EP1685237A2 - Nouvelles cellules souches multipotentes - Google Patents

Nouvelles cellules souches multipotentes

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Publication number
EP1685237A2
EP1685237A2 EP04796882A EP04796882A EP1685237A2 EP 1685237 A2 EP1685237 A2 EP 1685237A2 EP 04796882 A EP04796882 A EP 04796882A EP 04796882 A EP04796882 A EP 04796882A EP 1685237 A2 EP1685237 A2 EP 1685237A2
Authority
EP
European Patent Office
Prior art keywords
cells
bone marrow
cell
hbmsc
graft
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Withdrawn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
EP04796882A
Other languages
German (de)
English (en)
Other versions
EP1685237A4 (fr
Inventor
Young-Sup Yoon
Douglas W. Losordo
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.)
Caritas St Elizabeth Medical Center of Boston
St Elizabeths Medical Center of Boston Inc
Original Assignee
Caritas St Elizabeth Medical Center of Boston
St Elizabeths Medical Center of Boston Inc
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 Caritas St Elizabeth Medical Center of Boston, St Elizabeths Medical Center of Boston Inc filed Critical Caritas St Elizabeth Medical Center of Boston
Publication of EP1685237A2 publication Critical patent/EP1685237A2/fr
Publication of EP1685237A4 publication Critical patent/EP1685237A4/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Withdrawn legal-status Critical Current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
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem 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
    • 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
    • C12N5/0607—Non-embryonic pluripotent stem cells, e.g. MASC
    • 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
    • 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
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • 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

Definitions

  • the invention generally relates to isolated bone marrow stem cells (BMSCs) having undetectable or low levels of selected cell markers, Importantly, the cells are multipotent and can be used to make a variety of desirable cell types.
  • BMSCs bone marrow stem cells
  • the invention has a wide spectrum of useful applications including use in the prevention and treatment of cardiovascular disease.
  • CHF congestive heart failure
  • MI myocardial infarction
  • Infarct size is a major determinant of morbidity and mortality in CHF.
  • large infarcts affect 40% or more of the left ventricle (LV) are typically associated with intractable cardiogenic shock or the rapid development of congestive heart failure.
  • the longstanding axiom has been that the myocardium has a limited capacity for self repair or regeneration, and the irreversible loss of muscle and accompanying contraction and fibrosis of myocardial scar, sets into play a series of events, namely progressive ventricular remodeling of non-ischemic myocardium, that ultimately leads to progressive HF.
  • tissue specific stem cells could only differentiate into cells of the tissue of origin.
  • tissue-specific stem cells can differentiate into lineages other than the tissue of origin.
  • plasticity has been referred to as trans-differentiation.
  • Bone marrow cells appear to have the capacity to repopulate many non-hematopoietic tissues such as neuroectodermal cells, skeletal myoblasts, CMCs, endothelium, hepatic and biliary duct epithelium, and lung, gut and skin epithelia.
  • MSCs multipotent adult progenitor cells
  • MAPCs are thought to proliferate extensively and differentiate into cells of all three germ layers. Therapeutic application of BM derived-stem cells has been attempted. Work has shown that endothelial progenitor cells (EPCs), angioblasts, or CD34(+) cells transplanted into ischemic myocardium incorporate into foci of neovascularization and have a favorable impact on LV function after ML The more versatile potential of BM derived hematopoietic stem cells(HSCs) has been documented in other studies.
  • EPCs endothelial progenitor cells
  • angioblasts or CD34(+) cells transplanted into ischemic myocardium incorporate into foci of neovascularization and have a favorable impact on LV function after ML
  • HSCs BM derived hematopoietic stem cells
  • MSCs non-hematopoietic mesenchymal stem cells
  • MSCs can be derived from adult BM and have multi-lineage differentiation capacity. In culture, MSCs can maintain an undifferentiated, stable phenotype over many generations. In animal models of MI, it has also been documented that murine, bovine and human MSCs can undergo cardiomyogenic differentiation.
  • BM cells that are convenient to isolate and maintain in tissue culture as clonal isolates. It would be especially desirable if such BM cells could be maintained as pluripotent clones particularly as a source for at least one and preferably all three of EC, SMC and CMC cells. It would be even more desirable if such BM cells could be transplanted into diseased or damaged heart issue to help prevent, treat or reduce associated symptoms.
  • BMSCs bone marrow stem cells
  • BMSCs bone marrow stem cells
  • BMSCs clonally expanded BMSCs obtained from human bone marrow (referred to as hBMSCs) and found that they do not belong to any known BM-derived SC population such as hematopoietic SCs, mesenchymal SCs or multipotent adult progenitor cells such as endothelial progentior cells (EPCs).
