents, ACE inhibitors, and β-adrenergic blockers.

In some methods, the stem cell recipient patient and the donor from which the cells are harvested are HLA-matched to reduce allotype rejection. In another method, the cells are administered under an immunosuppressive regimen to reduce the risk of rejection. Immunosuppressants that can be used include cyclosporine, corticosteroids and OKT3. In other methods, the immune response is circumvented by obtaining stem cells from the patient to be treated. Stem cells are obtained by biopsy of heart tissue and can be expanded in vitro before readministration. Alternatively, where provided for the present invention of isolated heart-derived stem cells, differentiation markers are identified for these cells and the cells can be isolated from the blood of the patient to be treated.

VIII. Use of Heart-Derived Stem Cells in Drug Screening The heart-derived stem cells described above can be used to test compounds for activity in promoting or inhibiting cell proliferation and / or differentiation. In general, the compound under test is contacted with a population of cardiac-derived stem cells, optionally in the presence of a metabolic pathway or other agent known to promote or inhibit the phenotype, and the compound to be tested. Phenotypic and metabolic changes are monitored as compared to controls in which no is present.

Compounds to be tested include known or predicted growth factors and analogs thereof, libraries of natural compounds not previously known to have activity in promoting growth or differentiation and live combinations of compounds. Rally. Large combinatorial libraries of compounds include Affymax, WO95 / 12608, Affymax, WO93 / 06121,
Columbia University, WO94 / 08051, Pharmacopeia, WO95 / 35503 and Scripps,
It can be constructed by the coded synthetic library (ESL) method described in WO95 / 30642, the contents of which are incorporated herein by reference. Peptide libraries can also be generated by phage display (see, eg, Devlin, WO91 / 18980).

Compounds that cause cardiac-derived stem cells to proliferate and / or differentiate into cardiac cells are useful as therapeutics under the same conditions where cardiac-derived stem cells are useful. Such compounds can be administered alone to stimulate the proliferation and differentiation of endogenous heart-derived stem cells, or can be administered together with exogenous heart-derived stem cells. Such compounds are screened for proliferative activity by contacting them with heart-derived stem cells in a growth medium and monitoring cell number expansion or ³ H-thymidine incorporation. Compounds are screened for promotion of differentiation into cardiac cells by monitoring cells having a characteristic morphological appearance and differentiation markers as described above.

Similarly, compounds can be monitored for activity in promoting differentiation of heart-derived stem cells into other differentiated cell types, such as smooth muscle cells, skeletal muscle cells, osteoblasts and chondrocytes. Activity is detected by detecting the characteristic morphological appearance and differentiation markers of one of these cell types. Compounds having activity in promoting differentiation into one of the above cell types are useful in treating patients with degenerative bone, muscle or cartilage disorders.

Compounds that inhibit the differentiation of stem cells into certain cell types, such as adipocytes, are also useful in some circumstances. For example, compounds that inhibit adipocyte differentiation
It can be used in treating obesity. Such a compound can contact the compound under test with a heart-derived stem cell under conditions that could otherwise result in the differentiation of the stem cell into a cell type, and compare the cell type relative to a control containing no compound. Identified by monitoring the reduction in frequency or degree of conversion to

Compounds identified as therapeutics from such screening using the cell lines of the present invention are formulated for therapeutic use as pharmaceutical compositions. The compounds may contain pharmaceutically acceptable, non-toxic carriers or diluents as described above, depending on the desired formulation.

Embodiment 1 Material a. Serum-free medium for muscle cells Dulbecco's modified Eagle's medium / ham nutrient mixture F12 (1: 1; Gibco Laboratorie
s) Insulin (JRH 5C2059), 1 μg / ml transferrin (JRH 6K2222), 5 μg / ml selenium (Aldrich Chem. Co. 20010/7), 1 nM thyroxine (Sigma T-0397), 1 nM ascorbic acid (Sigma A- 4034), 25μg / ml LiCl (Sigma L-0505), 1 nM

B. Plated medium (PM) Modified Dulbecco's Eagle's medium / ham nutrient mixture F12 (1% ) containing 15% HIA FBS
: 1; Gibco Laboratories) c. Variant of medium Dulbecco for myocyte isolation Eagle's medium / Ham Nutrient Mixture F12 (1: 1; Gibco Laboratori
es) DF 20 (containing 20% HIA FBS) DF 10 (containing 10% HIA FBS) DF 5 (containing 5% HIA FBS) HIA FBS (HyClone lot number AEL4614)

D. Enzyme collagenase for heart tissue digestion (Worthington LS004176), 2% solution DNase (Worthington LS002007), 0.5% solution Enzyme is dissolved in PBS (Gibco Laboratories) w / 1% glucose

E. Percoll Purification Percoll (Pharmacia # 17-0891-01) Add buffer (g / L for 1X; or g / 100ml for 10X), pH7 6.8NaCl 1.0 Dextrose 1.5NaH ₂ PO ₄ 0.4KCL 0.1MgSO ₄ 4.76HEPES 0.02 phenol red-Sigma

