EP1315510A2 - Procedes et compositions permettant la reparation et/ou la regeneration de myocarde endommage - Google Patents

Procedes et compositions permettant la reparation et/ou la regeneration de myocarde endommage

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Publication number
EP1315510A2
EP1315510A2 EP01961855A EP01961855A EP1315510A2 EP 1315510 A2 EP1315510 A2 EP 1315510A2 EP 01961855 A EP01961855 A EP 01961855A EP 01961855 A EP01961855 A EP 01961855A EP 1315510 A2 EP1315510 A2 EP 1315510A2
Authority
EP
European Patent Office
Prior art keywords
cells
stem cells
hematopoietic stem
myocardium
heart
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Withdrawn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
EP01961855A
Other languages
German (de)
English (en)
Other versions
EP1315510A4 (fr
Inventor
Piero Anversa
Donald Orlic
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.)
New York Medical College
US Government
Original Assignee
New York Medical College
US Department of Health and Human Services
US Government
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by New York Medical College, US Department of Health and Human Services, US Government filed Critical New York Medical College
Publication of EP1315510A2 publication Critical patent/EP1315510A2/fr
Publication of EP1315510A4 publication Critical patent/EP1315510A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • 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
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood cells
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/12—Hepatocyte growth factor [HGF]

Definitions

  • the present invention relates generally to the field of cardiology, and more particularly relates to methods and cellular compositions for treatment of a patient suffering from a cardiovascular disease, including, but not limited to, atherosclerosis, ischemia, hypertension, restenosis, angina pectoris, rheumatic heart disease, congenital cardiovascular defects and arterial inflammation and other disease of the arteries, arterioles and capillaries.
  • a cardiovascular disease including, but not limited to, atherosclerosis, ischemia, hypertension, restenosis, angina pectoris, rheumatic heart disease, congenital cardiovascular defects and arterial inflammation and other disease of the arteries, arterioles and capillaries.
  • the present invention relates to any one or more of:
  • Methods and/or pharmaceutical composition comprising a therapeutically effective amount of somatic stem cells alone or in combination with a cytokine such as a cytokine selected from the group consisting of stem cell factor (SCF), granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), stromal cell-derived factor- 1, steel factor, vascular endothelial growth factor, macrophage colony stimulating factor, granulocyte- macrophage stimulating factor or Interleukin-3 or any cytokine capable of the stimulating and/or mobilizing stem cells.
  • a cytokine selected from the group consisting of stem cell factor (SCF), granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), stromal cell-derived factor- 1, steel factor, vascular endothelial growth factor, macrophage colony stimulating factor, granulocyte- macrophage stimulating factor or Interle
  • Cytokines may be administered alone or in combination of with any other cytokine capable of: the stimulation and/or mobilization of stem cells; the maintenance of early and late hematopoiesis (see below); the activation of monocytes (see below), macrophage/monocyte proliferation; differentiation, motility and survival (see below) and a pharmaceutically acceptable carrier, diluent or excipient (including combinations thereof).
  • the stem cells are advantageously adult stem cells, such as hematopoietic or cardiac stem cells or a combination thereof or a combination of cardiac stem cells and any other type of stem cells.
  • the implanting, depositing, administering or causing of implanting or depositing or administering of stem cells such as adult stem cells, for instance hematopoietic or cardiac stem cells or a combination thereof or any combination of cardiac stem cells (e.g., adult cardiac stem cells) and stem cells of another type of (e.g., adult stem cells of another type), alone or with a cytokine such as a cytokine selected from the group consisting of stem cell factor (SCF), granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), stromal cell-derived factor- 1, steel factor, vascular endothelial growth factor, macrophage colony stimulating factor, granulocyte-macrophage stimulating factor or Interleukin-3 or any cytokine capable of the stimulating and/or mobilizing stem cells (wherein "with a cytokine " can include sequential implanting, depositing administering or causing of implanting or depositing or administering of
  • This implanting, depositing, or administering or causing of implanting, depositing or administering can be in conjunction with grafts.
  • Such implanting, depositing or administering or causing of implanting, depositing or administering is advantageously employed in the treatment or therapy or prevention of cardiac conditions, such as to treat areas of weakness or scarring in the heart or prevent the occurrence or further occurrence of such areas or to treat conditions which cause or irritate such areas, for instance myocardial infarction or ischemia or other e.g., genetic, conditions that impart weakness or scarring to the heart (see also cardiac conditions mentioned infra).
  • stem cells alone or in combination with said cytokine(s), in the formulation of medicaments for such treatment, therapy or prevention.
  • Medicaments for use in such treatment, therapy or prevention comprising the stem cells and optionally the cytokine(s).
  • Kits comprising the stem cells and optionally the cytokine(s) for formulations for use in such treatment, therapy or prevention.
  • Compositions comprising such stem cells and optionally at least one cytokine and kits for preparing such compositions.
  • kits and compositions described herein Methods of making the kits and compositions described herein. Methods of implanting or depositing stem cells or causing the implanting or depositing of stem cells. BACKGROUND OF THE INVENTION
  • Cardiovascular disease is a major health risk throughout the industrialized world.
  • Atherosclerosis the most prevalent of cardiovascular diseases, is the principal cause of heart attack, stroke, and gangrene of the extremities, and thereby the principal cause of death in the United States.
  • Atherosclerosis is a complex disease involving many cell types and molecular factors (for a detailed review, see Ross, 1993, Nature 362: 801-809).
  • Ischemia is a condition characterized by a lack of oxygen supply in tissues of organs due to inadequate perfusion. Such inadequate perfusion can have number of natural causes, including atherosclerotic or restenotic lesions, anemia, or stroke, to name a few. Many medical interventions, such as the interruption of the flow of blood during bypass surgery, for example, also lead to ischemia. In addition to sometimes being caused by diseased cardiovascular tissue, ischemia may sometimes affect cardiovascular tissue, such as in ischemic heart disease. Ischemia may occur in any organ, however, that is suffering a lack of oxygen supply.
  • MI myocardial infarction
  • This area of necrotic tissue is referred to as the infarct site, and will eventually become scar tissue.
  • Current treatments for MI focus on reperfiision therapy, which attempts to start the flow of blood to the affected area to prevent the further loss of tissue.
  • the main choices for reperfiision therapy include the use of anti-thrombolytic agents, or performing balloon angioplasty, or a coronary artery bypass graft.
  • Anti- thrombolytic agents solubilize blood clots that may be blocking the artery, while balloon angioplasty threads a catheter into the artery to the site of the occlusion, where the tip of the catheter is inflated, pushing open the artery.
  • Still more invasive procedures include the bypass, where surgeons remove a section of a vein from the patient, and use it to create a new artery in the heart, which bypasses the blockage, and continues the supply of blood to the affected area.
  • bypass there were an estimated 553,000 coronary artery bypass graft surgeries and 539,000 percutaneous transluminal coronary angioplastys. These procedures average $27,091 and $8,982 per patient, respectively (American Heart Association, 2000).
  • progenitors very immature cells
  • stem cells progenitor cells themselves derive from a class of progenitor cells called stem cells.
  • stem cells have the capacity, upon division, for both self-renewal and differentiation into progenitors. Thus, dividing stem cells generate both additional primitive stem cells and somewhat more differentiated progenitor cells.