  • EPCs endothelial progentior cells
  • preferred BMSCs of the invention are capable of differentiating into at least three different cell types when provided with suitable cues, particularly endothelial cells (ECs), smooth muscle cells (SMCs) and cardiomyocytes (CMCs).
  • ECs endothelial cells
  • SMCs smooth muscle cells
  • CMCs cardiomyocytes
  • the invention is well-suited to provide a potentially unlimited source of cells and tissue (including grafts) needed, for instance, to prevent, treat or reduce suffering associated with a range of cardiovascular disorders including those associated with infarcts.
  • the invention provides an isolated population of bone marrow stem cells (BMSCs) that have undetectable or low (negligible) levels of at least one and preferably all of the following cell markers: CD90, CD 117, CD34, CD113, FLK-1, tie-2, Oct 4, GATA-4, NKx2.5, Rex-1, CD105, CD117, CD133, MHC class I receptor and MHC class II receptor as determined by standard cell marker detection assay.
  • BMSCs have important uses and advantages.
  • the BMSCs are highly plastic and can be used to provide a potentially unlimited source of cells and tissues to address cardiovascular disorders particularly those in which regenerative therapies are indicated.
  • BMSCs that can be readily converted into one or a variety of cardiovascular cells and particularly ECs, SMCs, and CMCs.
  • hBMSCs human patient
  • such cells can be maintained and/or treated ex vivo (eg., with one or more mitogens) and given back (transplanted) to the patient (or immunologically related individuals such as family members) to treat the cardiovascular disease.
  • the BMSCs can be isolated from the patient and treated ex vivo (with or without mitogens) to produce tissue grafts that in particular embodiments are at least allogeneic and preferably syngeneic with respect to the patient.
  • tissue grafts can be used, for example, to address cardiovascular disorders in which relatively large amounts of the BMSCs or cells derived therefrom are needed.
  • the method includes at least one and preferably all of the following steps: a) collecting bone marrow cells from a mammal which cells have a size of less than about 100 microns, preferably less than about 50 microns, more preferably about 40 microns or less, b) culturing (expanding) the collected cells in medium under conditions that select for adherent cells, c) selecting the adherent cells and expanding those cells in medium to semi-confluency, d) serially diluting the cultured cells into chambers with conditioned medium, the dilution being sufficient to produce a density of less than about 1 cell per chamber to make clonal isolates of the expanded cells; and e) culturing (expanding) each of the clonal isolates and selecting chambers having expanded cells to make the population of isolated bone marrow cells.
  • the invention provides tissue or a graft that includes the isolated BMSCs described herein such as, but not limited to, hBMSCs. Further provided is a cell culture, tissue or organ comprising the graft.
  • a method for preventing, treating or reducing the severity of a cardiovascular (heart) disorder includes administering to a mammal in need of such treatment at least one of the isolated BMSCs described herein (eg., hBMSCs).
  • the administration is sufficient to prevent, treat or reduce the severity of the disorder in the mammal.
  • the method includes administering to the mammal at least one graft of the invention under conditions that can help augment the heart disorder.
  • a pharmaceutical product for preventing, treating or reducing the severity of a heart disorder is provided by the invention.
  • the product includes at least one of the following components: a population of isolated BMSCs as described herein (eg., hBMSCs) and/or optionally directions for isolating the cells from a mammal; a graft of the invention and/or optionally directions for preparing, maintaining and/or using the graft; the cell culture, tissue or organ of the invention and/or optionally directions for preparing same from a mammal.
  • a population of isolated BMSCs as described herein eg., hBMSCs
  • optionally directions for isolating the cells from a mammal e.g., hBMSCs
  • Also provided by the present invention is a population of isolated bone marrow cells obtained by at least one of and preferably all of the following process steps: a) collecting bone marrow cells from a mammal which cells have a size of less than about 100 microns, preferably less than about 50 microns, more preferably about 40 microns or less, b) culturing (expanding) the collected cells in medium under conditions that select for adherent cells, c) selecting the adherent cells and expanding those cells in medium to semi-confluency, d) serially diluting the cultured cells into chambers with conditioned medium, the dilution being sufficient to produce a density of less than about 1 cell per chamber to make clonal isolates of the expanded cells; and e) culturing (expanding) each of the clonal isolates and selecting chambers having expanded cells to make the population of isolated bone marrow cells.
  • FIGS. 1A-E show characteristics of the Human Bone Marrow Stem Cells (hBMSC) of the invention.
