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EP1064356A2 - Cellules souches d'origine cardiaque - Google Patents

Cellules souches d'origine cardiaque

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Publication number
EP1064356A2
EP1064356A2 EP99912853A EP99912853A EP1064356A2 EP 1064356 A2 EP1064356 A2 EP 1064356A2 EP 99912853 A EP99912853 A EP 99912853A EP 99912853 A EP99912853 A EP 99912853A EP 1064356 A2 EP1064356 A2 EP 1064356A2
Authority
EP
European Patent Office
Prior art keywords
cells
cell
cardiac
nonadherent
derived stem
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99912853A
Other languages
German (de)
English (en)
Inventor
Theresa A. Deisher
Birgit Hanson
Emma E. Moore
Tamara L. Robertson
Deborah L. Thompson
Karen D. Lum
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Zymogenetics Inc
Original Assignee
Zymogenetics Inc
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Filing date
Publication date
Application filed by Zymogenetics Inc filed Critical Zymogenetics Inc
Publication of EP1064356A2 publication Critical patent/EP1064356A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0668—Mesenchymal stem cells from other natural sources
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0657—Cardiomyocytes; Heart cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/90—Serum-free medium, which may still contain naturally-sourced components

Definitions

  • the invention resides in the technical fields of cell biology, drug discovery and medicine.
  • the terminal state of cell differentiation is incompatible with cell division.
  • the cell nucleus is disintegrated, in red blood cells, there is no nucleus, and in muscle cells, myofibrils obstruct mitosis and cytokinesis.
  • the ability of such cells and tissues to develop, regenerate and repair is dependent on the existence of stem cells that on division form additional differentiated progeny cells.
  • Stem cells are not terminally differentiated, can divide without limit, and give rise to progeny, which can continue to divide or can differentiate.
  • Stem cells can be totipotent, pluripotent or unipotent.
  • Totipotent stem cells e.g., embryonic stem cells
  • Pluripotent stem cells can give rise to more than one differentiated cell type.
  • a unipotent stem cell can give rise to a single differentiated cell type.
  • Stem cells are generally characterized by small size, low granularity, low cytoplasmic to nuclear ratio and no expression of osteopontin, collagens and alkaline phosphatase.
  • Hemopoietic stem cells are present in circulation as well as bone marrow. Circulating hemopoietic stem cells can colonize organs such as spleen. Stem cells for cells of bone, cartilage, fat and three types of muscle (smooth, skeletal and cardiocyte) are thought to be a common mesenchymal stem cell precursor, but neither mesenchymal stem cells or the more committed tissue specific progenitors have been characterized (Owen et al., Ciba Fdn. Symp. 136, 42-46, 1988); Owen et al., J. Cell Sci.
  • An isolated cell is a cell that has been at least partially purified from other cell types with which it is naturally associated. Often an isolated cell exists in a population of cells at least 25, 50, 75, 90, 95 or 99% of which are the isolated cell type.
  • an isolated cell gives rise to a cell line in which all cells are essentially identical except for spontaneous mutations that may arise in propagation of the cell line.
  • an isolated cell undergoes spontaneous differentiation to generate a mixed population of mature cell types.
  • a set of differentiation markers means one or more phenotypic properties that can be identified and are specific to a particular cell type. Differentiation markers are transiently exhibited at various stages of cell lineage. Pluripotent stem cells that can regenerate without commitment to a lineage express a set of differentiation markers that may be lost when commitment to a cell lineage is made. Precursor cells express a set of differentiation markers that may or may not continue to be expressed as the cells progress down the cell lineage pathway toward maturation. Differentiation markers that are expressed exclusively by mature cells are usually functional properties such as cell products, enzymes to produce cell products and receptors.
  • Adherence capacity of cells in culture is one marker of progression from an undifferentiated to differentiated state.
  • Adherent cells form a monolayer on plastic substrate.
  • a cell lineage refers to a differentiated cell type and the ancestor cell types from which the differentiated cell type was derived. For example, cardiocytes and myoblasts are two cell types in the same lineage.
  • the invention provide isolated nonadherent pluripotent cardiac-derived human stem cells.
  • progeny cells comprising a cell type selected from the group consisting of an adherent cardiac-derived stem cell, a fibroblast, a smooth muscle cell, a skeletal muscle cell, a cardiocyte, a keratinocyte, an osteoblast and a chondrocyte.
  • Some such stem cells generate all of the cell types selected from the group.
  • Nonadherent cardiac-derived stem cells of the invention are producible by propagating a population of heart tissue-derived cells in a liquid medium on a substrate; and discarding cells from the population adhering to the substrate and leaving a suspension of the nonadherent cardiac-derived stem cells.
  • the invention further provides an isolated nonadherent pluripotent cardiac- derived stem cell, which on proliferation and differentiation produces progeny cells comprising cardiocytes and either chondrocytes or keratinocytes.
  • Some such stem cells on proliferation and differentiation produce progeny cells further comprising at least one cell type selected from the group consisting of an adherent cardiac-derived stem cell, a fibroblast, a smooth muscle cell, and a skeletal muscle cell.
  • Such stem cells can be human or mouse stem cells, for example.
  • mouse stem cells can be obtained from a p53 deficient mouse.
  • the invention further provides an isolated adherent human cardiac-derived stem cell, which proliferates and differentiates to produce progeny cells comprising a cell type selected from the group consisting of a fibroblast, a smooth muscle cell, skeletal muscle cell, a cardiocyte, a chondrocyte, a keratinocyte and an osteoblast.
  • a cell type selected from the group consisting of a fibroblast, a smooth muscle cell, skeletal muscle cell, a cardiocyte, a chondrocyte, a keratinocyte and an osteoblast.
  • the invention further provides an isolated adherent cardiac-derived stem cell, which proliferates and differentiates to produce progeny cells comprising a cardiocyte and either a chondrocyte or a keratinocyte.