  • stem cells also give rise to cells found in other tissues, including but not limited to the liver, brain, and heart.
  • Stem cells have the ability to divide indefinitely, and to specialize into specific types of cells.
  • Totipotent stem cells which exist after an egg is fertilized and begins dividing, have total potential, and are able to become any type of cell. Once the cells have reached the blastula stage, the potential of the cells has lessened, with the cells still able to develop into any cell within the body, however they are unable to develop into the support tissues needed for development of an embryo.
  • the cells are considered pluripotent, as they may still develop into many types of cells. During development, these cells become more specialized, committing to give rise to cells with a specific function.
  • These cells, considered multipotent are found in human adults and referred to as adult stem cells. It is well known that stem cells are located in the bone marrow, and that there is a small amount of peripheral blood stem cells that circulate throughout the blood stream (National Institutes of Health, 2000).
  • stem cells Due to the regenerative properties of stem cells, they have been considered an untapped resource for potential engineering of tissues and organs. It would be an advance to provide uses of stem cells with respect to addressing cardiac conditions.
  • U.S. Patent No. 6,001,934 involving the development of functional islets from islets of Langerhans stem cells.
  • U.S. Patents Nos.5,906,934 and 6,174,333 pertaining to the use of mesenchymal stem cells for cartilage repair, and the use of mesenchymal stem cells for regneration of ligaments; for instance, wherein the stem cells are embedded in a gel matrix, which is contracted and then implanted to replace the desired soft tissue.
  • PCT/US00/08353 (WO 00/57922) and PCT US99/17326 WO 00/06701) involving intramyocardial injection of autologous bone marrow and mesenchymal stem cells which fails to teach or suggest administering, implanting, depositing or the use of hematopoietic stem cells as in the present invention, especially as hematopoietic stem.
  • cells as in the present invention are advantageously isolated and/or purified adult hematopoietic stem cells.
  • stem cells in medicine are for the treatment of cancer.
  • bone marrow is transplanted into a patient whose own marrow has been destroyed by radiation, allowing the stem cells in the transplanted bone marrow to produce new, healthy, white blood cells.
  • stem cells are transplanted into their normal environment, where they continue to function as normal.
  • any particular stem cell line was only capable of producing three or four types of cells, and as such, they were only utilized in treatments where the stem cell was required to become one of the types of cells for which their ability was already proven.
  • researchers are beginning to explore other options for treatments of myriad disorders, where the role of the stem cell is not well defined. Examples of such work will be presented in support of the present invention.
  • Organ transplantation has been widely used to replace diseased, nonfunctional tissue. More recently, cellular transplantation to augment deficiencies in host tissue function has emerged as a potential therapeutic paradigm.
  • One example of this approach is the well publicized use of fetal tissue in individuals with Parkinsonism (reviewed in Tompson, 1992), where dopamine secretion from transplanted cells alleviates the deficiency in patients.
  • transplanted myoblasts from uneffected siblings fused with endogenous myotubes in Duchenne's patients; importantly the grafted myotubes expressed wild-type dystrophin (Gussoni et al., 1992).
  • Figure 1 shows a log-log plot showing Lin " bone marrow cells from EGFP transgenic mice sorted by FACS based on c-kit expression (The fraction of c-kit F0S cells (upper gate) was 6.4%. c-Att NBG cells are shown in the lower gate. c-J ⁇ t ?os cells were 1-2 logs brighter than c-fcft NEG cells)
  • Figure 2 A shows a photograph of a tissue section from a MI induced mouse
  • Figure 2B shows a photograph of the same tissue section of Figure 2 A at a higher magnification, centering on the area of the MI with magnification being 5 OX;
  • Figures 2C, D show photographs of a tissue section at low and high magnifications of the area of MI, injected with Lin " c-f ⁇ ' t pos cells, with the magnification of 2C being 25X, and the magmfication of 2D being 50 X;
  • Figures 3 A-C show photographs of a section of tissue from a MI induced mouse, showing the area of MI injected with Lin " c-kit P0S cells (Visible is a section of regenerating myocardium from endocardium (EN) to epicardium (EP). All photographs are labeled to show the presence of infarcted tissue in the subendocardium (IT) and spared myocytes in the subendocardium (SM).
  • Figure 3A is stained to show the presence of EGFP (green). Magnification is 250X.
  • Figure 3B is stained to show the presence of cardiac myosin (red).
  • Magnification is 250X.
  • Figure 3C is stained to show the presence of both EGFP and myosin (red-green), as well as Pi-stained nuclei (blue). Magnification is 250X);
  • Figure 4 A shows of grafts depicting the effects of myocardial infarction on left ventricular end-diastolic pressure (LNEDP), developed pressure (LNDP), LN + rate of pressure rise (dP/dt), and LN - rate of pressure decay (dP/dt)
  • LNEDP left ventricular end-diastolic pressure
  • LNDP developed pressure
  • dP/dt LN + rate of pressure rise
  • dP/dt LN - rate of pressure decay
  • Figures 5A-I show photographs of a tissue sections from a MI induced mouse depicting regenerating myocardium in the area of the MI which has been injected with Lin " c-kit ?os cells
  • Figure 5A is stained to show the presence of EGFP (green). Magnification is 300X.
  • Figure 5B is stained to show the presence of ⁇ - smooth muscle actin in arterioles (red). Magnification is 300X.
  • Figure 5C is stained to show the presence of both EGFP and ⁇ -smooth muscle actin (yellow-red), as well as Pi-stained nuclei (blue). Magnification is 300X.
  • Figures 5D-F and G-I depict the presence of MEF2 and Csx/Nkx2.5 in cardiac myosin positive cells.
  • Figure 5D shows Pi-stained nuclei (blue). Magnification is 300X.
  • Figure 5E is stained to show MEF2 and Csx/Nkx2.5 labeling (green). Magnification is 300X.
  • Figure 5F is stained to show cardiac myosin (red), as well as MEF2 or Csx/Nkx2.5 with PI (bright fluorescence in nuclei). Magmfication is 300X.
  • Figure 5G shows Pi-stained nuclei (blue). Magnification is 300X.
  • Figure 5H is stained to show MEF2 and Csx/Nkx2.5 labeling (green). Magnification is 300X.
  • Figure 51 is stained to show cardiac myosin (red), as well as MEF2 or Csx/Nkx2.5 with PI (bright fluorescence in nuclei). Magnification is 300X);
  • Figure 6 shows photographs of tissue sections from MI induced mice, showing regenerating myocardium in the area of the MI injected with Lin " c-kit F s cells
  • Figures 6A-C show tissue which has been incubated in the presence of antibodies to BrdU.
  • Figure 6A has been stained to show Pi-labeled nuclei (blue).
  • Magnification is 900X.
  • Figure 6B has been stained to show BrdU- and Ki67-labeled nuclei (green).
  • Magnification is 900X.
  • Figure 6C has been stained to show the presence of ⁇ -sarcomeric actin (red).
  • Magnification is 900X.
  • Figures 6D-F shows tissue that has been incubated in the presence of antibodies to Ki67.
  • Figure 6D has been stained to show Pi-labeled nuclei (blue). Magnification is 500X.
  • Figure 6E has been stained to show BrdU- and Ki67-labeled nuclei (green). Magnification is 500X.