  • Figure 2A-D show in vitro differentiation of hBMSC into endothelial cell (EC) and smooth muscle cell (SMC) lineages.
  • EC endothelial cell
  • SMC smooth muscle cell
  • Figures 3A-M show in vitro differentiation of hBMSC into neural and endodermal lineages.
  • Figures 4A-X show in vitro trans-differentiation and fusion of hBMSC to cardiomyocytes (CMC), EC and SMC cells.
  • FIGS 5A-F show that hBMSC transplantation attenuates adverse cardiac remodeling in a rat model of myocardial infarction (MI).
  • Figure 6A-T show engraftment and multilineage differentiation of transplanted hBMSC in infarcted myocardium.
  • Figure 7A-U show that human BMSC transplantation augments myocardial cell proliferation and reduces myocardial apoptosis.
  • Figure 8A-B shows that hBMSC transplantation upregulates mRNA expression of angiogenic cytokines and cardiac transcription.
  • Figure 9A-L shows human BMSC transplantation increased capillary and CMC density and increased myocardial fibrosis.
  • FIG. 10 shows results of FACS analysis using multiple surface epitopes
  • the invention provides an isolated population of novel and multipotent BMSCs that have undetectable or low (negligible) levels of at least one and preferably all of the following cell markers: CD90, CD117, CD34, CD113, FLK-1, tie-2, Oct 4, GATA-4, NKx2.5, Rex-1, CD105, CD117, CD133, MHC class I receptor and MHC class II receptor.
  • cell markers CD90, CD117, CD34, CD113, FLK-1, tie-2, Oct 4, GATA-4, NKx2.5, Rex-1, CD105, CD117, CD133, MHC class I receptor and MHC class II receptor.
  • Such markers can be readily determined by what is referred to herein as a standard cell marker detection assay.
  • the BMSCs of the invention have a wide spectrum of important uses including use in the prevention, treatment or alleviation of symptoms associated with a cardiovascular disorders, particularly those coronary diseases directly or indirectly associated with ischemia (myocardial ischemia), an infarct (myocardial infarction), congestive heart failure (CHF) and related coronary indications.
  • ischemia myocardial ischemia
  • infarct myocardial infarction
  • CHF congestive heart failure
  • standard cell marker detection assay is meant a conventional immunological or molecular assay formatted to detect and optionally quantitate one of the foregoing cell markers (ie., CD90, CD 117, CD34 etc.).
  • conventional immunological assays include Western blotting, ELIS A, and RIA.
  • Preferred antibodies for use in such assays are provided below. See generally, Harlow and Lane in Antibodies: A Laboratory Manual, CSH Publications, N.Y. (1988), for disclosure relating to these and other suitable assays.
  • Particular molecular assays suitable for such use include polymerase chain reaction (PCR) type assays using oligonucleotide primers disclosed herein (see Table 1, for instance).
  • PCR polymerase chain reaction
  • the BMSCs of the invention are at least 80% or 90% to 95% pure (w/w).
  • BMSC and particularly hBMSC having at least 98 to 99% homogeneity (w/w) are most preferred for many pharmaceutical, clinical and research applications.
  • the BMSC would be substantially free of unwanted marrow contaminants.
  • the BMSC are suited for therapeutic or other uses such as those provided herein. Purity can be determined by a variety of standard techniques such as cell culture, microscopical and centrifugation techniques (eg., Ficoll gradient). Particular disclosure relating to isolating and manipulating endothelial cells
  • EPCs endothelial progenitor cells
  • mammal is meant a primate, domesticated or other mammal such as a rodent or rabbit.
  • a preferred primate is a chimpanzee, monkey or a human patient in need of treatment.
  • Suitable domesticated animals include gerbils, horses, dogs, cats, goats, sheep, pigs, chickens and the like.
  • a preferred rodent is a rat or mouse.
  • a preferred BMSC is isolated from a primate and particularly a human subject such as those in need of cardiovascular therapy.
  • BMSC in accord with the invention are essentially spherical as determined by inspection (typically microscopy) and have a diameter of less than about 25 to 35 micrometers, preferably less than about 15 micrometers.
  • Other particular cells of the invention will have a mean telomere restriction fragment length (TRF) of less than about 30 to 40 kilobases, preferably less than about 20 kilobases such as about 17 kilobases.
  • TRF mean telomere restriction fragment length
  • BMSC of the invention are essentially euploid which euploidy is essentially maintained for at least about 10 passages in cell culture, preferably between from about 20 to about 200 passages. Methods for determining cell ploidy are known and are provided below.