All reagents were passed through a 0.22 μM filter before use. 9 parts (45 ml) Percoll + 1 part (5 ml) 10xAds (no phenol red) gradient steps were made as follows:

[0069]

[Table 1]

F. Human fibronectin plate stock solution is 1 mg / ml (1000 µg / ml) Coating concentration 1 µg / cm ² diluted stock solution to 10 µg / ml (100X: 200 µl in 20 ml SFM) Plate 3 ML / 60 mm tissue culture dish Incubate for 1 hour at room temperature Aspirate residual material Carefully rinse plate with dH ₂ O-do not scrape bottom Prepare plate for use and store at 4 ° C

2. Isolation of non-adherent heart-derived stem cells from p53-deficient mice In this example, the code is used to indicate the month. Thus, A, B and C are A
Indicates consecutive months starting with. On A / 19, hearts from 10 p53-/-female mice (about 2 months old) were digested as follows: 1) Hearts were minced with scissors in a minimum volume of PBS (-) . 2) PBS (-) + 1% glucose was added to bring the final volume to 18 ml. 3) 1 ml of DNase and 1 ml of collagenase were added.

4) The suspension was stirred at 37 ° C. for 30 minutes in a 50 ml Falcon tube on a rotary shaker. 5) This first supernatant was discarded as it mainly contained RBC debris and fibroblasts. 6) Steps 2-5 were repeated until the heart was completely digested. 7) After a stirring time of 30 minutes each (except for the first digest that was discarded), the supernatant was removed and added to 20 ml of DF20. The supernatant containing DF20 was spun at 1650 rpm for 10 minutes at 4 ° C. The resulting pellet was saved, resuspended in 10 ml of DF10, and kept on ice.

8) After complete digestion of the heart, the pellets were combined (5 pellets / 50 ml Falcon tube) and spun at 1650 rpm for 10 minutes at 4 ° C. 9) The resulting final pellet was resuspended in 1.082 gm / ml Percoll (12 ml). 10) A Percoll gradient was prepared in a 50 ml Falcon tube as follows: 12 ml of 1.050 gm / ml percoll were charged, followed by 12 ml of 1.060 gm / ml percoll from the bottom, followed by 1.082 gm. Cell suspension in / ml Percoll was injected from the bottom. 50 ml Falcon tubes were spun in a Beckman CS-6R centrifuge tube at 3000 rpm for 30 minutes at room temperature.

11) The resulting myocyte fraction in the band between the lower 1.082 gm / ml Percoll and the intermediate 1.06 gm / ml Percoll layer was removed and an equal volume of DF10 was added. 12) The myocyte fraction was spun at 1650 rpm for 10 minutes at room temperature. 13) The resulting pellet contained 3.75 × 10 ⁶ cells, which were plated equally in 5 wells in 5 ml DF5. 14) At A / 20, the medium was replaced with plating medium (PM) in a 6-well tissue culture plate, which was replaced with fresh PM at A / 22 and A / 25. Adherent beating heart cells appeared at A / 20-A / 25, but the number was reduced. In addition, minimal suspended cells were present in the wells.

15) In A / 26, these suspension cells were mixed with PM, PM + 1% horse serum (HS) and PM + 10%
1: 3 split in HS (HyClone, Logan, Utah). 16) In B / 01, suspend cells in PM + 1% horse serum (HS) and PM + 10% HS in a T25 flask (0.5 ml of cell suspension + 4.5 ml of 1% HS or 10% HS). 1) in fresh PM containing
: 10 divided. For B / 15, cells in suspension in PM + 1% HS were split 1: 1. The flask was not operated again until C / 19. The flask was incubated at 37 ° C. in 95% O ₂ and 5% CO ₂ .

At C / 19, cells retained in PM + 10% HS were still viable by photorefractive and trypan blue exclusion. Only cells in PM were not viable. Cells in PM and 1% HS became adherent cells. This will be described in detail below. The suspended cell density was determined to be 6 × 10 ⁶ / ml using a hemocytometer.

Further, on C / 19, cells grown in PM + 10% HS were spun at 1500 rpm for 10 minutes, serum-free medium (SFM), SFM + 1% HS, SFM + 10% HS, PM 0.5 in PM + 1% HS or PM + 10% HS
and resuspended in x 10 ⁶ / ml. At C / 26, cells suspended in PM + 10% HS were 8.5 × 10 ⁵ / ml, while cells suspended in SFM + 10% HS were 8.75 × 105 / ml. Half of these suspension cells (both SFM and PM cells) are resuspended in PM + 10% HS or SFM + 10% HS and re-suspended on methylcellulose in the presence of various cytokines and growth factors. Plated. The suspended cells did not form colonies even after 12 days in culture, suggesting that the cells are primitive stem cells.

At this point, culturing suspension cells in 10% HS does not appear to have a significant effect on the cells, and therefore cells were subcultured in PM only or SFM only . Determined by short pulse tritium-thymidine incorporation, zFGF-5 (150 pg / ml) enhanced cell proliferation by 2.5-5.0 fold and stem cell factor (SCF 10 ng / ml) by about 1.0-2.0 fold.