  • Figure 6F has been stained to show the presence of ⁇ -smooth muscle actin (red). Magnification is 500X. Bright fluorescence: combination of PI with BrdU (C) or Ki67 (F));
  • Figure 7 shows photographs of tissue sections from MI induced mice, showing the area of MI injected with Lin " c-f ⁇ ' t P0S cells (Depicted are the border zone, viable myocardium (NM) and the new band ( ⁇ B) of myocardium separated by an area of infarcted non-repairing tissue (arrows).
  • Figure 7A is stained to show the presence of EGFP (green). Magnification is 280X.
  • Figure 7B is stained to show the presence of cardiac myosin (red).
  • Magnification is 280X.
  • Figure 7C is stained to show the presence of both EGFP and myosin (red-green), as well as PI- stained nuclei (blue).
  • Figure 8 shows photographs of tissue sections from MI induced mice, showing regenerating myocardium in the area of MI injected with Lin " c-kit ?os cells
  • Figure 8 A is stained to show the presence of EGFP (green).
  • Magnification is 650X.
  • Figure 8B is stained to show the presence of cardiac myosin (red).
  • Magnification is 650X.
  • Figure 8C is stained to show both the presence of EGFP and myosin (yellow), as well as Pi-stained nuclei (blue).
  • FIG. 8D is stained to show the presence of EGFP (green). Magnification is 65 OX.
  • Figure 8E is stained to show the presence of ⁇ -smooth muscle actin in arterioles (red). Magnification is 65 OX.
  • Figure 8F is stained to show the presence of both EGFP and ⁇ -smooth muscle actin (yellow-red) as well as Pi-stained nuclei (blue). Magnification is 650X);
  • Figure 9 shows photographs of tissue sections from MI induced mice, showing the area of MI injected with Lin " c-f ⁇ ' t pos cells and showing regenerating myocardium (arrowheads).
  • Figure 9A is stained to show the presence of cardiac myosin (red) Magnification is 400X.
  • Figure 9B is stained to show the presence of the Y chromosome (green).
  • Magnification is 400X.
  • Figure 9C is stained to show both the presence of the Y chromosome (light blue) and Pi-labeled nuclei (dark blue). Note the lack of Y chromosome in infarcted tissue (IT) in subendocardium and spared myocytes (SM) in subepicardium. Magnification is 400X);
  • Figure 10 shows photographs of tissue sections from MI induced mice, showing GATA-4 in cardiac myosin positive cells ( Figure 10A shows Pi-stained nuclei (blue). Magnification is 650X.
  • Figure 10B shows the presence of GATA-4 labeling (green). Magnification is 650X.
  • Figure IOC is stained to show cardiac myosin (red) in combination with GATA-4 and PI (bright fluorescence in nuclei). Magnification is 650X);
  • Figure 11 shows photograph of tissue sections from a MI induced mouse ( Figure 11 A shows the border zone between the infarcted tissue and the surviving tissue. Magnification is 500X. Figure 11B shows regenerating myocardium. Magnification is 800X. Figure 11C is stained to show the presence of connexin 43 (yellow-green), and the contacts between myocytes are shown by arrows. Magnification is 800X. Figure 11D is stained to show both ⁇ -sarcomeric actin (red) and Pi-stained nuclei (blue).
  • Figure 12 shows photographs of tissue sections from a MI induced mouse showing the area of MI that was injected with Lin " c-kit ?os cells and now shows regenerating myocytes (Figure 12A is stained to show the presence of cardiac myosin (red) and Pl-labeled nuclei (yellow-green). Magnification is 1,000.
  • Figure 12B is the same as Figure 12A at a magnification of 700X;
  • Figures 13A-B show photographs of tissue sections from MI induced mice (Figure 13 A shows a large infarct (MI) in a cytokine-treated mouse with forming myocardium (arrowheads) (Magnification is 50X) at higher magnification (80X - adjacent panel).
  • Figure 13B shows a MI in a non-treated mouse.
  • Healing comprises the entire infarct (arrowheads) (Magnification is 50X). Scarring is seen at higher magnification (80X - adjacent panel).
  • LNFW left ventricular free wall
  • Figures 15E-M show M-mode echocardiograms of SO (e-g), MI (h-j) and MI-C (k-m) (Newly formed contracting myocardium (arrows));
  • Figures 16A-G show grafts depicting aspects of myocardial infarction, cardiac anatomy and ventricular function
  • Figures 16H-P show two dimensional (2D) images and M-mode tracings of SO (h-j), MI (k-m) and MI-C (n-p);
  • Figure 18A-E shows graphs of aspects of myocardial regeneration ( Figure 17
  • FIG. 18A classifies the cells in the tissue as remaining viable (Re), lost (Lo) and newly formed (Fo) myocardium in LVFW at 27 days in MI and MI-C; SO, myocardium without infarct.
  • Figure 18B shows the amount of cellular hypertrophy in spared myocardium.
  • Figure 18C shows cell proliferation in the regenerating myocardium.
  • Myocytes (M), EC and SMC labeled by BrdU and Ki67; n l 1. *'**p ⁇ 0.05 vs M and EC.
  • Figure 19 shows photographs of tissue sections from MI induced mice that were incubated with antibodies to Ki67 (A,B) and BrdU (C,D)
  • Figure 19A shows labeling of myocytes by cardiac myosin. Bright fluorescence of nuclei reflects the combination of PI and Ki67. Magnification is 800X.
  • Figure 19B shows labeling of SMC by ⁇ -smooth muscle actin. Bright fluorescence of nuclei reflects the combination of PI and Ki67. Magnification is 1,200X.
  • Figure 19C shows labeling of SMC by ⁇ -smooth muscle actin. Bright fluorescence of nuclei reflects the combination of PI and BrdU. Magnification is 1,200X.
  • Figure 19D shows labeling of EC in the forming myocardium by factor NIII. Bright fluorescence of nuclei reflects the combination of PI and BrdU. Magnification is 1.600X;
  • Figure 20 shows photographs of tissue sections from MI induced mice showing markers of differentiating cardiac cells (Figure 20A is stained to show labeling of myocytes by nestin (yellow)). Red fluorescence indicates cardiac myosin. Magnification is 1,200X. Figure 20 B is stained to show labeling of desmin (red). Magnification is 800X. Figure 20C is stained to show labeling of connexin 43 (green). Red fluorescence indicates cardiac myosin. Magnification is 1,400X.
  • Figure 20D shows NE-cadherin and yellow-green fluorescence reflects labeling of EC by flk-1 (arrows). Magnification is 1,800X.
  • Figure 20E shows red fluorescence indicating factor NIII in EC and and yellow- green fluorescence reflects labeling of EC by flk-1 (arrows). Magnification is
  • Figure 20F shows green fluorescence labeling of SMC cytoplasms by flk-1 and endothelial lining labeled by flk-1. Red fluorescence indicates ⁇ -smooth muscle actin. Blue fluorescence indicates PI labeling of nuclei. Magnification is 800X; and Figure 21A-C show tissue sections from MI induced mice (Figure 21A uses bright fluorescence to depict the combination of PI labeling of nuclei with Csx ⁇ kx2.5. Magnification is 1,400X.
  • Figure 21B uses bright fluorescence to depict the combination of PI labeling of nuclei with GATA-4. Magnification is 1,200X.
  • Figure 21 C uses bright fluorescence to depict the combination of PI labeling of nuclei with MEF2.