  • More specific BMSCs in accord with the invention are capable of forming endothelial cells (ECs), for instance, after contact with EC promoting conditions as determined by a standard EC differentiation assay.
  • EC promoting conditions are known in the field and include contact with certain angiogenic factors and cell mitogens such as those disclosed by the US Patent No. 5,980,887; PCT/US99/05130 (WO 99/45775) and references cited therein.
  • Such disclosed factors and mitogens include acidic and basic fibroblast growth factors (aFGF and bFGF), vascular endothelial growth factor (VEGF-1), VEGF165, epidermal growth factor (EGF), transforming growth factor ⁇ and ⁇ (TGF- ⁇ and TFG- ⁇ ), platelet-derived endothelial growth factor (PD-ECGF), platelet-derived growth factor (PDGF), tumor necrosis factor ⁇ (TNF- ⁇ ), hepatocyte growth factor (HGF), insulin like growth factor (IGF), erythropoietin, colony stimulating factor (CSF), macrophage-CSF (M-CSF), granulocyte/macrophage CSF (GM-CSF), angiopoetin-1 (Angl) and nitric oxidesynthase (NOS); and functional fragments thereof.
  • aFGF and bFGF acidic and basic fibroblast growth factors
  • VEGF-1 vascular endothelial growth factor
  • a preferred EC promoting condition includes contact with VEGF, particularly VEGF-1, VEGF 165, or both. Additionally preferred EC promoting conditions include contact with certain cell matrix proteins such as fibronectin. See Example 2 below.
  • standard EC differentiation assay any of the assays used to detect and monitor function of ECs such as those disclosed by the US Pat. No. 5,980,887 and WO 99/45775.
  • a preferred assay involves detection of EC specific markers such as reported in Example 2.
  • BMSCs that can form smooth muscle cells (SMCs), particularly after contact with SMC promoting conditions as determined by a standard SMC differentiation assay.
  • SMC promoting conditions are known and include contact with at least one angiogenic factor or cell mitogen as discussed previously.
  • a preferred SMC promoting condition involves contact with a platelet derived growth factor (PDGF) including muteins or active fragments thereof.
  • PDGF platelet derived growth factor
  • standard SMC differentiation assay is meant an immunological or molecular test (eg., ELISA, Western blot or PCR) that is capable of detecting and optionally quantitating at least one and preferably all of the following SMC specific markers: ⁇ SMA, PDGF ⁇ receptor, SM22 ⁇ , and SMI.
  • An illustration of an acceptable assay is provided by Example 2.
  • Still further BMSCs in accord with the invention can form neuronal cells especially after contact with neuronal cell promoting conditions as determined by a standard neuronal cell differentiation assay.
  • a variety of neuronal cell promoting conditions are known and include contact with one or more of the forgoing factors and mitogens. Preferred are conditions that involve contact with hepatocyte growth factor (HGF) alone or preferably in combination with fibroblast growth factor 4 (FGF-4).
  • Still further preferred neuronal cell promoting conditions involve further contact with DMSO or a pharmaceutically acceptable butyrate salt such as sodium or potassium salts thereof.
  • Still further preferred neuronal cell promoting conditions include contact with a suitable cell matrix protein such as poly-L-ornithine-laminin.
  • standard neuronal cell differentiation assay is meant an immunological or molecular test (eg., ELISA, Western blot or PCR) that is capable of detecting and optionally quantitating at least one and preferably all of the following neuronal specific markers: GFAP, GalC, NF200, a tubulin, myelin basic protein, MAP2, GAD and Tau. See Example 2 (disclosing a particularly preferred neuronal cell promoting condition).
  • immunological or molecular test eg., ELISA, Western blot or PCR
  • BMSCs of the invention include those cells that can fomi cardiomyocytes after contact with accessory cardiomyocytes.
  • accessory cardiomyocytes are meant cells that were cardiomyocytes prior to the contact with the BMSCs.
  • accessory cardiomyocytes include established cultures of such cells as well as cardiomyocytes inhabiting cardiac tissue.
  • the formation of cardiomyocytes by the BMSCs will be assisted by cell fusion between the stem cells and the accessory cardiomyocytes.
  • Such cardiomyocytes (and particularly the accessory cells) can be maintained by one or a combination of stratagies including those involving maintenance in vitro, ex vivo or in vivo.
  • graft that includes (or in some embodiments consists of) the isolated bone marrow stem cells described herein.
  • graft is meant a cell or tissue preparation that includes the BMSCs and optionally other cells such as ECs, SMCs and CMCs from a mammal.