One month treatment of suspension cells with 10 ng / ml SCF in PM resulted in the appearance of an adherent layer of a mixed lineage of cells called SCF / PM derived cells. In contrast, treatment of suspension cells with SCF in SFM did not give rise to adherent cell lines, which, in addition to SCF, did not contain any other present in 15% FBS found in PM. Growth factors are essential. Addition of zFGF-5 (150 pg / ml) to flasks containing SCF in PM blocked induction of adherent cell lines, suggesting that zFGF-5 blocked the differentiation activity of SCF.

By light microscopy, the morphology of the SCF / PM-derived cells suggested a lineage including cardiac cells, chondrocytes, fibroblasts, smooth muscle cells and skeletal muscle cells. Cardiac-like cells had the appearance of cigars or driftwood. The chondrocyte-like cells had the appearance of a nest or cobblestone pavement. Fibroblasts were long and elongated. Cells with the smooth muscle cell phenotype were amorphous or star-shaped, and skeletal muscle cells had the shape of a pen. Myoblasts are
It was intermediate between fibroblasts and skeletal muscle cells. Other lines are probably present and can be characterized using immunohistochemistry, immunofluorescence and FACS analysis.

[0081] 3. Cells retained in PM + 1% HS in adherent heart-derived stem cells C / 19 were differentiated into adherent cells having a mixed morphology. After trypsinizing these cells and pelleting, SFM, SFM + 1% HS,
1: 6 split in SFM + 10% HS, PM, PM + 1% HS or PM + 10% HS. At C / 26, adherent cells were expanded in PM alone, as different media or HS concentrations did not have any significant effect on adherent cell morphology. By light microscopy, the adherent cells appear to be a mixed population comprising pericytes, fibroblasts, heart cells, smooth muscle cells, skeletal muscle cells.

The adherent cells were subcloned into C / 08. From subcloning, 11 clones were obtained and expanded on plated media at 37 ° C. in 95% O ₂ /5% CO ₂ . Cells were characterized according to the following criteria. (1) Light microscopic morphology (2) Alkaline phosphatase staining (identifies pericytes, osteoblast precursors and chondrocyte precursors and mature osteoblasts and chondrocytes) (3) Acetylated LDL uptake (endothelial cells) marker)

(4) MF-20Ab (mouse anti-adult chicken breast myosin; cross-acts with mouse myosin to detect both adult skeletal and cardiac myocyte myosin) (5) M0636 (Dako # 5/56 mouse Human muscle actin; recognizes α-actin in heart, skeletal and smooth muscle cells and γ-actin in smooth muscle cells. (6) Mitotic response to zFGF, acidic FGF and basic FGF. This test is
Inspect for growth potential. Cells were plated in 96-well plates in PM and expanded to confluence before switching to serum-free medium and 24 hours later growth factors and tritiated thymidine were added.

(7) Production of zFGF-5 mRNA identified by Northern blotting. It is expressed at high concentrations by cardiac cell lineage cells. (8) Differentiation induced by contact inhibition or passage through SFM to identify proliferation and / or differentiation potential. (9) Ascorbic acid / □ -glycerophosphate for identifying osteoblasts
(AA / □ -GP) induced differentiation.

(A) Mixed population grown in PM + 1% HS prior to clone isolation Cells in this population include pericytes, fibroblasts, heart cells, smooth muscle cells, skeletal muscle cells And different morphological characteristics of other cells. About 20-25% of the cells were positive for alkaline phosphatase, indicating cells of the osteoblast or chondrocyte lineage. All cells were negative for acetylated LDL uptake, indicating the absence of endothelial cells. All cells are MF-20
Negative for binding, indicating the absence of terminally differentiated bone cells. All cells were positive for M0636 binding, indicating the presence of precursors of heart, muscle, skeletal and smooth muscle cells.

The cells showed a mitotic response to zFGF-5 (cFGF), acidic FGF (aFGF) and basic FGF. bFGF was most effective in inducing a mitotic response. The response is
Increased from 1 pg / ml to 1 □ g / ml in a concentration dependent manner. Upon treatment with serum-free medium, cells exhibited moderate growth and differentiated into cells with a fried egg-like appearance. This appearance suggests early stage myocytes. Upon treatment with ascorbic acid / β-GP, the cells differentiated into chondroid cells by day 6 in morphology. Mineralization did not occur even after day 25, indicating the absence of osteoblast lineage cells.

Clone 1D9 : Clone 1D9 was isolated from a single cell of the mixed population described above in 3a. Some cells grown from 1D9 exhibited cardiac cell morphology, and others exhibited different morphologies. 2
~ 3 cells stained slightly positive for alkaline phosphatase. Cells did not take up acetylated LDL. MF-20 did not bind to the cells. M06
36 bound cells, indicating that some cells were of the smooth muscle, skeletal muscle and / or cardiac cell lineage.

[0088] zFGF, acidic FGF and basic FGF all induced a mitotic response, with bFGF being the most potent. Serum-free medium induced slow growth, but no obvious morphological changes. Contact-dependent differentiation has produced chondrocytes, heart cells and other cell types, recognizing in morphology. Ascorbic acid / β-GP treatment resulted in the appearance of chondrocyte-like cells on day 6. Mineralization did not occur even after 25 days. These results
Figure 9 shows that clone 1D9 gives rise to a different lineage of differentiated cells.