  • the present invention provides methods and/or pharmaceutical composition comprising a therapeutically effective amount of hematopoietic stem cells
  • the pharmaceutical composition of the present invention is delivered via injection.
  • routes for administration include, but are not limited to subcutaneous or parenteral including intravenous, intraarterial, intramuscular, intraperitoneal, intramyocardial, transendocardial, trans- epicardial, intranasal administration as well as intrathecal, and infusion techniques.
  • the pharmaceutical composition is in a form that is suitable for injection.
  • a therapeutic of the present invention When administering a therapeutic of the present invention parenterally, it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion).
  • the pharmaceutical formulations suitable for injection include sterile aqueous solutions or dispersions and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • the carrier can be a solvent or dispersing medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like) , suitable mixtures thereof, and vegetable oils.
  • Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Nonaqueous vehicles such as cottonseed oil, sesame oil, olive oil, soybean oil, corn oil, sunflower oil, or peanut oil and esters, such as isopropyl myristate, may also be used as solvent systems for compound compositions
  • various additives which enhance the stability, sterility, and isotonicity of the compositions including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added.
  • antibacterial and antifungal agents for example, parabens, chlorobutanol, phenol, sorbic acid, and the like.
  • isotonic agents for example, sugars, sodium chloride, and the like.
  • Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. According to the present invention, however, any vehicle, diluent, or additive used would have to be compatible with the compounds.
  • Sterile injectable solutions can be prepared by incorporating the compounds utilized in practicing the present invention in the required amount of the appropriate solvent with various amounts of the other ingredients, as desired.
  • composition of the present invention e.g., comprising a therapeutic compound
  • any compatible carrier such as various vehicles, adjuvants, additives, and diluents
  • the compounds utilized in the present invention can be administered parenterally to the patient in the form of slow-release subcutaneous implants or targeted delivery systems such as monoclonal antibodies, iontophoretic, polymer matrices, liposomes, and microspheres.
  • the pharmaceutical composition utilized in the present invention can be administered orally to the patient.
  • Conventional methods such as administering the compounds in tablets, suspensions, solutions, emulsions, capsules, powders, syrups and the like are usable.
  • Known techniques which deliver the compound orally or intravenously and retain the biological activity are preferred.
  • a composition of the present invention can be administered initially, and thereafter maintained by further administration.
  • a composition of the invention can be administered in one type of composition and thereafter further administered in a different or the same type of composition.
  • a composition of the invention can be administered by intravenous injection to bring blood levels to a suitable level. The patient's levels are then maintained by an oral dosage form, although other forms of administration, dependent upon the patient's condition, can be used.
  • mice are treated generally longer than the mice or other experimental animals which treatment has a length proportional to the length of the disease process and drug effectiveness.
  • the doses may be single doses or multiple doses over a period of several days, but single doses are preferred.
  • animal experiments e.g., rats, mice, and the like
  • the treatment generally has a length proportional to the length of the disease process and drug effectiveness and the patient being treated.
  • the quantity of the pharmaceutical composition to be administered will vary for the patient being treated.
  • 2 x 10 4 - l x l 0 5 stem were administered to the patient. While there would be an obvious size difference between the hearts of a mouse and a human, it is possible that 2 x 10 4 - l x l 0 5 stem cells would be sufficient in a human as well.
  • the precise determination of what would be considered an effective dose may be based on factors individual to each patient, including their size, age, size of the infarct, and amount of time since damage. Therefore, dosages can be readily ascertained by those skilled in the art from this disclosure and the knowledge in the art.
  • any additives in addition to the active stem cell(s) are present in an amount of 0.001 to 50 wt% solution in phosphate buffered saline, and the active ingredient is present in the order of micrograms to milligrams, such as about 0.0001 to about 5 wt%, preferably about 0.0001 to about 1 wt%, most preferably about 0.0001 to about 0.05 wt% or about 0.001 to about 20 wt%, preferably about 0.01 to about 10 wt%, and most preferably about 0.05 to about 5 wt%.
  • any composition to be administered to an animal or human it is preferred to determine therefore: toxicity, such as by determining the lethal dose (LD) and LD 50 in a suitable animal model e.g., rodent such as mouse; and, the dosage of the composition(s), concentration of components therein and timing of administering the composition(s), which elicit a suitable response.
  • toxicity such as by determining the lethal dose (LD) and LD 50 in a suitable animal model e.g., rodent such as mouse
  • LD 50 lethal dose
  • LD 50 low-d dose
  • suitable animal model e.g., rodent such as mouse
  • the dosage of the composition(s), concentration of components therein and timing of administering the composition(s) which elicit a suitable response.
  • compositions comprising a therapeutic of the invention include liquid preparations for orifice, e.g., oral, nasal, anal, vaginal, peroral, intragastric, mucosal (e.g., perlingual, alveolar, gingival, olfactory or respiratory mucosa) etc., administration such as suspensions, syrups or elixirs; and, preparations for parenteral, subcutaneous, intradermal, intramuscular or intravenous administration (e.g., injectable administration), such as sterile suspensions or emulsions.
  • Such compositions may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose or the like.
  • compositions can also be lyophilized.
  • the compositions can contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as "REMINGTON'S PHARMACEUTICAL SCIENCE", 17th edition, 1985, incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation.
  • compositions of the invention are conveniently provided as liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions or viscous compositions which may be buffered to a selected pH. If digestive tract absorption is preferred, compositions of the invention can be in the "solid" form of pills, tablets, capsules, caplets and the like, including “solid” preparations which are time-released or which have a liquid filling, e.g., gelatin covered liquid, whereby the gelatin is dissolved in the stomach for delivery to the gut. If nasal or respiratory (mucosal) administration is desired, compositions may be in a form and dispensed by a squeeze spray dispenser, pump dispenser or aerosol dispenser. Aerosols are usually under pressure by means of a hydrocarbon. Pump dispensers can preferably dispense a metered dose or, a dose having a particular particle size.
  • compositions of the invention can contain pharmaceutically acceptable flavors and/or colors for rendering them more appealing, especially if they are administered orally.
  • the viscous compositions may be in the form of gels, lotions, ointments, creams and the like (e.g., for transdermal administration) and will typically contain a sufficient amount of a thickening agent so that the viscosity is from about 2500 to 6500 cps, although more viscous compositions, even up to 10,000 cps may be employed.
  • Viscous compositions have a viscosity preferably of 2500 to 5000 cps, since above that range they become more difficult to administer. However, above that range, the compositions can approach solid or gelatin forms which are then easily administered as a swallowed pill for oral ingestion.
  • Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection or orally. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with mucosa, such as the lining of the stomach or nasal mucosa.
  • suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, e.g., liquid dosage form (e.g., whether the composition is to be formulated into a solution, a suspension, gel or another liquid form), or solid dosage form (e.g., whether the composition is to be formulated into a pill, tablet, capsule, caplet, time release form or liquid-filled form) .
  • liquid dosage form e.g., whether the composition is to be formulated into a solution, a suspension, gel or another liquid form
  • solid dosage form e.g., whether the composition is to be formulated into a pill, tablet, capsule, caplet, time release form or liquid-filled form
  • Solutions, suspensions and gels normally contain a major amount of water (preferably purified water) in addition to the active compound. Minor amounts of other ingredients such as pH adjusters (e.g., a base such as NaOH), emulsifiers or dispersing agents, buffering agents, preservatives, wetting agents, jelling agents, (e.g., methylcellulose), colors and/or flavors may also be present.