  • the invention provides a method of preparing an isolated nonadherent cardiac-derived stem cell.
  • a method entails centrifuging a suspension of cells from heart tissue of a subject on a density gradient; isolating a band of cells comprising myocytes; propagating the cells until adherent cardiocytes have died or been discarded leaving suspension cells; and culturing the suspension cells until a population of nonadherent cardiac cells is detectable.
  • the invention also provides methods of preparing adherent cardiac derived stem cells. Such methods start with a nonadherent cardiac-derived stem cell as described above.
  • the stem cell is the propagated until adherent progeny cells appears.
  • An adherent cell lacking markers of a cell selected from the group consisting of myoblasts, smooth muscle cells, skeletal muscle cells, cardiocytes, osteoblasts, keratinocytes and chondroblasts, which on proliferation and differentiation produces progeny cells comprising at least one cell type from the group is then identified.
  • the invention further provides methods of preparing a cardiocyte. Some such methods entail providing a nonadherent cardiac-derived stem cell as described above; propagating the cell under conditions in which the cell proliferates and differentiates to produce progeny cells comprising adherent cells; and identifying an adherent cell with differentiation markers characteristic of a cardiocyte. Alternatively, such methods can start with a nonadherent cardiac-derived stem cell as described above. The cell is propagated under conditions in which the cell proliferates and differentiates to produce adherent progeny cells. An adherent cell is then identified with differentiation markers characteristic of a cardiocyte.
  • the invention provides an isolated population of cells comprising smooth muscle cells, skeletal muscle cells, cardiocytes, fibroblasts, keratinocytes, osteoblasts and chondrocytes.
  • the invention provides methods of treating a patient suffering from necrotic heart tissue. Such methods entail administering to the patient an effective dose of nonadherent or adherent stem cells as described above, whereby the stem cells proliferate and differentiate to produce cardiocytes, which replace the necrotic tissue.
  • the nonadherent stem cells are administered directly to the heart of the patient.
  • the nonadherent stem cells are administered intravenously.
  • fibroblast growth factor is also administered to the patient to stimulate proliferation and/or differentiation of the nonadherent cells.
  • stem cell factor is administered to the patient to stimulate differentiation of the nonadherent cells to cardiocytes.
  • the patient has a congestive heart defect.
  • the stem cells are obtained from the blood of the patient, and propagated in vitro before readministering to the patient.
  • the invention provides methods of screening potential agents for activity in promoting proliferation and/or differentiation of cardiac-derived stem cells. Such methods entail propagating nonadherent or adherent cardiac-derived stem cell in the presence of a potential agent, and monitoring a change in differentiation state of progeny cells relative to the nonadherent or adherent cardiac-derived stem cells.
  • the change of differentiation state is adhesion of progeny cells.
  • the change in differentiation state is monitored by detecting the appearance of cardiocytes.
  • the appearance of adherent cardiac-derived stem cell is monitored.
  • Fig. 1 shows the differentiation pathways from cardiac tissue-derived stem cells to various differentiated cell types.
  • Fig. 2. shows a density gradient of cells obtained by digesting heart tissue. The myocyte band is third from top.
  • the invention provides at least two classes of pluripotent cardiac-derived stem cells.
  • One class of cells is nonadherent and the other is adherent.
  • the adherent cells are partially differentiated progeny of the nonadherent cells.
  • Both classes of stem cells can be propagated and differentiated into a variety of differentiated cell types.
  • the cardiac-derived stem cells can be isolated from humans and other vertebrate animals.
  • the cells are isolated from a fraction of a suspension of cardiac- derived cells that has typically been discarded as debris by previous workers in the field.
  • a band of myocyte cells is typically isolated from a density gradient, and the cells are propagated in a culture medium on a substrate.
  • Previous workers have retained cells adhering to the substrate and discarded the culture medium in the mistaken belief that it contained only debris.
  • the present inventors have kept the culture medium and discarded the adherent cells. After propagation of the culture medium a population of nonadherent suspension cells becomes apparent.
  • the cells have several applications in therapy and drug discovery.
  • the cells are administered to patients suffering from heart defect, such as necrotic tissue resulting from a myocardial infarction.
  • the administered stem cells colonize the heart of a patient and give rise to myocardial progeny cells that replace necrotic tissue and/or supplement preexisting heart tissue.
  • the stem cells are used to screen compounds for activity in promoting or inhibiting differentiation of stem cells to cardiocytes or other mature differentiated cell types.
  • Compounds that promote differentiation of stem cells to cardiocytes can be used for therapy of patients with heart defects, optionally in conjunction with the stem cells of the invention.
  • Other compounds find uses in other therapeutic application in which promotion or inhibition of stem cell differentiation is desired.
  • Another use for stem cells of the present invention is screening for compounds which expand the stem cell population in culture. Some such compounds also induce differentiation and others do not.
  • Another therapeutic use for cells of the present invention is to provide a method to screen for compounds that promote mobilization of cardiac-derived stem cells from the heart to the circulatory system.
  • In vivo assays for evaluating cardiac neogenesis include treating neonatal and mature animals with the cells of the present invention.
  • the animals' cardiac function is measured as heart rate, blood pressure, LV pressure-rate production (tdp/dt)and cardiac output to determine left ventricular function.
  • Post-mortem methods for assessing cardiac improvement include: increased cardiac weight, nuclei/cytoplasmic volume, staining of cardiac histology sections to determine proliferating cell nuclear antigen (PCNA) vs. cytoplasmic actin levels (Quaini et al., Circulation Res. 75:1050-1063, 1994 and Reiss et al, Proc. Natl Acad. Sci. 93:8630-8635, 1996.)