Clone 2E7 : Cells expanded from 2E7 have the form of myoblasts (skeletal muscle cells / fibroblasts / adipocyte precursors). Cells were negative for alkaline phosphatase staining, acetylated LDL uptake and MF-20 binding. Cells were positive for M0636 binding, indicating the presence of precursors of smooth muscle, skeletal muscle and / or cardiomyocytes.

Cells proliferated in response to zFGF-5, acidic FGF and basic FGF, with basic FGF being the most potent. Upon treatment with serum-free medium, cells proliferated, changed morphology, and appeared as large polygonal cells, which may be early stage myocytes. Cells are MF
Was negative for -20. Upon treatment with ascorbic acid / β-GP, cells differentiated into a “ridge-like” structure on day 11. No mineralization was observed after 25 days.

Clone 1G11 : Clone 1G11 was also isolated as a single cell from the mixed population of adherent P53-deficient cells. Cells expanded from 1G11 had skeletal muscle cells, smooth muscle cells, heart cells and other cell types. About 20-25% of the cells stained positive for alkaline phosphatase. Cells showed a mitotic response to zFGF-5, acidic FGF and basic FGF, with bFGF being the most potent and bFGF and aFGF being equally active. Addition of serum-free medium resulted in slow growth rates and the appearance of astrocytes, which are myocyte precursors. Cells did not bind to MF-20. Ascorbic acid / β-GP
Treatment did not result in mineralization even after 25 days.

Clone 2B6 : This clone exhibited the morphology of skeletal muscle cells, smooth muscle cells, heart cells and other cell types. Cells were negative for alkaline phosphatase staining as well as acetylated LDL uptake and MF-20 binding. Cells showed a mitotic response to bFGF and aFGF, but not zFGF-5. SFM-induced differentiation and slower growth rates resulted in thicker, rod-shaped cells. Treatment with ascorbic acid / β-GP resulted in cells that collected together on day 11 in a “ridge-like” structure. No mineralization occurred even after 25 days.

Clone 2A7 : This clone exhibited the morphology of myoblasts (skeletal muscle cells / fibroblasts / adipocyte precursors). Cells were negative for alkaline phosphatase staining, acetylated LDL uptake, and MF-20 binding. Cells were slightly positive for M0636 binding. Cells showed a mitotic response to zFGF-5, acidic FGF and basic FGF. aFGF and bFGF were the most potent, with all three FGFs equally active. SFM did not cause any changes in growth rate or morphology. Ascorbic acid / β-GP treatment did not result in mineralization even after 25 days.

Clone 2G7 : This clone had the morphology of multinucleated heart cells and other cell types. Some cells were positive for alkaline phosphatase staining. Cells were negative for acetylated LDL uptake and MF-20 binding. Cells were positive for M0636 binding. Cells showed a mitotic response to zFGF-5, acidic FGF and basic FGF, with bFGF being the most potent and aFGF and bFGF equally active. SFM induced a moderate growth rate but no obvious morphological changes. Cells were negative for MF-20 binding. Ascorbic acid / β-GP treatment is 25
No mineralization occurred after the day.

Clone 2F11 : This clone had the morphology of myoblasts (skeletal muscle cells / fibroblasts / adipocyte precursors). 5% of the cells stained with alkaline phosphatase. Cells were negative for acetylated LDL uptake, MF-20 binding and M0636 binding. SFM induced a modest growth rate, resulting in astrocytes. Ascorbic acid / β
-GP produced cells that gathered in a “ridge-like” structure on day 11. After 25 days, no mineralization occurred.

Clone 2A6 : These cells had the myoblast star-shaped appearance. Cells were negative for alkaline phosphatase staining, negative for acetylated LDL uptake, negative for MF-20 binding, and negative for M0636 binding. Cells are zFGF-5, acidic FG
It showed a mitotic response to F and basic FGFs, with bFGF being the most potent and all three FGFs being equally active. SFM treatment resulted in moderate proliferation and a somewhat larger stellate cell appearance. Ascorbic acid / β-GP treatment induced differentiation with cells that clustered in “ridge-like” structures on day 11 (many structures were evident). No mineralization occurred even after 25 days.

Clone 2G12 : These cells exhibited the morphology of heart cells, skeletal muscle cells, smooth muscle cells and other cell types. Cells were stained for alkaline phosphatase, acetylated LDL uptake, MF
-20 binding, negative for M0636 binding.

The cells showed a mitotic response to bFGF and aFGF. bFGF and cF
GF was equally potent, active from 10 fg / ml to 1 □ g / ml. FGF stimulation regenerated the original suspension cell progenitor population and produced zFGF-5 mRNA. Stimulation of zFGF-5 at 150 pg / ml (1 day) resulted in adult cardiac cells morphologically apparent. 5 days treatment, stack structure,
A ridge and trough morphology resulted. Contact-dependent differentiation produced heart cells, chondrocytes and other cell types. Ascorbic acid / β-GP treatment resulted in differentiation, producing large cells on day 6. No mineralization occurred even after 25 days.