  • pH adjusters e.g., a base such as NaOH
  • emulsifiers or dispersing agents e.g., a base such as NaOH
  • buffering agents e.g., preservatives
  • wetting agents e.g., methylcellulose
  • jelling agents e.g., methylcellulose
  • colors and/or flavors e.g., methylcellulose
  • compositions of this invention may be accomplished using sodium chloride, or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes.
  • sodium chloride is preferred particularly for buffers containing sodium ions.
  • Viscosity of the compositions may be maintained at the selected level using a pharmaceutically acceptable thickening agent.
  • Methylcellulose is preferred because it is readily and economically available and is easy to work with.
  • suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the like. The preferred concentration of the thickener will depend upon the agent selected. The important point is to use an amount which will achieve the selected viscosity. Viscous compositions are normally prepared from solutions by the addition of such thickening agents.
  • a pharmaceutically acceptable preservative can be employed to increase the shelf-life of the compositions.
  • Benzyl alcohol may be suitable, although a variety of preservatives including, for example, parabens, thimerosal, chlorobutanol, or benzalkonium chloride may also be employed.
  • a suitable concentration of the preservative will be from 0.02% to 2% based on the total weight although there may be appreciable variation depending upon the agent selected.
  • compositions should be selected to be chemically inert with respect to the active compound. This will present no problem to those skilled in chemical and pharmaceutical principles, or problems can be readily avoided by reference to standard texts or by simple experiments (not involving undue experimentation), from this disclosure and the documents cited herein.
  • inventive compositions of this invention are prepared by mixing the ingredients following generally accepted procedures. For example the selected components may be simply mixed in a blender, or other standard device to produce a concentrated mixture which may then be adjusted to the final concentration and viscosity by the addition of water or thickening agent and possibly a buffer to control pH or an additional solute to control tonicity. Generally the pH may be from about 3 to 7.5.
  • compositions can be administered in dosages and by techniques well known to those skilled in the medical and veterinary arts taking into consideration such factors as the age, sex, weight, and condition of the particular patient, and the composition form used for administration (e.g., solid vs. liquid). Dosages for humans or other mammals can be determined without undue experimentation by the skilled artisan, from this disclosure, the documents cited herein, and the knowledge in the art.
  • Suitable regimes for initial administration and further doses or for sequential administrations also are variable, may include an initial administration followed by subsequent administrations; but nonetheless, may be ascertained by the skilled artisan, from this disclosure, the documents cited herein, and the knowledge in the art.
  • compositions of the present invention are used to treat cardiovascular diseases, including, but not limited to, atherosclerosis, ischemia, hypertension, restenosis, angina pectoris, rheumatic heart disease, congenital cardiovascular defects and arterial inflammation and other diseases of the arteries, arterioles and capillaries or related complaint.
  • cardiovascular diseases including, but not limited to, atherosclerosis, ischemia, hypertension, restenosis, angina pectoris, rheumatic heart disease, congenital cardiovascular defects and arterial inflammation and other diseases of the arteries, arterioles and capillaries or related complaint.
  • the invention involves the administration of stem cells as herein discussed, alone or in combination with one or more cytokine, as herein discussed, for the treatment or prevention of any one or more of these conditions or other conditions involving weakness in the heart, as well as compositions for such treatment or prevention, use of stem cells as herein discussed, alone or in combination with one or more cytokine, as herein discussed, for formulating such compositions, and kits involving stem cells as herein discussed, alone or in combination with one or more cytokine, as herein discussed, for preparing such compositions and/or for such treatment, or prevention.
  • advantageous routes of administration involves those best suited for treating these conditions, such as via injection, including, but are not limited to subcutaneous or parenteral including intravenous, intraarterial, intramuscular, intraperitoneal, intramyocardial, transendocardial, trans-epicardial, intranasal administration as well as intrathecal, and infusion techniques.
  • subcutaneous or parenteral including intravenous, intraarterial, intramuscular, intraperitoneal, intramyocardial, transendocardial, trans-epicardial, intranasal administration as well as intrathecal, and infusion techniques.
  • compositions of the present invention may be used as therapeutic agents - i.e. in therapy applications.
  • treatment and “therapy” include curative effects, alleviation effects, and prophylactic effects.
  • patient may encompass any vertebrate including but not limited to humans, mammals, reptiles, amphibians and fish.
  • the patient is a mammal such as a human, or an animal mammal such as a domesticated mammal, e.g., dog, cat, horse, and the like, or production mammal, e.g., cow, sheep, pig, and the like.
  • stem cell or “stem cell” or “hematopoietic cell” refers to either autologous or allogenic stem cells, which may be obtained from the bone marrow, peripheral blood, or other source.
  • adult stem cells refers to stem cells that are not embryonic in origin nor derived from embryos or fetal tissue.
  • ischemic myocardium refers to myocardium which has been damaged within one week of treatment being started. In a preferred embodiment, the myocardium has been damaged within three days of the start of treatment. In a further preferred embodiment, the myocardium has been damaged within 12 hours of the start of treatment. It is advantageous to employ stem cells alone or in combination with cytokine(s) as herein disclosed to a recently damaged myocardium.
  • damaged myocardium refers to myocardial cells which have been exposed to ischemic conditions. These ischemic conditions may be caused by a myocardial infarction, or other cardiovascular disease or related complaint. The lack of oxygen causes the death of the cells in the surrounding area, leaving an infarct, which will eventually scar.
  • home refers to the attraction and mobilization of somatic stem cells towards damaged myocardium and/or myocardial cells.
  • assemble refers to the assembly of differentiated somatic stem cells into functional structures i.e., myocardium and/or myocardial cells, coronary arteries, arterioles, and capillaries etc. This assembly provides functionality to the differentiated myocardium and/or myocardial cells, coronary arteries, arterioles and capillaries.
  • the invention involves the use of somatic stem cells. These are present in animals in small amounts, but methods of collecting stem cells are known to those skilled in the art.
  • the stem cells are selected to be lineage negative.
  • lineage negative is known to one skilled in the art as meaning the cell does not express antigens characteristic of specific cell lineages.
  • the lineage negative stem cells are selected to be c-kit positive.
  • c-kit is known to one skilled in the art as being a receptor which is known to be present on the surface of stem cells, and which is routinely utilized in the process of identifying and separating stem cells from other surrounding cells.
  • the invention further involves a therapeutically effective dose or amount of stem cells applied to the heart.
  • An effective dose is an amount sufficient to effect a beneficial or desired clinical result. Said dose could be administered in one or more administrations.
  • 2 x 10 4 - l x l 0 5 stem cells were administered in the mouse model. While there would be an obvious size difference between the hearts of a mouse and a human, it is possible that this range of stem cells would be sufficient in a human as well. However, the precise determination of what would be considered an effective dose may be based on factors individual to each patient, including their size, age, size of the infarct, and amount of time since damage.
  • One skilled in the art specifically a physician or cardiologist, would be able to determine the number of stem cells that would constitute an effective dose without undue experimentation.
  • the stem cells are delivered to the heart, specifically to the border area of the infarct.
  • the infarcted area is visible grossly, allowing this specific placement of stem cells to be possible.