  • PCNA proliferating cell nuclear antigen
  • the cardiac-derived mesenchymal stem cells of the present invention can be used in treatment of disorders associated with heart disease, i.e., myocardial infarction, coronary artery disease, congestive heart failure, hypertrophic cardiomyopathy, myocarditis, congenital heart defects and dilated cardiomyopathy.
  • Cells of the present invention are useful for improving cardiac function, either by inducing cardiac myocyte neogenesis and/or hyperplasia, by inducing coronary collateral formation, or by inducing remodeling of necrotic myocardial area.
  • Cells of the present invention are also be useful for promoting angiogenesis and wound healing following angioplasty or endarterectomy, to develop coronary collateral circulation, for revascularization in the eye, for complications related to poor circulation such as diabetic foot ulcers, for stroke, following coronary reperfusion using pharmacologic methods and other indications where angiogenesis is of benefit.
  • An ischemic event is the disruption of blood flow to an organ, resulting in necrosis or infarct of the non-perfused region.
  • Ischemia-reperfusion is the interruption of blood flow to an organ, such as the heart or brain, and subsequent restoration (often abrupt) of blood flow. While restoration of blood flow is essential to preserve functional tissue, the reperfusion itself is known to be deleterious. In fact, there is evidence that reperfusion of an ischemic area compromises endothelium-dependent vessel relaxation resulting in vasospasms, and in the heart compromised coronary vasodilation, that is not seen in an ischemic event without reperfusion (Cuevas et al., Growth Factors 15:29-40, 1997).
  • the cells of the present invention have therapeutic value to reduce damage to the tissues caused by ischemia or ischemia-reperfusion events, particularly in the heart or brain.
  • Other therapeutic uses for the present invention include induction of skeletal muscle neogenesis and/or hyperplasia, cartilage regeneration, bone formation, tendon regeneration, neural neogenesis, neogenesis in the pancreas and kidney, and/or for treatment of systemic and pulmonary hypertension.
  • the various specialized cell functions are attained by stimulating the differentiation of cardiac-derived mesenchymal stem cells (MSCs) down the appropriate cell pathway, e.g.
  • MSCs cardiac-derived mesenchymal stem cells
  • Cardiac-derived MSC induced benefits for treatment of stroke are tested in vivo in rats utilizing bilateral carotid artery occlusion and measuring histological changes, as well as maze performance (Gage et al., Neurobiol. Aging 9:645- 655, 1988).
  • Cardiac-derived MSC-induced efficacy in hypertension is tested in vivo utilizing spontaneously hypertensive rats (SHR) for systemic hypertension (Marche et al., Clin. Exp. Pharmacol Physiol Suppl 1:S114-116, 1995).
  • SHR spontaneously hypertensive rats
  • These cells are characterized by being highly refractory to light, having small size (typically less than 10 microns and often less than 5 microns), round shape and slow growth.
  • the cells are further characterized by their capacity to proliferate (i.e., self- renew) in a medium containing a carbon source, a nitrogen source, insulin and transferrin. Addition of stem cell factor (commercially available from Amgen), acid or basic fibroblast growth factor, zFGF-5, leukemia inhibitory factor (commercially available) or increasing concentration of serum stimulates propagation.
  • stem cell factor commercially available from Amgen
  • acid or basic fibroblast growth factor zFGF-5
  • leukemia inhibitory factor commercially available
  • increasing concentration of serum stimulates propagation.
  • the cells are further characterized by capacity to differentiate into several cell types.
  • the cell types include cells in various states of differentiation more advanced than the nonadherent cardiac-derived stem cells. These cell types include adherent cardiac-derived stem cells (see below), fibroblasts, myoblasts, smooth muscle cells, skeletal muscle cells, cardiocytes, chondroblasts, keratinocytes and chondrocytes. Probably, adipoblasts, adipocytes, osteoblasts and osteocytes are also present in progeny cells. Tendocytes may also be present. The relationship of the nonadherent cardiac- derived stem cells to differentiated progeny cell lineages and types is shown in Fig. 1.
  • Cardiac-derived stem cells can be stimulated to differentiate by propagation in FBS-supplemented media. Differentiation can be stimulated by treatment with growth factors, such as stem cell growth factor, and by contact inhibition. Higher concentrations of horse serum, although stimulating proliferation, have a tendency to inhibit differentiation.
  • the type of growth factor used to induce differentiation can bias differentiation toward a selected lineage. Retinoic acid, TGF- ⁇ , bone morphogenic proteins (BMP), ascorbic acid, and ⁇ -glycerophosphate lead to production of osteoblasts. Indomethacin, IBMX (3-isobutyl-l-methylxanthine), insulin, and triiodithyrocine (T3) lead to production of adipocytes.
  • aFGF, bFGF, vitamin D3, TNF- ⁇ and retinoic acid lead to production of myocytes.
  • zFGF-5 leads to expansion of cardiocytes progenitors although may also be effective later in the pathway from adherent cells to cardiocytes.
  • Adherent pluripotent cardiac-derived stem cell results from proliferation and partial differentiation of the nonadherent stem cells described in the previous section.
  • the cells are characterized by amorphous shape and lack of differentiation markers tested to-date. Differentiation markers tested include alkaline phosphatase expression, which is 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 types as the nonadherent stem cells.
  • the rate of proliferation and the lineage to which differentiation can be induced and can be controlled by supplementation of media with growth factors as described above.
  • the starting material is a heart or heart biopsy from a human or animal subject.
  • the subject can be embryonic beyond the mesoderm stage, neonatal, infant or adult. If immortalized cells are desired, the starting material can be obtained from the heart of a transgenic animal that is deficient in one or both copies of a tumor suppressor gene.
  • tumor suppressor genes include p53, p21, and the retinoblastoma gene.
  • Transgenic mice with a homozygous mutation in p53 are commercially available from Taconic Farms or Jackson Labs.