  • a donor is defined as the source of the BMSCs whereas the recipient is the subject that receives the graft.
  • Immunological relationship between the donor and recipient can be allogenic, autologous, or xenogeneic as needed, preferred invention embodiments, the donor and recipient will be genetically identical and usually will be the same individual (syngeneic). In this instance, the graft will be syngeneic with respect to the donor and recipient.
  • graft is also meant BMSCs of the invention that have been administered to a recipient and become part of one or more tissues or organs of that recipient.
  • engraftment will be used to denote intended assimilation of the BMSCs into a targeted tissue or organ (either as BMSCs or differentiated cells).
  • Preferred engraftment involves a cardiovascular tissue such as veins, arteries and particularly cardiac tissue.
  • a graft of the invention may also take the form of a tissue culture preparation in which BMSCs of the invention have been combined with other cells and or mitogens to promote differentiation and/or cell replication that produces an intended graft. If desired, the preparation can be combined with synthetic or semi synthetic fibers to give structure to the graft.
  • a particular example of a graft of the invention is a preparation of hBMSCs that have been isolated from a donor to prevent, treat or reduce the severity of a cardiovascular disorder such as myocardial ischemia or an infarct.
  • the preparation can include a pharmaceutically acceptable carrier such as saline and optionally may include at least one of mitogens, angiogenic factors, CMCs, ECs, EPCs, and SMCs to assist an intended engraftment result.
  • Particular CMCs of the invention express CMC specific protein (cTnl).
  • ECs that ILB-4.
  • SMCs that express ⁇ -SMA and related markers.
  • the invention further provides a method for making the BMSCs described herein including those cells obtained from human patients (hBMSCs).
  • the method includes at least one and preferably all of the following steps: a) collecting bone marrow cells from a mammal (preferably a young adult) which cells have a size of less than about 100 microns, preferably less than about 50 microns, more preferably about 40 microns or less, b) culturing (expanding) the collected cells in medium under conditions that select for adherent cells, c) selecting the adherent cells and expanding those cells in medium to semi-confluency, d) serially diluting the cultured cells into chambers with conditioned medium, the dilution being sufficient to produce a density of less than about 1 cell per chamber to make clonal isolates of the expanded cells; and e) culturing (expanding) each of the clonal isolates and selecting chambers having expanded cells to make the population of isolated bone marrow cells.
  • the hBMSC can be obtained by taking fresh unprocessed bone marrow (BM) cells from young male donors. Alternatively, such cells can be purchased. The cells are typically separated from blood cells by centrifugation, hemolysis and related standard procedures. The BMs are washed in an acceptable buffer such as DPBS and filtered to collect cells having a size less that about 100 microns, preferably less than about 50 microns, more preferably about 40 microns. A standard nylon filter, for instance, can be used . Once isolated, the BMs are grown on a complete culture medium with low glucose (eg., DMEM) that contains a rich source of growth factors and cytokines.
  • DMEM low glucose
  • Fetal bovine serum is preferred.
  • Cells are cultured (ie. expanded) for less than about two weeks, preferably about a week or less such as four to six days.
  • the conditioned medium is then replaced with fresh medium, adherent cells are removed from the culture dishes and resuspended in fresh medium to select cells that can be expanded.
  • the selected cells are grown to semiconfluency (between 50% to 90% confluent) and again, adherent cells are selected.
  • Such cells are then reseeded in complete medium in a tissue culture flask at a density of about 10 4 cells per centimeter. After the cells reach semiconfluency, they are reseeded (serially) into the flasks at the same or similar density.
  • the cultures are preferably passaged more than one time, typically less than five times and preferably about two times to continue selection for expanding cells.
  • Selected cells are then serially diluted into single well chambers (eg., standard 96 well plate) at a density of less than about 1 cell per chamber, preferably VT. a cell per chamber.
  • the cells are cultured with conditioned media to promote growth to sub confluence (ie. less then 50% confluent).
  • Wells with expanded cell clones were expanded and replated as needed.
  • the method further includes selected cell clones that do express detectable levels of at least one of the following markers: CD90, CD117, CD34, CD113, FLK-1, tie-2, Oct 4, GATA-4, NKx2.5, Rex-1, CD105, CD117, CD133, MHC class I receptor, MHC class II receptor or other cell marker as described herein.
  • Methods for performing the selection include any of the suitable assays disclosed herein. In embodiments in which larger amounts of stem cells are needed a more automated or semi-automated method will often be preferred such as fluorescence activated cell sorting (FACS). See the Examples provided below.