  • the stem cells are advantageously administered by injection, specifically an intramyocardial injection. As one skilled in the art would be aware, this is the preferred method of delivery for stem cells as the heart is a functioning muscle. Injection of the stem cells into the heart ensures that they will not be lost due to the contracting movements of the heart.
  • the stem cells are administered by injection transendocardially or trans-epicardially.
  • This preferred embodiment allows the stem cells to penetrate the protective surrounding membrane, necessitated by the embodiment in which the cells are injected inrramyocardially .
  • a preferred embodiment of the invention includes use of a catheter-based approach to deliver the trans-endocardial injection.
  • the use of a catheter precludes more invasive methods of delivery wherein the opening of the chest cavity would be necessitated.
  • optimum time of recovery would be allowed by the more minimally invasive procedure, which as outlined here, includes a catheter approach.
  • stem cells to migrate into the infarcted region and differentiate into myocytes, smooth muscle cells, and endothelial cells. It is known in the art that these types of cells must be present to restore both structural and functional integrity. Other approaches to repairing infarcted or ischemic tissue have involved the implantation of these cells directly into the heart, or as cultured grafts, such as in U.S. Patent No. 6,110,459, and 6,099,832.
  • Another embodiment of the invention includes the proliferation of the differentiated cells and the formation of the cells into cardiac structures including coronary arteries, arterioles, capillaries, and myocardium. As one skilled in the art is aware, all of these structures are essential for proper function in the heart. It has been shown in the literature that implantation of cells including endothelial cells and smooth muscle cells will allow for the implanted cells to live within the infarcted region, however they do not form the necessary structures to enable the heart to regain full functionality. The ability to restore both functional and structural integrity is yet another aspect of this invention. The restoration or some restoration of both functional and structural integrity of cardiac tissue - advantageously over that which has occurred previously - is yet another aspect of this invention.
  • stem cell factor is available under the name SCF (multiple forms of recombinant human, recombinant mouse, and antibodies to each), from R & D Systems (614 McKinley Place N.E., Minneapolis, MN 55413); granulocyte-colony stimulating factor is available under the name G-CSF (multiple forms of recombinant human, recombinant mouse, and antibodies to each), from R & D Systems; stem cell antibody-1 is available under the name SCA-1 from MBL
  • c-kit antibody is available under the name c-kit (Ab-1) Polyclonal Antibody from CN Biosciences Corporate (Affiliate of Merck KgaA, Darmstadt, Germany. Corporate headquarters located at 10394 Pacific Center Court, San Diego, CA 92121).
  • Bone marrow was harvested from the femurs and tibias of male transgenic mice expressing enhanced green fluorescent protein (EGFP). After surgical removal of the femurs and tibias, the muscle was dissected and the upper and lower surface of the bone was cut on the surface to allow the collecting buffer to infiltrate the bone marrow. The fluid containing buffer and cells was collected in tubes such as 1.5 ml Epindorf tubes.
  • EGFP enhanced green fluorescent protein
  • Bone marrow cells were suspended in PBS containing 5% fetal calf serum (FCS) and incubated on ice with rat anti-mouse monoclonal antibodies specific for the following hematopoietic lineages: CD4 and CD8 (T -lymphocytes), B-220 (B-lymphocytes), Mac-1 (macrophages), GR-1 (granulocytes) (Caltag
  • Myocardial infarction was induced in female C57BL/6 mice at 2 months of age as described by Li et al. (1997). Three to five hours after infarction, the thorax of the mice was reopened and 2.5 ⁇ l of PBS containing Lin " c-f ⁇ ' t PGS cells were injected in the anterior and posterior aspects of the viable myocardium bordering the infarct ( Figure 2). Infarcted mice, left uninjected or injected with Lin " c-£z ' t- NE cells, and sham-operated mice i.e., mice where the chest cavity was opened but no infarction was induced, were ⁇ sed as controls. All animals were sacrificed 9 ⁇ 2 days after surgery.
  • Protocols were approved by institutional review board. Results are presented as mean ⁇ SD. Significance between two measurements was determined by the Student's t test, and in multiple comparisons was evaluated by the Bonferroni method (Scholzen and Gerdes, 2000). PO.05 was considered significant.
  • mice were anesthetized with chloral hydrate (400 mg/kg body weight, i.p.), and the right carotid artery was cannulated with a microtip pressure transducer (model SPR-671, Millar) for the measurements of left ventricular (LV) pressures and LV + and -dP/dt in the closed-chest preparation to determine whether developing myocytes derived from the HSC transplant had an impact on function.
  • LV left ventricular
  • LV + and -dP/dt left ventricular
  • Infarcted mice non-injected or injected with Lin " c-Ait NEG cells were combined in the statistics. In comparison with sham-operated groups, the infarcted groups exhibited indices of cardiac failure ( Figure 3).
  • LV end- diastolic pressure (LVEDP) was 36% lower, and developed pressure (LVDP) and LV+ and -dP/dt were 32%, 40%, and 41 % higher, respectively ( Figure 4A).
  • LVEDP LV end- diastolic pressure
  • LVDP developed pressure
  • LV+ and -dP/dt were 32%, 40%, and 41 % higher, respectively.
  • Figure 4A D. Determination of Cell Proliferation and EGFP Detection
  • the abdominal aorta was cannulated, the heart was arrested in diastole by injection of cadmium chloride (CdCl 2 ), and the myocardium was perfused retrogradely with 10% buffered formalin.
  • CdCl 2 cadmium chloride
  • the infarcted portion of the ventricle was easily identifiable grossly and histologically (see Fig. 2A).
  • the lengths of the endocardial and epicardial surfaces delimiting the infarcted region, and the endocardium and epicardium of the entire left ventricle were measured in each section. Subsequently, their quotients were computed to yield the average infarct size in each case. This was accomplished at 4X magnification utilizing an image analyzer connected to a microscope.
  • BrdU 50 mg/kg body weight, i.p.
  • BrdU 50 mg/kg body weight, i.p.
  • Sections were incubated with anti-BrdU antibody and BrdU labeling of cardiac cell nuclei in the S phase was measured.
  • expression of Ki67 in nuclei was evaluated by treating samples with a rabbit polyclonal anti-mouse Ki67 antibody (Dako Corp.).
  • FITC-conjugated goat anti-rabbit IgG was used as secondary antibody.
  • Figure 5 and 6 EGFP was detected with a rabbit polyclonal anti-GFP (Molecular Probes).
  • Myocytes were recognized with a mouse monoclonal anti- cardiac myosin heavy chain (MAB 1548; Chemicon) or a mouse monoclonal anti- ⁇ - sarcomeric actin (clone 5C5; Sigma), endothelial cells with a rabbit polyclonal anti- human factor VIII (Sigma) and smooth muscle cells with a mouse monoclonal anti- ⁇ -smooth muscle actin (clone 1 A4; Sigma). Nuclei were stained with propidium iodide (PI), 10 ⁇ g/ml. The percentages of myocyte (M), endothelial cell (EC) and smooth muscle cell (SMC) nuclei labeled by BrdU and Ki67 were obtained by confocal microscopy.
  • MAB 1548 mouse monoclonal anti- cardiac myosin heavy chain
  • clone 5C5 mouse monoclonal anti- ⁇ - sarcomeric actin
  • endothelial cells with a rabbit polyclonal anti
  • Myocyte proliferation was 93% (p ⁇ 0.001) and 60% (pO.OOl) higher than in endothelial cells, and 225% (pO.OOl and 176% (pO.OOl) higher than smooth muscle cells, when measured by BrdU and Ki67, respectively.