  • Cells are grown at about 37DC in an atmosphere of 95% 0 2 and 5% CO 2 on a plastic substrate. Initially, most cells are adherent myocytes. However, most of the adherent myocytes (e.g., at least 75 or 90%) die within 5-30 days and small nonadherent cells appear in suspension in increasing concentration. The time period is the shorter end of the above range for p53 deficient cells and the longer end of the range for normal cells. The small nonadherent cells are pluripotent cardiac-derived stem cells.
  • Differentiated cell progeny of stem cells are recognized in part by the presence of differentiation markers.
  • muscle cells There are three types of muscle cells, cardiocytes (i.e., cardiac muscle cells), striated muscle and smooth muscle cells.
  • the three types of cells derive from a common precursor, termed a myoblast.
  • Cardiac myosin isozyme expression and the cardiac specific pattern of creatine kinase isozyme expression when identified together on the same cell or a clonal population of cells are markers for cardiac muscles cells.
  • Cardiocytes can also be recognized by their bifurcated appearance and capacity to form gap junctions by light microscopy. Such cells can be recognized by forming an electric potential across confluent cells and detecting transfer of signal across the cells.
  • Muscle ⁇ -actin mRNA, and smooth muscle cell actin are differentiation markers of myocytes.
  • Myosin isozyme expression and a muscle-specific pattern of creatine kinase isozyme expression when identified in a cell or clonal population are markers for skeletal muscle cells.
  • ALP, osteocalcin expression, PTH-induced cAMP expression, and bone mineralization capacity identified together in a cell or clonal population of cells are markers of differentiation for osteoblasts.
  • Aggrecan and collagen Type IIB identified in a cell or clonal population of cells, Alcian Blue staining, which detects production of chondroitin sulfate, are markers for chondrocytes.
  • Basic FGF also known as FGF-2
  • FGF-2 Basic FGF
  • endothelial cells vascular smooth muscle cells, fibroblasts, and generally for cells of mesoderm or neuroectoderm origin, including cardiac and skeletal myocytes (Gospodarowicz 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 FGF include: increased endothelial release of tissue plasminogen activator, stimulation of extracellular matrix synthesis, chemotaxis for endothelial cells, induced expression of fetal contractile genes in cardiomyocytes (Parker et al., J. Clin. Invest. 85, 507-514, 1990), and enhanced pituitary hormonal responsiveness (Baird et al., J. Cellular Physiol 5, 101-106, 1987.)
  • 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 decreased levels in fetal lung, skeletal muscle, smooth muscle tissues such as small intestine, colon and trachea.
  • the high level of expression of zFGF-5 in fetal and adult heart and its effects in cardiac tissue-derived cells suggests that ZFGF-5 has particular potency in stimulating proliferation and/or differentiation of cardiac-derived stem cells to cardiocytes.
  • the isolation of zFGF-5 and its properties are described in more detail in commonly owned PCT US97/18635, filed October 16, 1997 (incorporated by reference).
  • the nucleotide sequence encoding zFGF-5 is described in SEQ ID NO. 1, and its deduced amino acid sequence is described in SEQ ID No. 2.
  • the sequence of zFGF-5 shows some sequence similarity with FGF-8.
  • Cells that can be continuously cultured and do not die after a limited number of cell generations are termed immortalized.
  • a cell that survives for only 20 to 80 population doublings is considered finite (Freshney, Culture of Animal Cells (Wiley- Liss, NY, 1994) herein incorporated by reference), and a cell that survives more than 80, preferably at least 100, cell generations is considered immortalized.
  • cells can be immortalized by using a transgenic animal deficient in a tumor suppressor gene as a donor of heart tissue.
  • Cells from other sources can be immortalized by other means. These means include transformation and expression by a gene whose product plays a role in cell senescence, or overexpression or mutation of one or more oncogenes that override the action of the senescence genes. Expression of genes that result in positive signals for cell proliferation include SV40 large T antigen (Linder et al., Exp. Cell Res.
  • Heart disease is the major cause of death in the United States, accounting for up to 30% of all deaths. More than 5 million people are diagnosed with coronary disease in the US. Coronary disease can be due to congestive heart failure, hypertrophic cardiomyopathy, cardiomyopathy, viral infection or myocardial infarction (MI). Myocardial infarction accounts for 750,000 hospital admissions per year in the US. In MI patients, ischemia stimulates growth of fibroblasts and promotes development of greater than normal quantities of fibrous tissue to replace necrotic muscular tissue. Risk factors for MI include diabetes mellitus, hypertension, truncal obesity, smoking, high levels of low density lipoprotein in the plasma or genetic predisposition.
  • MI myocardial infarction
  • Such conditions are treated by administration of cardiac-derived stem cells as described above.
  • Either adherent or nonadherent cells can be used.
  • the cells can be administered either intravenously, intracoronary or intraventricularly.
  • a catheter can be used for the latter two routes of administration, which are more usual for adherent cells.
  • Cells are administered in a therapeutically effective dosage.
  • Such a dosage is sufficient to generate significant numbers of new cardiocytes cells in the heart, and/or at least partially replace necrotic heart tissue, and/or produce a clinically significant change in heart function.
  • a clinically significant improvement in heart performance can be determined by measuring the left ventricular ejection fraction, prior to, and after administration of cells, and determining at least a 5% increase, preferably 10% or more, in the total ejection fraction.
  • Cells can be administered as pharmaceutical compositions, which can also include, depending on the formulation desired, pharmaceutically-acceptable, typically sterile,, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration.
  • diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solutions, dextrose solution, and Hank's solution.
  • the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like
  • stem cells can be preceded, accompanied or followed by administration of growth factor(s) that stimulate proliferation and/or differentiation of the stem cells into cardiocytes.