  • FACS fluorescence activated cell sorting
  • Example 1 A preferred example of the method for making the BMSCs is provided by Example 1 below.
  • the invention also provides a method for preventing, treating or reducing the severity of a heart disorder that in one embodiment includes administering to a mammal in need of treatment at least one of the isolated BMSCs, grafts, or both disclosed herein. Preferably, the administration is sufficient to prevent, treat or reduce the severity of the disorder in the mammal.
  • the method further includes incubating the cells or graft in the mammal for at least about a week, preferably between from about two to eight weeks. It will be apparent to those working in the field that the incubation period is flexible and can be extended or shorten to address a particular indication or with respect to the health or age of the individual in need of treatment. Typical amounts of BMSC to use will depend on these and other recognized parameters including the disease to be treated and the speed of recovery needed. However for most applications between from about 10 3 to about 10 7 BMSCs will suffice, typically about 10 5 of such cells.
  • Cells may be administered by any acceptable route including suspending the cells in saline and administering same with a needle, stent, catheter or like device. In embodiments in which myocardial ischemia or an infarct is to be addressed, the administration will be a bolus injection near or directly into the site of injury.
  • Examples 3 and 4 A particular method for regenerating heart tissue following myocardial ischemia is provided by Examples 3 and 4. See also Examples-5-6 (showing engraftment of hBMSC into cardiac tissue). As discussed, transplantation of the
  • the method further includes administering to the mammal in need of treatment at least one angiogenic factor or mitogen (or functional fragment of the factor or mitogen).
  • angiogenic factors and mitogens are disclosed herein as well as US Pat. No. 5,980,887 and WO 99/45775.
  • the method can include administering to the mammal at least one nucleic acid encoding at least one angiogenic factor or functional fragment thereof. Method for administering such nucleic acids to the mammal have been disclosed by US Pat. No. 5,980,887 and WO 99/45775, for instance. Treatment with the angiogenic factor/mitogen protein or nucleic acid encoding same can precede use of the BMSCs or it can be used during or after such treatment as needed.
  • the method further includes administering to the mammal endothelial progenitor cells (EPCs).
  • EPCs mammal endothelial progenitor cells
  • This invention embodiment especially finds use where good vascular growth is needed to address a cardiovascular disorder.
  • Methods for making and using EPCs have been disclosed. See U.S. Pat. No. 5,980,887, for example.
  • Typical methods can include isolating the EPCs from the mammal and contacting the EPCs with at least one angiogenic factor and/or mitogen ex vivo.
  • the invention is applicable to the prevention and treatment of a wide variety of cardiovascular disorders including congestive heart failure (CHF), ischemic cardiomyopathy, myocardial ischemia, and an infarct.
  • CHF congestive heart failure
  • ischemic cardiomyopathy myocardial ischemia
  • infarct cardiovascular disorders
  • such methods can further include monitoring cardiac function in the mammal seeking treatment, eg., by monitoring at least one of echocardiography, ventricular end- diastolic dimension (LVEDD), end-systolic dimension (LVESD), fractional shortening (FS), wall motion score index (WMSI) and LV systolic pressure (LVSP).
  • Prefereed invention methods involving prevention or treatment of a particular cardiovascular disease will manifest good cardiac function as exemplified by one or more of these tests.
  • good is meant at least a 10% improvement, preferably at least 20% or 30% compared to a control that does not receive an invention composition (or receives a placebo). Particular methods for performing
  • a pharmaceutical product for preventing, treating or reducing the severity of a heart disorder that includes, for instance, at least one of the following components: BMSCs, preferably hBMSCs and optionally directions for isolating the cells from a mammal; a graft as disclosed herein, preferably one with hBMSCs or cells or tissue derived from same and optionally directions for preparing, maintaining and/or using the graft; the cell culture, tissue or organ and optionally directions for preparing same.
  • the product further includes at least one angiogenic factor, mitogen; or functional fragment thereof, h another embodiment, the product further comprises at least one nucleic acid encoding the angiogenic factor, mitogen or functional fragment thereof.
  • the invention provides isolated BMSCs such as hBMSCs that are made by a method disclosed herein.
  • Such cells possess advantages such as desirable plasticity and ability to propagate for long periods of time without significant culture problems (eg., undesired polyploidy and loss of muhpotency).
  • such cells are further made by collecting cells that do not express detectable levels of at least one of the following markers: CD90, CD 117, CD34, CD113, FLK-1, tie-2, Oct 4, GATA-4, NKx2.5, Rex-1, CD105, CD117, CD133, MHC class I receptor, MHC class II receptor or other marker as provided herein.