Clone 2D6 : The cells had a star-shaped or stacked appearance when subcloned, suggesting myoblasts (skeletal muscle cells / fibroblasts / adipocyte precursors). Cells were negative for acetylated LDL uptake, negative for alkaline phosphatase staining, negative for MF-20 binding, and positive for M0636 binding.
SFM treatment resulted in rapid growth as well as loss of the straw or star appearance. Ascorbic acid / β-GP treatment resulted in the appearance of cartilage-like cells on day 6. No mineralization occurred even after 25 days.

Clone 2H6 : The cells showed the morphology of heart cells and other cells. Cells were negative for alkaline phosphatase staining, acetylated LDL uptake, MF-20 binding, and M063
6 Positive for binding. Cells showed a mitotic response to zFGF-5, acidic FGF and basic FGF. bFGF was the most potent and all three FGFs were equally active. SFM treatment resulted in reduced growth rate, reduced adherence to plastic, and the appearance of large polygonal cells. Treatment with ascorbic acid / β-GP resulted in cells that gathered together on day 11 in a “ridge-like” structure. No mineralization occurred even after 25 days.

[0101] 5. Isolation of heart-derived stem cells from neonatal mice 1) 1-4 day old neonates (mouse) were killed by cervical dislocation and placed in a 70% ETOH bath. If 10-15 newborns are collected, one is placed back-up and a midline sternotomy is performed. When pressure is applied to the thoracic cavity using forceps and the heart is ejected, the ventricles can be easily opened. Ventricles are placed in ice-cold PBS (50 ml Falcon tubes containing PBS on ice), and each tube contains approximately 100 ventricles. 2) Once the whole heart has been collected, rinse several times in PBS. The heart tissue is then minced with scissors in a minimum volume of PBS and rinsed several times in PBS again (rinsing until all red blood cells and debris are removed). From step 3), PBS is supplemented with 1% glucose.

3) a) To initiate dissociation, grow hearts in 18 ml PBS. b) Add 1 ml of each stock solution of DNase / collagenase. 4) Stir on a rotary shaker at 37 ° C. for 30 minutes (1. digest). 5) 1. The supernatant from the digest is discarded, which contains mainly fibroblasts, red blood cells and debris. 6) a) To initiate dissociation, grow hearts in 18 ml PBS. b) Remove 1 ml of each stock solution of DNase / collagenase.

7) Stir on a rotary shaker at 37 ° C. for 20 minutes. 8) Transfer the supernatant (20 ml) from the tube to a 50 ml Falcon tube containing 20 ml DF20. Mix carefully (rotate tube up and down). 9) Spin tube (s) at 1650 rpm for 10 minutes at 4 ° C. 10) Keep the pelleted cells at the bottom and discard the supernatant (〜40 ml). 11) Add 10 ml of DF10 to the pellet, mix well up and down (use a sterile pipette) and keep the cell suspension (s) on ice.

Steps 6) to 11) are repeated until all the tissues are digested. 12) When dissociation is completed, all of the steps 11) are combined. 13) Pellet cells (rotate tube at 1650 rpm for 10 minutes at 4 ° C and discard supernatant). 14) Resuspend myocytes in 1.082 g / ml Percoll (12 ml Percoll / 100 newborns). 15) 1050 g / ml, 1.060 g / ml and then 1.082 g / ml (containing cells) of 12 ml each of percoll were added from the bottom into the required number of 50 ml Falcon tubes, from the bottom.
Using a ml pipette, prepare a Percoll gradient (1 tube Percoll gradient / 100 newborns).

16) Centrifuge at 3000 rpm for 30 minutes at room temperature. Beckman CS-6R centrifuges are suitable. 18) After the centrifugation step in a Percoll gradient, four bands appear (FIG. 2).
reference). 19) After aspirating the upper band and collecting (if the fibroblast layer is desired to be retained first, transfer it to a 50 ML Falcon tube and add the same amount of the collected DF10 cell suspension), Collect the layers and transfer to a 50 ml Falcon tube and add the same amount of DF10 as the volume of the collected cell suspension.

20) Spin the cells at 1650 rpm for 10 minutes at room temperature with thorough but careful mixing. 21) Discard the supernatant. Add 5 ml DF5 / "100 newborns". 22) Incubate the myocytes on human fibronectin (HFn) for 1 hour at 37 ° C. The remaining fibroblasts adhere to the coated plate within one hour. 23) Collect non-adherent cells and wash dishes several times with DF5 to ensure that all non-adherent cells are collected. 24) Spin cells at 1650 rpm for 10 minutes, remove supernatant and resuspend cells in DF5.

6. MSCs from bone marrow Assays were performed to determine the frequency of fibroblast colony forming units from monkey low density non-adherent cells isolated from expanded bone marrow. This assay indicates mesenchymal stem cell frequency. Half of the 96-well microtiter plates were seeded with cells at a density of 10,000 cells / well, and the other half of the plates were seeded with cells at a density of 1,000 cells / well. The medium was αMEM (GIBCO-BRL, Gaithersburg, MD), 2% bovine serum albumin, 10μ
g / ml insulin, 200 μg / ml transferrin, antibiotics and 50 μM β-mercaptoethanol.