  • Growth factors can be administered intravenously or intraventricularly. Growth factors are administered in a dosage sufficient to cooperate with administered stem cells in generating significant numbers of new cardiocytes cells in the heart, and/or at least partially replace necrotic heart tissue, and/or produce a clinically significant change in heart function
  • Suitable growth factors include zFGF-5 and stem cell growth factor.
  • cardiac-derived stem cells are administered in combination with anti-inflammatory agents that arrest, reverse or partially ameliorate inflammation associated with coronary disease.
  • Suitable antiinflammatory agents include antibodies to Mac-1, and L-, E and P-selectin (see Springer, Nature 346, 425-433 (1990). Osborn, Cell 62, 3 (1990); Hynes, Cell 69, 11 (1992)).
  • Cardiac-derived stem cells can also be administered with diuretics, ACE inhibitors and ⁇ -adrenergic blockers.
  • the recipient patient of stem cells and the donor from which the cells are obtained are HLA-matched to reduce allotypic rejections.
  • cells are administered under cover of an immunosuppressive regime to reduce the risk of rejection.
  • Immunosuppressive agents that can be used include cyclosporin, corticosteroids, and OKT3.
  • immune responses are avoided by obtaining stem cells from the patient that is to be treated.
  • Stem cells can be obtained by biopsy of heart tissue, and expanded in vitro before readministration.
  • differentiation markers can be identified for these cells, and the cells can be isolated from the blood of the patient to be treated.
  • the cardiac-derived stem cells described above can be used to test compounds for activity in promoting or inhibiting proliferation and/or differentiation of the cells.
  • a compound being tested is contacted with a population of cardiac- derived stem cells, optionally, in the presence of other agents known to promote or inhibit the metabolic pathway or phenotype of interest, and phenotypic and metabolic changes are monitored in comparison with a control in which the compound being tested is absent.
  • Compounds to be tested include known or suspected growth factors, and analogs thereof, libraries of natural compounds not previously known to have activity in promoting proliferation or differentiation and combinatorial libraries of compounds.
  • Peptide libraries can also be generated by phage display methods. See, e.g., Devlin, W0 91/18980.
  • Compounds that cause cardiac-derived stem cells to proliferate and/or differentiate into cardiocytes are useful as therapeutic agents in the same conditions as the cardiac-derived stem cells are useful. Such compounds can be administered alone to stimulate proliferation and differentiation of endogenous cardiac-derived stem cells, or can be administered in conjunction with exogenous cardiac-derived stem cells. Such compounds are screened for proliferating activity by contacting them with cardiac- derived stem cells in growth media, and monitoring an increase in cell count, or incorporation of 3 H-thymidine. Compounds are screened for promoting differentiation to cardiocytes by monitoring cells with the characteristic morphological appearance and differentiation markers of cardiocytes as note above.
  • compounds can be monitored for activity in promoting differentiation of cardiac-derived stem cells to 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 with activity in promoting differentiation to one of the above cell types are useful in treating patients with degenerative diseases of bone, muscle or cartilage.
  • adipocytes Compounds that inhibit differentiation of stem cells to certain cell types such as adipocytes can also be useful in some circumstances.
  • compounds that inhibit differentiation of stem cells to adipocytes can be used in treating obesity.
  • Such compounds are identified by contacting a compound under test with cardiac-derived stem cells under conditions that would otherwise lead to differentiation of the stem cells to a certain cell type, and monitoring a decreased frequency or extent of conversion to the cell type relative to a control in which the compound is omitted.
  • compositions can also include, depending on the formulation desired, pharmaceutically-acceptable, non-toxic carriers or diluents, as described above.
  • DF 10 contains 10% HIA FBS DF 5 , contains 5% HIA FBS
  • Enzymes are dissolved in PBS (Gibco Laboratories) w/ 1% Glucose
  • Steps 2-5 were repeated until the hearts were completely digested.
  • the myocyte fraction was spun at 1650 rpm for 10 min at RT. 13) The resulting pellet contained 3.75 x 10 6 cells, which were plated equally into 5 wells in 5 ml DF5. 14) On A/20 the media was changed to plating media (PM) in 6-well tissue culture plates, which was replaced again with fresh PM on A/22 and A/25. Adherent beating cardiocytes were apparent from A 20 through A/25, although in diminishing numbers. Additionally, very small suspension cells were present in the wells. 15) On A/26 these suspension cells were split 1 :3 into PM, PM plus 1 % horse serum (HS), and PM plus 10% HS (HyClone, Logan, Utah).
  • PM plating media
  • the cells maintained in PM plus 10% HS were still viable by light refraction and trypan blue exclusion. Cells in PM only were not viable. Cells in PM and 1% HS had become adherent cells and are described in more detail below.
  • the suspension cell density was determined, using a hemacytometer, to be 6 x 10 6 /ml. Also, on C/19, the cells grown in PM + 10% HS were spun at 1500 rpm for
  • the suspension cells in PM plus 10% HS were at 8.5 x 10 5 /ml, while the suspension cells in SFM plus 10% HS were at 8.75 x 10 5 /ml.
  • Half of these suspension cells (both the SFM and PM cells) were resuspended in PM plus 10% HS or SFM plus 10% HS, and were plated on methylcellulose in the presence of various cytokines and growth factors.
  • the suspension cells did not form colonies on methylcellulose, even after 12 days in culture suggesting that the cells could be primitive stem cells. At this time, culturing the suspension cells in 10% HS no longer appeared to impact the cells significantly, so the cells were passaged in PM only or SFM only.
  • zFGF-5 150 pg/ml
  • stem cell factor SCF 10 ng/ml
  • SCF 10 ng/ml stem cell factor
  • a one month treatment of the suspension cells with 10 ng/ml SCF in PM resulted in the appearance of an adherent layer of cells of mixed lineage designated SCF/PM-derived cells.