  • FISH fluorescence in situ hybridization
  • Y-chromosomes were not detected in cells from the surviving portion of the ventricle. However, the Y-chromosome was detected in the newly formed myocytes, indicating their origin as from the injected bone marrow cells (Fig. 9).
  • MEF2 myocyte enhancer factor 2
  • GATA-4 cardiac specific transcription factor 4
  • GATA-4 the cardiac specific transcription factor 4
  • the early marker of myocyte ⁇ development Csx/Nkx2.5 was examined.
  • MEF2 proteins are recruited by GATA-4 to synergistically activate the promoters of several cardiac genes such as myosin light chain, troponin T, troponin I, ⁇ -myosin heavy chain, desmin, atrial natriuretic factor and ⁇ -actin (Durocher et al., 1997; Morin et al., 2000).
  • Csx/Nkx2.5 is a transcription factor restricted to the initial phases of myocyte differentiation (Durocher et al., 1997). In the reconstituting heart, all nuclei of cardiac myosin labeled cells expressed MEF2 (Figs. 7D-7F) and GATA-4 (Fig. 10), but only 40 ⁇ 9% expressed Csx/Nkx2.5 (Figs. 7G-7I). To characterize further the properties of these myocytes, the expression of connexin 43 was determined.
  • This protein is responsible for intercellular connections and electrical coupling through the generation of plasma membrane channels between myocytes (Beardsle et al., 1998; Musil et al., 2000); connexin 43 was apparent in the cell cytoplasm and at the surface of closely aligned differentiating cells (Figs. 11A-1 ID). These results were consistent with the expected functional competence of the heart muscle phenotype. Additionally, myocytes at various stages of maturation were detected within the same and different bands (Fig. 12).
  • EXAMPLE 2 Mobilization of Bone Marrow Cells to Repair Infarcted Myocardium
  • SCF rat stem cell factor
  • G-CSF human granulocyte colony stimulating factor
  • Amgen ⁇ g/kg/day
  • mice were anesthetized with chloral hydrate (400 mg/kg body weight, ip) and a microtip pressure transducer (SPR-671, Millar) connected to a chart recorder was advanced into the LV for the evaluation of pressures and + and - dP/dt in the closed-chest preparation (Orlic et al, 2001; Li et al., 1997; Li et al, 1999).
  • a microtip pressure transducer SPR-671, Millar
  • EF was 48%o, 62% and 114% higher in treated than in non-treated mice at 9, 16 and 26 days after coronary occlusion, respectively (Fig. 15D).
  • Figs. 15E-M In mice exposed to cytokines, contractile function developed with time in the infarcted region of the wall (Figs. 15E-M; Figs. 16H-P, www.pnas.org).
  • LVEDP LV end-diastolic pressure
  • the changes in LV systolic pressure (not shown), developed pressure (LVDP), + and -dP/dt were also more severe in the absence of cytokine treatment (Figs. 17A-D).
  • LVESD LV end-systolic
  • tissue regeneration decreased the expansion in cavitary diameter, -14%, longitudinal axis, -5% (Figs.l6F-G), and chamber volume,- 26% (Fig. 15B).
  • ventricular mass-to-chamber volume ratio was 36% higher in treated animals (Fig. 15C). Therefore, BMC mobilization that led to proliferation and differentiation of a new population of myocytes and vascular structures attenuated the anatomical variables which define cardiac decompensation.
  • C. Cardiac Anatomy and Determination of Infarct Size Following hemodynamic measurements, the abdominal aorta was cannulated, the heart was arrested in diastole with CdCl 2 and the myocardium was perfused with 10% formalin.
  • the LV chamber was filled with fixative at a pressure equal to the in vivo measured end-diastolic pressure (Li et al., 1997; Li et al., 1999).
  • the LV intracavitary axis was measured and three transverse slices from the base, mid-region and apex were embedded in paraffin. The mid-section was used to measure LV thickness, chamber diameter and volume (Li et al., 1997; Li et al., 1999).
  • Infarct size was determined by the number of myocytes lost from the LVFW (Olivetti et al., 1991 ; Beltrami et al., 1994).
  • the volume of the LVFW was determined in each group of mice.
  • the volume of newly formed myocardium was detected exclusively in cytokine-treated mice and found to be 14 mm 3 (Fig. 18A).
  • the spared portion of the LVFW at 27 days was 41 and 37 mm 3 in non-treated and treated mice (see above)
  • the remaining myocardium, shown in Fig. 18 ⁇ underwent 95% (pO.OOl) and 85% (pO.OOl) hypertrophy, respectively. Consistently, myocyte cell volume increased 94% and 77% (Fig. 18B).
  • the volume of regenerating myocardium was determined by measuring in each of three sections the area occupied by the restored tissue and section thickness. The product of these two variables yielded the volume of tissue repair in each section. Values in the three sections were added and the total volume of formed myocardium was obtained. Additionally, the volume of 400 myocytes was measured in each heart. Sections were stained with desmin and laminin antibodies and propidium iodide (PI). Only longitudinally oriented cells with centrally located nuclei were included. The length and diameter across the nucleus were collected in each myocyte to compute cell volume, assuming a cylindrical shape (Olivetti et al., 1991; Beltrami et al., 1994).
  • Myocytes were divided in classes and the number of myocytes in each class was calculated from the quotient of total myocyte class volume and average cell volume (Kajstura et al., 1995; Reiss et al., 1996). Number of arteriole and capillary profiles per unit area of myocardium was measured as previously done (Olivetti et al., 1991; Beltrami et al., 1994).
  • M Myocytes
  • EC endothelial cells
  • SMC smooth muscle cells
  • BrdU was injected daily between days 14 to 26 to measure the cumulative extent of cell proliferation while Ki67 was assayed to determine the number of cycling cells at sacrifice. Ki67 identifies cells in Gl, S, G2, prophase and metaphase, decreasing in anaphase and telophase (Orlic et al., 2001).
  • the percentages of BrdU and Ki67 positive myocytes were 1.6- and 1.4-fold higher than EC, and 2.8- and 2.2-fold higher than SMC, respectively (Fig. 18C, 19).
  • the forming myocardium occupied 76 ⁇ 11% of the infarct; myocytes constituted 61 ⁇ 12%, new vessels 12 ⁇ 5% and other components 3X2%.
  • the band contained 15xl0 6 regenerating myocytes that were in an active growing phase and had a wide size distribution (Figs. 18D-E).
  • EC and SMC growth resulted in the formation of 15 ⁇ 5 arterioles and 348 ⁇ 82 capillaries per mm of new myocardium.
  • Thick wall arterioles with several layers of SMC and luminal diameters of 10-30 ⁇ m represented vessels in early differentiation.
  • arterioles and capillaries containing erythrocytes Figs. 18F-H.
  • Cytoplasmic and nuclear markers were used.
  • Myocyte nuclei rabbit polyclonal Csx/Nkx2.5, MEF2, and GATA4 antibodies (Orlic et al, 2001; Lin et al., 1997; Kasahara et al., 1998);
  • cytoplasm mouse monoclonal nestin (Kachinsky et al., 1995), rabbit polyclonal desmin (Hermann and Aebi, 1998), cardiac myosin, mouse monoclonal ⁇ -sarcomeric actin and rabbit polyclonal connexin 43 antibodies (Orlic et al., 2001).