Cells were incubated for 14 days at 37 ° C. in 5% CO ₂ , then stained with toluidine blue to improve cell visibility and microscopically. Positive wells have at least 50 cells exhibiting "stromal" morphology, ie, large expanded cells. The positive control is a medium containing 20% fetal calf serum. The results showed that zFGF was
Increased to a level equivalent to 20% FBF positive control.

[0109] 7. Identification of cardiac MSCs for zFGF5 FITC-labeled protein and neonatal mouse or fetal sheep (Third trimester)
Heart tissue was used to identify putative mesenchymal stem cells as targets (ie, having receptors) for zFGF. ZFGF purified as described above was dialyzed in 0.1 M sodium bicarbonate, pH 9.0.
Fluorescein isothiocyanate (FITC; Molecular Probes, Eugene, OR)
It was dissolved at 1 mg / ml in the same buffer without exposure to strong light.

A mixture containing 1 mg FITC / 1 mg zFGF5 was prepared and reacted at room temperature in the dark for 1-2 hours. The reaction was stopped by adding 1 M glycine to a final concentration of 0.1 M and then allowed to react for 1 hour at room temperature. The mixture was then dialyzed against 0.1 M sodium bicarbonate and diluted 1: 500 to 1: 1000 for 3 hours. The dialysis solution was changed and the process was repeated for 3-18 hours to remove unlabeled FITC.

[0111] Neonatal mouse or fetal sheep heart ventricles were isolated, minced, and repeatedly washed in phosphate buffered saline (PBS) until erythrocytes and debris were removed. The minced ventricle was placed in a solution containing 18 ml PBS and 1% glucose and 2% DNase /
1 ml of collagenase solution was added. The mixture was incubated on a shaker at 37 ° C. for 30 minutes. The supernatant was discarded and the process was repeated once.

After incubation, the supernatant (（20 ml) was mixed with 20 ml DF20 (Dulbecco's modified Eagle medium / ham nutrient mixture F12, 1: 1 (GIBCO-BRL, Gaithesburg, MD) and
(20% fetal calf serum). After mixing, transfer the test tube to Beckman CS-
Centrifuged in a 6R centrifuge (Beckman, Fullerton, CA) at 1650 rpm for 10 minutes at 4 ° C. The supernatant was discarded and the pellet was resuspended in DF10 (10% FBS). Keep cells cool and
Spin again and resuspend in 40 ml DF10. The cell mixture was passed through a 40 μm filter (Becton Dickinson, Detroit, MI) and counted using a hemocytometer.

Cells were incubated in FITC-labeled zFGF5 at a concentration of 2 × 10 ⁶ cells / 1 μg zFGF5 at 4 ° C. for 30 minutes. After incubation, cells were spun at 1650 rpm in a Beckman CS-6R centrifuge (Beckman). Discard supernatant and pellet with 10 ml DF
Washed once in 10 and resuspended in 4 ml DF10. 10 μl of MACS anti-FITC microbeads (Miltenyi Biotech, Auburn, Calif.) Were mixed with 10 ⁷ cells in 4 ml of DF10 and incubated at 4 ° C. for 30 minutes.

The MACS positive selection LS type + separation column (Miltenyi Biotech) was mixed with 3 ml of MAC buffer (P
The cell / bead mixture was washed in 10 ml MAC buffer, then resuspended in 6 ml MAC buffer, washing with BS, 0.5% BSA, 2 mM EDTA). The cell / bead mixture was transformed between the two columns and the first negative fraction discarded. 1.5 ml of 0.6 M NaCl was added to each column and eluted but not collected. The column was then washed with 1.5 ml of MAC buffer. Cells bound to FITC-labeled zFGF5 were collected by adding 3 ml of MAC buffer, the column was removed from the magnet and positive cells were washed off using a plunger. Positive cell fractions were placed in T75 flasks and 50 ml of plating media was added (DF containing 15% FBS and antibiotics). Cells were incubated at 37 ° C. for 1 week and counted. The yield of positive cells was about 0.1% of the original layer cells counted.

Cells binding FITC-labeled zFGF5 were observed by transmission electron microscopy (TEM). Cells were 3-5μ in diameter. The cell nucleus occupies the majority of the cell volume and a few cytoplasmic organelles were observed. The phenotype identified by TEM identifies zFGF5 isolated cells as primitive mesenchymal stem cells.

[0116] 8. In vivo study of rats with cardiomyopathy Rats injected subcutaneously with epinephrine for 2 weeks develop cardiomyopathy exactly the same as human idiopathic flaccid cardiomyopathy (Deisher et al., Am. J. Cardiovasc. Pathol. ): 79-88, 1994 and Deisher et al., J. Pharmacol. Exp. Ther. 266 (1): 262.
-269,1993). Intracardiac, intracoronary, intraarterial, intraventricular or intravenous infusions into rats receiving subcutaneous infusions of epinephrine or saline were used to isolate cardiac-derived MSCs from healthy rats with similar genetic background. The effects of cardiac MSCs on the initiation and progression of catecholamine-induced cardiomyopathy by administration are evaluated.