  • treating the suspension cells with SCF in SFM did not give rise to adherent cell lineages, implying that some other growth factor present in the 15% FBS found in PM is essential, in addition to the SCF.
  • Cells with a phenotype of smooth muscle myocytes were amorphous or star-shaped, skeletal muscle cells had the shape of a pen. Myoblasts were intermediate between fibroblasts and skeletal muscle cells. Other lineages are probably present, and can be characterized by using immunohistochemistry, immunofluorescence and FACS analysis.
  • the cells maintained in PM plus 1% HS had differentiated into adherent cells of mixed morphology. These cells were trypsinized, pelleted, and then split 1:6 into SFM, SFM plus 1% HS, SFM plus 10% HS, PM, PM plus 1% HS or PM plus 10% HS.
  • adherent cells were expanded in PM only as the different media or HS concentrations did not have any noticeable affect on the adherent cell morphology.
  • the adherent cells appear to be a mixed population including pericytes, fibroblasts, cardiocytes, smooth muscle myocytes, skeletal muscle myocytes.
  • the adherent cells were subcloned on C/08. From the subcloning, 11 clones were obtained and expanded on plating media in 95% O 2 / 5% CO 2 at 37 DC. The cells were characterized by the following criteria.
  • Alkaline phosphatase staining identifies pericytes, osteoblast precursors and chondrocyte precursors and mature osteoblasts and chondrocytes
  • Acetylated LDL uptake endothelial cell marker
  • MF-20 Ab Muse anti-adult chicken pectoralis myosin, which crossreacts with mouse myosin and detects both adult skeletal and cardiac myocyte myosin
  • M0636 Dako #5/56 mouse anti-human muscle actin; recognizes alpha actin in cardiac, skeletal and smooth muscle myocytes, and gamma actin in smooth muscle myocytes
  • Mitogenic response to zFGF-5, acidic FGF and basic FGF This test tests for proliferation capacity.
  • the cells were plated in 96-well plates in PM and grown to confluence and then switched to serum-free media for 24 hr after which growth factor and tritiated thymidine was added. (7) Production of zFGF-5 rnRNA identified by Northern blotting which is expressed at high concentration by cardiocyte lineage cells.
  • Cells in this population showed different morphologies characteristic of pericytes, fibroblasts, cardiocytes, smooth muscle myocytes, skeletal muscle myocytes, and other cells.
  • the cells showed a mitogenic response to zFGF-5 (cFGF), acidic FGF (aFGF) and basic FGF.
  • cFGF zFGF-5
  • aFGF acidic FGF
  • basic FGF basic FGF
  • bFGF was most potent in inducing a mitogenic response.
  • the response increased in a concentration dependent manner from 1 pg/ml up to 1 Dg/ml.
  • serum free media the cells showed moderate growth and differentiated into cells with an appearance akin to frying eggs. This appearance suggests early stage myocytes.
  • Clone 1D9 was isolated from a single cell of the mixed population described above in 3a. Some cells propagated from 1D9 showed morphology of cardiocytes, and other cells showed different morphology. A few cells stained faintly positive for alkaline phosphatase. The cells did not take up acetylated LDL. MF-20 did not bind to the cells. M0636 did bind to cells indicting that some cells were of smooth muscle, skeletal muscle, and/or cardiocyte lineage. zFGF-5, acidic FGF and basic FGF all induced a mitogenic response and bFGF was most potent. Serum-free media induced slow growth but not obvious morphological changes.
  • Cells expanded from 2E7 have the morphology of myoblast (skeletal myocyte/fibroblast/adipocyte precursor).
  • the cells were negative for alkaline phosphatase staining, acetylated LDL uptake and MF-20 binding.
  • the cells were positive for M0636 binding indicating the presence of precursors of smooth, skeletal and/or cardiocyte muscle cells.
  • the cells proliferated in response to zFGF-5, acidic FGF and basic FGF, with basic FGFbeing the most potent.
  • the cells proliferated and changed morphology to appear as large polygonal cells, which may be early stage myocytes.
  • the cells were negative for MF-20 binding.
  • On treatment with ascorbic acid/ ⁇ -GP cells differentiated to "ridge-like" formations on Day 11. No mineralization was observed even after day 25.
  • Clone 1G11 was also isolated as a single cell from the mixed population of adherent p53 deficient cells described above. Cells expanded from 1G11 had the morphology of skeletal myocytes, smooth muscle myocytes, cardiocytes, and other cell types. About 20-25% of cells stained positive for alkaline phosphatase. The cells showed a mitogenic response to zFGF-5, acidic FGF and basic FGF, with bFGF the most potent and bFGF and aFGF equally active. Addition of serum free media caused a slow growth rate and the appearance of star-shaped cells, which are myocyte precursors. The cells did not bind MF-20. Treatment with ascorbic acid/ ⁇ -GP did not result in mineralization even after Day 25.
  • This clone showed the morphology of skeletal myocytes, smooth muscle myocytes, cardiocytes, and other cell types.
  • Cells were negative for alkaline phosphatase staining and acetylated LDL uptake, MF-20 binding.
  • the cells showed a mitogenic response to bFGF and aFGF, but not to zFGF-5.
  • Treatment with ascorbic acid/ ⁇ -GP induced resulted in cells gathering together in 'ridge-like' formations on Day 11. No mineralization occurred even after Day 25.
  • This clone showed the morphology of myoblasts (skeletal myocyte/fibroblast/adipocyte precursors).
  • the cells were negative for alkaline phosphatase staining, acetylated LDL uptake, MF-20 binding.
  • the cells were slightly positive for M0636 binding.
  • the cells showed a mitogenic response to zFGF-5, acidic
  • FGF FGF and basic FGF. aFGF and bFGF were most potent, all 3 FGFs were equally active.