  • the present disclosure particularly shows that the invention can be used to isolate, from a single cell, BMSCs and especially hBMSCs that possess potentially unlimited proliferation capacity and are capable of triple lineage differentiation including ECs, SMCs and CMCs.
  • BMSCs and especially hBMSCs that possess potentially unlimited proliferation capacity and are capable of triple lineage differentiation including ECs, SMCs and CMCs.
  • the transplantation of hBMSC into acutely infarcted myocardium attenuates cardiac dysfunction both by de novo differentiation into myocardial tissues and favorable paracrine effects on the adjacent cells.
  • the present disclosure including the Examples show that a novel stem cell (SC) population(pop) has been identified within adult human bone marrow(BM). Further shown is isolation of these cells at the single cell level and proliferation without obvious senescence or loss of multi-potency for > 140 population doublings (PDs).
  • SC stem cell
  • PDs population doublings
  • hBMSC clonally expanded human BM-derived multipotent SC
  • the hBMSC that we have isolated do not belong to any known BM-derived SC pop such as hematopoietic SCs, mesenchymal SCs or multipotent adult progenitor cells.
  • hBMSC exhibit a potential for differentiation(Diff) into cells of all three germ layers in vitro.
  • hBMSC Co-culture of hBMSC with neonatal rat cardiomyocytes(CMC), rat aortic endothelial cells(EC) or rat smooth muscle cells(SMC) revealed phenotypic changes consisting of both true Diff and cell to cell fusion.
  • CMC neonatal rat cardiomyocytes
  • EC rat aortic endothelial cells
  • SMC rat smooth muscle cells
  • Myoc function measured by echo and pressure transducer was significantly better in the hBMSC xplnt rats than in the total BM cell xplnt or saline injected control rats 28 days later. Robust engraftment of xplnt'd cells was observed, which exhibited Diff into CMCs, ECs and SMCs. In the hBMSC transplanted Myoc, multiple angiogenic cytokines and essential cardiac transcription factors were significantly upregulated and higher rates of EC and CMC proliferation were observed.
  • a novel multipotent SC population exists within the human BM, and locally xplnt'd hBMSC can ameliorate the outcome of acute MI by de novo vasculogenesis and myogenesis as well as augmenting proliferation and preservation of host myocardial tissues.
  • human BM-derived SCs can differentiate into all the elements required for the regeneration of ischemic myocardium both in vitro and in vivo.
  • the disclosure shows that a novel adult human stem cell population, hBMSC, which does not belong to the known population of adult stem cells, was isolated beginning at the single cell level from mixed total BM cell culture and expanded clonally in culture for >140 PDs without loss of plasticity or onset of replicative senescence.
  • clonally derived hBMSC differentiated into cells of three germ layers (endoderm, mesoderm, neuroectoderm) in vitro.
  • transplantation of hBMSC into an animal model of MI ameliorates the functional and pathologic changes following MI.
  • the mechanism of improved cardiac function consists not only of differentiation into essential myocardial tissues such as CMCs, ECs and SMCs, but also involves apparent paracrine effects of the transplanted stem cells, which stimulate the proliferation of host myocardial tissues and prevent apoptosis of endangered cells following ischemic injury.
  • hBMSC that we have identified are a unique population of adult BM derived multipotent SCs in that ⁇ 1% of hBMSC expresses CD90 and GDI 17. All the common marker panels defining the HSC, MSC, and MAPC do not match the profile of hBMSC. hBMSC do not express the well known MSC marker proteins, CD105(SH2) and CD73(SH3 and SH4) even though the media used is similar to those of MSC cultures ⁇ Barry, 1999; Barry, 2001 ⁇ . The absence of or minimal expression of surface molecules is a pre-requisite for the plasticity of hBMSC as shown in other adult multipotent stem cells[Jiang, 2002] ⁇ Colter, 2001 ⁇ .
  • hBMSC unique surface phenotype results from the use of total BM cell population for initial culture and the clonal isolation/selection procedure.
  • Our invention has addressed one important issue in stem cell biology, i.e. whether multipotent stem cells could be culture-expanded from a single cell.
  • stem cell biology i.e. whether multipotent stem cells could be culture-expanded from a single cell.
  • hBMSC do not express genetic markers of ESCs, such as Oct-4 and Rex-1, which are believed to be the factors essential for MAPCs.
  • the hBMSC of the invention provide advantages for regenerative therapy of cardiac diseases.