In one protocol, rats (300 g) were implanted with epinephrine in an osmotic pump, injected with cardiac-derived MSCs or control cells, and monitored for mortality for two weeks, at which time rats were sacrificed. The heart was then wet weighed and fixed in 10% neutral buffered formalin for tissue preparation. Before sacrifice, cardiac function was measured.

Measurements include body weight, heart weight, cardiac fibrosis and cardiac histomorphometry of epinephrine injected rats. Cardiac fibrosis is determined by scoring Masson trichrome stained heart sections. Three sections were scored for each heart and an average score was generated. Positive results indicate that infusion of cardiac MSCs may result in heart failure of various etiologies, including myocardial infarction (MI), idiopathic flaccid cardiomyopathy (IDCM), hypertrophic cardiomyopathy, viral myocarditis, congenital abnormalities and obstructive Indicates that it may be beneficial in controlling the disease.

It is clear from the foregoing that the present invention has a number of uses, some of which may be briefly described as follows. The invention provides for the use of non-adherent or adherent heart-derived stem cells in the treatment of a disease or in the discovery of an agent for use therein. The invention further provides for the use of non-adherent or adherent heart-derived stem cells in the manufacture of a medicament for the treatment of a disease.

Although the invention has been described above in some detail for clarity and understanding, it is understood that various changes in form and detail may be made without departing from the true scope of the invention. Upon reading, it will be apparent to those skilled in the art. All publications and patent documents in this application are incorporated herein by reference, to the same extent as if each were individually indicated.

[Brief description of the drawings]

FIG. 1 shows the differentiation pathway of stem cells derived from heart tissue into various differentiated cell types.

FIG. 2 shows the density gradient of cells obtained by digesting heart tissue. The monocyte band is the third from the top.

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[Submission date] October 26, 2000 (2000.10.26)

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Abstract: The present invention relates to a cardiac-derived differentiated cell that can produce various cell types during proliferation and differentiation, including heart cells, fibroblasts, smooth muscle cells, skeletal muscle cells, keratinocytes, osteoblasts and chondrocytes. Provide competent stem cells. The cells can be used in methods of treating patients suffering from cardiac cell necrosis. Stem cells proliferate and differentiate to produce heart cells that replace necrotic tissue. Cells can also be used to screen compounds for activity in promoting the growth and / or differentiation of heart-derived stem cells.

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY , CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP , KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Hanson, Birgit United States of America, Washington 98102, US Attle, East Brain Street 210 # 304 (72) Inventor Moore, Emma. United States, Washington 98199, Seattle, Surtees Avenue North East 3507 (72) Inventor Robertson, Tamara El. United States, Washington 98115, Seattle, Twenty First Avenue North East 6523 (72) Inventors Thompson, Deborah El. United States, Washington 98107, Seattle, 9th Avenue Northwest 5808 (72) Inventor Lam, Calendy. USA, Washington 98108, Seattle, Columbia Drive South 5110 F-term (reference) 2G045 BA13 BA20 BB10 BB20 BB24 CB01 CB26 DA20 DA36 DA80 FA16 4B065 AA90X AA91X AA93X AC20 BA06 BB21 BB32 CA24 CA44 4C087 BB47 BB64 NA14

Claims (39)