  • This clone had the morphology of multinucleated cardiocytes, and other cell types. Some cells were positive for alkaline phosphatase staining. The cells were negative for acetylated LDL uptake, and MF-20 binding. The cells were positive for M0636 binding. The cells showed a mitogenic response to zFGF-5, acidic FGF and basic
  • FGF FGF
  • bFGF bFGF
  • aFGF and bFGF being equally active.
  • SFM induced moderate growth rate, and no distinct morphology change.
  • Cells were negative for MF-20 binding.
  • Ascorbic acid/ ⁇ -GP treatment did not result in mineralization even after Day 25.
  • This clone had the morphology of myoblasts (skeletal myocyte/fibroblast/adipocyte precursors). 5% of cells stained with alkaline phosphatase.
  • the cells were negative for acetylated LDL uptake, MF-20 binding, and M0636 binding. SFM induced moderate growth rate resulting in star-shaped cells.
  • Ascorbic acid/ ⁇ -GP-induced differentiation resulted in cells gathering in a 'ridge-like' formation on Day 11.
  • the cells had a stellate appearance characteristic of myoblasts.
  • the cells were negative for alkaline phosphatase staining, negative for acetylated LDL, negative for MF-20 binding and negative for M0636 binding.
  • the cells showed a mitogenic response to zFGF-5, acidic FGF and basic FGF, with bFGF being the most potent, and all 3 FGFs being equally active.
  • SFM treatment caused moderate growth and the appearance of some large star-shaped cells.
  • Treatment with ascorbic acid/ ⁇ -GP induced differentiation with cells gathering together in 'ridge-like' formations on Day 11 (many formations were evident). No mineralization occurred even after Day 25.
  • the cells showed a mitogenic response to zFGF-5, acidic FGF and basic FGF.
  • bFGF and cFGF were equally potent and active from 10 fg/ml up to 1 Dg/ml.
  • FGF stimulation regenerated the original suspension cell progenitor population and produced zFGF-5 mRNA.
  • zFGF-5 150 pg/ml (1 day) stimulation produced clear adult cardiocytes by morphology.
  • Treatment for five days resulted in formation of haystack structure, ridges and troughs.
  • SFM treatment resulted in strong growth, loss of multinucleation, and appearance of large polygonal cells.
  • Contact-dependent differentiation produced cardiocytes, chondrocytes, and other cell types. Ascorbic acid/ ⁇ -GP-treatment resulted in differentiation and larger cells by Day 6. No mineralization occurred even after Day 25.
  • the cells had a stellate or haystack appearance when sub-confluent suggesting myoblasts (skeletal myocyte/fibroblast/adipocyte precursor).
  • the cells were negative for acetylated LDL uptake, negative for alkaline phosphatase staining, negative for MF-20 binding and positive for M0636 binding.
  • SFM treatment caused rapid growth, and loss of haystack or stellate appearance.
  • Ascorbic acid/ ⁇ -GP treatment resulted in appearance of chondrocyte-like cells on Day 6. No mineralization occurred even after Day 25.
  • the cells showed the morphology of cardiocytes and other cells.
  • the cells were negative for alkaline phosphatase staining, acetylated LDL uptake, MF-20 binding and positive for M0636 binding.
  • the cells showed a mitogenic response to zFGF-5, acidic FGF and basic FGF.
  • bFGF was the most potent, and all 3 FGFs were equally active.
  • SFM treatment caused reduced growth rate, reduced adherence to plastic, and appearance of large polygonal cells.
  • Treatment with ascorbic acid/ ⁇ -GM resulted in cells gathering together in 'ridge-like' formations on Day 11. No mineralization occurred even after Day 25.
  • PBS PBS several times. (Rinse until all red blood cells and debris is removed). From step 3) PBS is supplemented with 1% Glucose.
  • Digestion which contains mainly fibroblasts, red blood cells and debris.
  • Assays were performed to measure the frequency of fibroblast colony forming units from monkey low density, non-adherent cells isolated from bone marrow. This assay is indicative of mesenchymal stem cell frequency.
  • One half of a 96 well microtiter plate is inoculated with cells at a density of 10,000 cells/well and the other half of the plate is inoculated with cells at a density of 1,000 cells/well.
  • the culture medium is ⁇ MEM (GIBCO-BRL, Gaithersburg, MD), 2% bovine serum albumin, 10 ⁇ g/ml insulin, 200 ⁇ g/ml transferrin, antibiotic and 50 ⁇ M ⁇ - Mercaptoethanol.
  • the cells are incubated at 37°C in 5% CO 2 for 14 days and then stained with toluidine blue to improve cell visibility and examined microscopically.
  • Positive wells have at least 50 cells exhibiting a "stromal" morphology, i.e., large, spread out cells.
  • the positive control is medium containing 20% fetal bovine serum. Results demonstrated that zFGF5, at a concentration of 100 ng/ml increased the frequency of CFU-F to levels equivalent to the positive control of 20% FBS.
  • a putative mesenchymal stem cell as a target i.e. having a receptor
  • zFGF5 was made using FITC-labeled protein and either neonatal mouse or fetal lamb (third trimester) heart tissue.
  • zFGF5 purified as described above, was dialyzed into 0.1 M sodium bicarbonate pH 9.0.
  • Fluorescein isothiocyanate (FITC; Molecular Probes, Eugene, OR) was dissolved at 1 mg/ml in the same buffer without exposure to strong light. The mixture was prepared containing 1 mg FITC/1 mg zFGF5, and reacted for 1-2 hours in the dark at room temperature.
  • the reaction was stopped by adding 1 M glycine to a final concentration of 0.1 M, then reacted for 1 hour at room temperature. The mixture was then dialyzed against 0.1 M sodium bicarbonate to make a 1:500-1 :1000 dilution for 3 hours. The dialysis solution was changed and the process repeated for 3-18 hours to remove unlabeled FITC.