  • EC cytoplasm mouse monoclonal flk-1, VE-cadherin and factor VIII antibodies (Orlic et al, 2001; Yamaguchi et al, 1993; Breier et al., 1996).
  • SMC cytoplasm flk-1 and ⁇ -smooth muscle actin antibodies (O ⁇ ic et al., 2001; Couper et al., 1997). Scar was detected by a mixture of collagen type I and type III antibodies.
  • cytoplasmic proteins Five cytoplasmic proteins were identified to establish the state of differentiation of myocytes (Orlic et al., 2001; Kachinsky et al., 1995; Hermann and Aebi, 1998): nestin, desmin, ⁇ -sarcomeric actin, cardiac myosin and connexin 43. Nestin was recognized in individual cells scattered across the forming band (Fig. 20A). With this exception, all other myocytes expressed desmin (Fig. 20B), ⁇ - sarcomeric actin, cardiac myosin and connexin 43 (Fig. 20C).
  • This tyrosine kinase receptor promotes migration of SMC during angiogenesis (Couper et al., 1997). Therefore, repair of the infarcted heart involved growth and differentiation of all cardiac cell populations resulting in de novo myocardium.
  • HGF hepatocyte growth factor
  • SCF stem cell factor
  • GM-CSF granulocyte monocyte colony stimulating factor
  • HGF did not mobilize a larger number of cells at a concentration of 100 ng/ml.
  • the cells that showed a chemotactic response to HGF consisted of 15% of c-kit positive (c-kit ?os ) cells, 50% of multidrug resistance -1 (MDR-1) labeled cells and 30% of stem cell antigen-1 (Sca-1) expressing cells.
  • MDR-1 multidrug resistance -1
  • Cardiac myosin positive myocytes constituted 50% of the preparation, while factor VIII labeled cells included 15%, alpha-smooth muscle actin stained cells 4%, and vimentin positive factor VIII negative fibroblasts 20%. The remaining cells were small undifferentiated and did not stain with these four antibodies.
  • the mouse heart possesses primitive cells which are mobilized by growth factors. HGF translocates cells that in vitro differentiate into the four cardiac cell lineages.
  • infarcted Fischer 344 rats were injected with these BrdU positive cells in the damaged region, 3-5 hours after coronary artery occlusion. Two weeks later, animals were sacrificed and the characteristics of the infarcted area were examined. Myocytes containing parallel arranged myofibrils along their longitudinal axis were recognized, in combination with BrdU labeling of nuclei. Moreover, vascular structures comprising arterioles and capillary profiles were present and were also positive to BrdU.
  • primitive c-kit positive cells reside in the senescent heart and maintain the ability to proliferate and differentiate into parenchymal cells and coronary vessels when implanted into injured functionally depressed myocardium.
  • the heart is not a post-mitotic organ but contains a subpopulation of myocytes that physiologically undergo cell division to replace dying cells.
  • Myocyte multiplication is enhanced during pathologic overloads to expand the muscle mass and maintain cardiac performance.
  • the origin of these replicating myocytes remains to be identified. Therefore, primitive cells with characteristics of stem/progenitor cells were searched for in the myocardium of of Fischer 344 rats. Young and old animals were studied to determine whether aging had an impact on the size population of stem cells and dividing myocytes.
  • the numbers of c-kit and MDR1 positive cells in rats at 4 months were 11 ⁇ 3, and 18 ⁇ 6/100 mm 2 of tissue, respectively. Values in rats at 27 months were 35 ⁇ 10, and 42 ⁇ 13/100 mm 2 .
  • Ki67 protein which is expressed in nuclei of cycling cells was detected in 1.3 ⁇ 0.3% and 4.1 ⁇ 1.5% of myocytes at 4 and 27 months, respectively.
  • the critical role played by resident primitive cells in the remodeling of the injured heart is well appreciated when organ chimerism, associated with transplantation of a female heart in a male recipient, is considered.
  • organ chimerism associated with transplantation of a female heart in a male recipient
  • 8 female hearts implanted in male hosts were analyzed.
  • Translocation of male cells to the grafted female heart was identified by FISH for Y chromosome (see Example IE).
  • FISH FISH for Y chromosome
  • the percentages of myocytes, coronary arterioles and capillary profiles labeled by Y chromosome were 9%, 14% and 7%, respectively.
  • the numbers of undifferentiated c-kit and multidrug resistance- 1 (MDRl) positive cells in the implanted female hearts were measured. Additionally, the possibility that these cells contained the Y chromosome was established.
  • Cardiac transplantation involves the preservation of portions of the atria of the recipient on which the donor heart with part of its own atria is attached. This surgical procedure is critical for understanding whether the atria from the host and donor contained undifferentiated cells that may contribute to the complex remodeling process of the implanted heart. Quantitatively, the values of c-kit and, MDRl labeled cells were very low in control non-transplanted hearts: 3 c-kit and 5 MDRl/100 mm 2 of left ventricular myocardium. In contrast, the numbers of c-kit and MDRl cells in the atria of the recipient were 15 and 42/100 mm 2 .
  • the number of MDRl positive cells was higher than those expressing c-kit, but followed a similar localization pattern; 43 ⁇ 14, 29 ⁇ 16, 14 ⁇ 7 and 12 ⁇ 10/100 mm 2 in the atria, apex, base and mid-section. Again, the values in the atria and apex were greater than in the other two areas. Sca-1 labeled cells showed the highest value; 150 ⁇ 36/100 mm 2 positive cells were found in the atria. Cells positive for c-kit, MDRl and Sca-1 were negative for CD45, and for myocyte, endothelial cell, smooth muscle cell and fibroblast cytoplasmic proteins.
  • Li et al. Cardiomyocyte Transplantation Improves Heart Function; 1996 by the Society of Thoracic Surgeons; 62: pp. 654-661. 50. Li et al. Human Pediatric and Adult Ventricular Cardiomyocytes in Culture: Assessment of Phenotypic Changes with Passaging; Feb. 20, 1996 Cardiovascular Research; pp. 1-12.
  • Betafectin® PGG-glucan alone and in combination with granulocyte colony- stimulating factor Stem Cells 1998 May; 16(3):208-217.

Abstract

La présente invention concerne des procédés, compositions, et trousses pour la réparation de myocarde et/ou de cellules de myocarde endommagés comprenant l'administration de cellules-souches, telles que des cellules-souches adultes, éventuellement avec des cytokines.
EP01961855A 2000-07-31 2001-07-31 Procedes et compositions permettant la reparation et/ou la regeneration de myocarde endommage Withdrawn EP1315510A4 (fr)

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US25856400P 2000-12-29 2000-12-29
US258564P 2000-12-29
US25880501P 2001-01-02 2001-01-02
US258805P 2001-01-02
PCT/US2001/024223 WO2002013760A2 (fr) 2000-07-31 2001-07-31 Procedes et compositions permettant la reparation et/ou la regeneration de myocarde endommage

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WO2002013760A3 (fr) 2002-04-18
CA2423592A1 (fr) 2002-02-21
AU2001283088A1 (en) 2002-02-25
WO2002013760A2 (fr) 2002-02-21
WO2002013760A9 (fr) 2003-09-04

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