[Claims] Claims 1. After proliferation and differentiation, adherent heart-derived stem cells, fibroblasts,
An isolated non-adherent pluripotent heart-derived human stem cell producing a progeny cell comprising a cell type selected from the group consisting of smooth muscle cells, skeletal muscle cells, heart cells, keratinocytes, osteoblasts and chondrocytes . 2. The isolated non-adherent heart-derived stem cell according to claim 1, which produces, after differentiation, progeny cells containing at least two cells selected from the group. 3. The isolated non-adherent heart-derived stem cell according to claim 1, which produces, when proliferating and differentiating, progeny cells including all cells selected from the group. 4. The isolated non-adherent heart-derived stem cell of claim 1, which is immortalized. 5. Growing a population of cells derived from heart tissue in a liquid medium on a support; discarding the cells from the population adhering to the support, leaving a suspension of non-adherent heart-derived stem cells; 2. The isolated non-adherent heart-derived stem cell of claim 1 produced by: 6. An isolated, non-adherent, pluripotent heart-derived stem cell that, after proliferation and differentiation, produces heart cells and progeny comprising either chondrocytes or keratinocytes. 7. After proliferation and differentiation, the adherent heart-derived stem cells, fibroblasts,
7. The isolated non-adherent pluripotent heart-derived stem cell of claim 6, which produces a progeny cell further comprising at least one cell type selected from the group consisting of a smooth muscle cell and a skeletal muscle cell. 8. The isolated non-adherent heart-derived stem cell according to claim 7, which produces, after differentiation, a progeny cell containing at least two cells selected from the group. 9. The isolated non-adherent heart-derived stem cell according to claim 7, which produces, after proliferation and differentiation, progeny cells including all cells selected from the group. 10. The isolated non-adherent heart-derived stem cell according to claim 7, which is a human stem cell. 11. The isolated non-adherent heart-derived stem cell according to claim 7, which is a mouse stem cell. 12. The isolated non-adherent heart-derived stem cell according to claim 11, which is derived from a P53-deficient mouse. 13. The method of claim 1 wherein said proliferating and producing a progeny cell comprising a cell type selected from the group consisting of fibroblasts, smooth muscle cells, skeletal muscle cells, heart cells, chondrocytes, keratinocytes and osteoblasts. An isolated adherent human heart-derived stem cell that differentiates. 14. The isolated human adherent heart-derived stem cell of claim 13, which proliferates and differentiates to produce progeny cells comprising at least two cell types selected from the group. 15. The isolated human adherent heart-derived stem cell of claim 13, which proliferates and differentiates to produce progeny cells encompassing all cell types selected from the group. 16. An isolated adherent heart-derived stem cell that proliferates and differentiates to produce progeny, including heart cells and chondrocytes or keratinocytes. 17. A method of growing and differentiating to produce progeny cells further comprising one or more cell types selected from the group consisting of fibroblasts, smooth muscle cells, skeletal muscle cells and osteoblasts. Item 18. The isolated adherent heart-derived stem cell according to Item 16. 18. A method for preparing an isolated non-adherent heart-derived stem cell, comprising:
The following: centrifuging a suspension of cells from the subject's heart tissue on a density gradient; isolating a band of cells, including muscle cells; suspended cells in which adherent heart cells have died or discarded Culturing the cells in suspension until a population of non-adherent heart cells is detectable. 19. The method of claim 18, further comprising: harvesting the heart tissue from the subject; digesting the heart tissue with collagenase to produce a cell suspension. 20. The stem cell derived from the non-adherent heart according to claim 1, wherein the stem cell is grown until adherent progeny cells appear; myoblasts, smooth muscle cells, skeletal muscle cells, heart cells, osteoblasts Identifying a adherent cell that lacks a marker for a cell selected from the group consisting of keratinocytes and chondrocytes, comprising the steps of: A method for producing a progeny cell encompassing two cell types. 21. The non-adherent heart-derived stem cell according to claim 1, wherein the cell is grown under conditions in which the cell proliferates and differentiates to produce a progeny cell including an adherent cell; Identifying a adherent cell with the indicated differentiation marker; a method for preparing heart cells. 22. Providing the non-adherent heart-derived stem cells of claim 13 or 16; allowing the cells to proliferate under conditions in which the cells proliferate and differentiate to produce adherent progeny cells; Identifying a adherent cell with a marker; a method for preparing a heart cell, comprising the steps of: 23. A method for preparing a non-adherent heart-derived stem cell, comprising culturing the cell in the presence of fibroblast growth factor and allowing the cell to grow. 24. An isolated population of cells comprising smooth muscle cells, skeletal muscle cells, heart cells, fibroblasts, keratinocytes, osteoblasts and chondrocytes. 25. A method of treating a patient suffering from cardiac tissue necrosis, comprising administering to the patient an effective dose of the non-adherent stem cells of claim 1, whereby the stem cells proliferate and differentiate to produce heart cells. And this replaces necrotic tissue. 26. The method of claim 25, wherein the non-adherent stem cells are administered directly to the patient's heart.
the method of. 27. The method of claim 25, wherein the non-adherent stem cells are administered intravenously. 28. The method of claim 25, further comprising administering FGF to the patient to stimulate proliferation and / or differentiation of non-adherent cells. 29. The method of claim 25, further comprising administering stem cell factor to the patient to stimulate differentiation of non-adherent cells into cardiac cells. 30. The method of claim 18, wherein the patient has a congenital heart defect. 31. Stem cells are obtained from the blood of the patient and prior to re-administration to the patient.
26. The method of claim 25, wherein the method is grown in vitro. 32. A method of treating a patient suffering from cardiac tissue necrosis, comprising administering to the patient an effective dose of the adherent stem cells of claim 13, whereby the stem cells proliferate and differentiate to produce cardiac cells, A method wherein this replaces necrotic tissue. 33. The method according to claim 32, wherein the non-adherent stem cells are administered directly to the patient's heart.
the method of. 34. The method of claim 32, further comprising administering FGF to the patient to stimulate proliferation of adherent cells. 35. The method of claim 32, further comprising administering stem cell factor to the patient to stimulate differentiation of adherent cells into cardiac cells. 36. A method for screening a potential agent for activity in promoting the proliferation and / or differentiation of heart-derived stem cells, wherein the method is in the presence of the potential agent. Proliferating the non-adherent heart-derived stem cells of item 6; and monitoring a change in the differentiation state of progeny cells as compared to the non-adherent heart-derived stem cells. 37. The method of claim 36, wherein the change in differentiation status is the attachment of progeny cells. 38. The method of claim 36, wherein monitoring comprises monitoring cardiac cell appearance. 39. The method of claim 36, wherein monitoring comprises monitoring the appearance of adherent heart-derived stem cells.