  • Neonatal mouse or fetal lamb heart ventricles were isolated, minced, and repeatedly washed in phosphate buffered solution (PBS) until all red blood cells and debris were removed.
  • PBS phosphate buffered solution
  • the minced ventricles were placed in a solution containing 18 ml PBS and 1% glucose and 1 ml of 2% DNAse/Collagenase solution was added. The mixture was incubated on a shaker for 30 minutes at 37°C. The supernatant was discarded and the process was repeated once more.
  • DF 20 Dulbecco's Modified Eagle's Medium/Ham's Nutrient Mixture F12, 1:1 (GIBCO-BRL, Gaithersburg, MD) and 20% fetal bovine serum.
  • DF 20 Dulbecco's Modified Eagle's Medium/Ham's Nutrient Mixture F12, 1:1 (GIBCO-BRL, Gaithersburg, MD) and 20% fetal bovine serum.
  • the tubes were centrifuged at 1650 rpms in a Beckman CS-6R centrifuge (Beckman, Fullerton, CA) at 4°C for 10 minutes.
  • the supernatant was discarded and the pellet was resuspended in DF 10 (10% FBS).
  • the cells were kept cold and spun again and resuspended in 40 ml of DF 10.
  • the cell mixture was passed over a 40 im filter (Becton Dickinson, Detroit, MI) and counted using a hemacytometer.
  • the cells were incubated in FITC-labeled zFGF5 at 4°C for 30 minutes at a concentration of 2 x 10 6 cells/1 _ ⁇ g zFGF5. After incubation, the cells were spun at 1650 rpms in a Beckman CS-6R centrifuge (Beckman) for 5 minutes. The supernatant was discarded and the pellet washed once in 10 ml of DF 10 and resuspended in 4 ml DF 10.
  • MACS positive selection type LS+ separation columns (Miltenyi Biotech) were washed with 3 ml of MAC buffer (PBS, 0.5% BSA, 2 mM EDTA) and the cell/bead mixture was washed in 10 ml MAC buffer and then resuspended in 6 ml MAC buffer. The cell/bead mixture was divided between the two columns and the first negative fraction was discarded. 1.5 ml of 0.6 M NaCl was added to each column and eluted but not collected. The columns were then washed with 1.5 ml MAC buffer.
  • MAC buffer PBS, 0.5% BSA, 2 mM EDTA
  • the cells bound with FITC-labeled zFGF5 were collected by adding 3 ml MAC buffer, removing the column from the magnet and flushing out the positive cells using the plunger.
  • the positive cell fraction was plated in a T75 flask and 50 ml of plating medium was added (DF with 15% FBS and antibiotics). The cells were incubated at 37°C for 1 week and counted. The yield of positive cells was approximately 0.1% of original total cells counted.
  • TEM transmission electronmicroscopy
  • MSCs isolated from healthy rats with a similar genetic background by intra-pericardial, intra-coronary, intra-arterial, intra- ventricular or intra- venous injection to rats receiving subcutaneous infusions of epinephrine or saline.
  • rats 300 gm are implanted with epinephrine in an osmotic pump and are injected with cardiac-derived MSCs or control cells and mortality is monitored for 2 weeks, at the end of which the rats are sacrificed, the hearts are weighed wet, and fixed in 10% neutral buffered formalin for histology. Prior to sacrificing, cardiac function is measured.
  • Measurement include body weight, heart weight, cardiac fibrosis and cardiohistomorphometry in epinephrine-infused rats.
  • Cardiac fibrosis is determined by scoring Masson's Trichrome stained heart sections. Three sections are scored for each heart, and the average score taken. Positive results indicate that infusion of cardiac- derived MSCs can be beneficial in the setting of heart failure of varying etiologies, of which can include myocardial infarct (MI), idiopathic dilated cardiomyopathy (IDCM), hypertrophic cardiomyopathy, viral myocarditis, congenital abnormalities, and obstructive diseases.
  • MI myocardial infarct
  • IDCM idiopathic dilated cardiomyopathy
  • hypertrophic cardiomyopathy viral myocarditis
  • congenital abnormalities congenital abnormalities
  • obstructive diseases include myocardial infarct (MI), idiopathic dilated cardiomyopathy (IDCM), hyper
  • the invention includes a number of uses, some of which can be expressed concisely as follows.
  • the invention provides for the use of nonadherent cardiac-derived stem or adherent cardiac-derived stem cells in the treatment of disease and or in the discovery of drugs for use in the same.
  • the invention further provides for the use of nonadherent or adherent cardiacderived stem cells in the manufacture of a medicament for treatment of disease.

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Abstract

L'invention concerne des cellules souches hématopoïétiques d'origine cardiaque, qui une fois soumises à la prolifération et à la différentiation, peuvent produire divers types de cellules, notamment des cardiocytes, des fibroblastes, des cellules musculaires lisses, des cellules musculaires squelettiques, des kératinocytes, des ostéoblastes, et des chondrocytes. Les cellules de cette invention peuvent être utilisées dans différentes méthodes permettant de traiter des patients souffrant d'un tissu cardiaque nécrotique, ces cellules souches subissant la prolifération et la différentiation de manière à produire des cardiocytes destinés à remplacer ledit tissu nécrotique. Ces cellules peuvent également être utilisées pour détecter des composés qui présentent une activité favorisant la prolifération et/ou la différentiation de cellules souches d'origine cardiaque.
EP99912853A 1998-03-23 1999-03-23 Cellules souches d'origine cardiaque Withdrawn EP1064356A2 (fr)

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WO1999049015A2 (fr) 1999-09-30
AU3112499A (en) 1999-10-18
JP2002507407A (ja) 2002-03-12
WO1999049015A3 (fr) 1999-12-16
CA2324350A1 (fr) 1999-09-30

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