  • stem cells shown to improve cardiac function are EPCs, CD34(+) cells, angioblasts, that have only shown to promote neovascularization in vivo.
  • Multipotent stem cells, such as MSCs, c-kit positive lineage negative cells, SP cells, and ESC have been tested in ischemic animal models.
  • ESCs have only demonstrated the potential to differentiate into CMCs but have not been shown to differentiate into ECs or SMCs in animal models of IHD.
  • the use of ESCs is hindered by certain ethical issues which remain to be resolved prior to clinical application.
  • Coronary heart disease accounts for 50% of all cardiovascular deaths and nearly 40% of the incidence of heart failure.
  • the current findings have provided compelling evidence that the invention can be used to prevent, treat or reduce the severity of a variety of heart disorders including those associated with infarcts and/or ischemia. More specifically, the invention promotes de novo vasculo-myogenesis and augments angiogenesis and proliferation of existent CMCs, reduces progressive fibrosis, and improves ventricular function in a rodent model of MI. We believe this is the first demonstration that adult human stem cells can promote successful treatment of acute Mis during the process of ischemic tissue damage.
  • the invention is also applicable to the prevention or treatment of IHD, particularly to improve the immediate and long-term outcome of IHD.
  • the following Examples are intended to be illustrative and not limit the invention. All references disclosed herein are incorporated by reference.
  • Example 1 Culture and Characteristics of hBMSC The clonality, surface epitopes, euploidy and proliferation of hBMSCs was evaluated as follows. Fresh unprocessed human BMs from young male donors were purchased. Three different marrow specimens from three different donors for SC cultures were used. After serial culture of total marrow cells in plastic dishes in
  • Dulbecco's modified eagle's medium with low(lg) glucose containing 17% of fetal bovine serum(FBS), cells were labeled with red fluorescent dye, Dil. After limiting dilution(l-2 cells per well in 96 well plate) wells containing a single cell visualized by fluorescent microscopy were selected. Of wells containing a single cell
  • hBMSC did not differentiate spontaneously and maintained their phenotype during culture expansion, i contrast, prior to clonal isolation the cultured hBMSC expressed low levels of CD105, CD90 and CD117 while mesenchymal stem cells expressed high levels of CD29, CD44, CD73, CD105 and CD90 (Fig. lb).
  • Major histocompatibility complex(MHC) class I(ABC) and II(DR) molecules and known hematopoietic stem markers(CD34, CD133, FLK-1, Tie2) were not expressed(Fig. IS).
  • telomere restriction fragment(TRF) length of hBMSC cultured for 5 PDs was about 17 kilobases (kb); when re-tested after 120 PDs, mean TRF remained unchanged(Fig. Id).
  • DNA ploidy(the number of DNA copies) was examined by FACS analysis after staining DNA with propidium iodide.
  • hBMSC cultured for 20 and 140 PDs from 3 different clones demonstrated no evidence for increased ploidy, suggesting that the euploidy is maintained during the culture-expansion.
  • TRF telomere restriction fragment
  • hBMSC before and after clonal selection were stained with propidium iodide and subjected to FACS analysis.
  • hBMSC cultured for 20(left panel) and 140(right panel) PDs demonstrated ⁇ 1% of over-diploid DNA content.
  • Example 2 Plasticity of hBMSC: in vitro Differentiation
  • the in vitro differentiation potential of hBMSC was tested by adopting and modifying previously published culture conditions for adult and embryonic stem cells, which primarily requires lineage-specific cytokines.
  • hBMSC were replated at 5 x 10 4 cells/cm 2 in either 0.1% gelatin or fibronectin coated glass chamber slides in DMEM or EBM- 2(Clonetics) with 2% FBS, 10 "8 dexamethasone and 10 ng/ml VEGF.
  • hBMSC exhibited EC specific phenotypes such as von WiUebrand factor(vWF), Flkl, VE-Cadherin, CD31, human-specific EC marker Ulex europaeus lectin type l(UAE-l) (Fig. 2a).
  • vWF von WiUebrand factor
  • Flkl Flkl
  • VE-Cadherin CD31
  • human-specific EC marker Ulex europaeus lectin type l(UAE-l) Fig. 2a
  • 63 ⁇ 8%(VE-Cadherin) to 85 ⁇ 12%(UAE-1) of hBMSC demonstrated EC phenotypes by immunocytochemistry.
  • RT- PCR of EC specific genes, VE-Cadherin, CD34, Flk-1, Tie2 and CD31 also confirmed the differentiation of hBMSC(pre-differentiation) into EC phenotypes(post- differentiation) (Fig.
  • hBMSC were replated at 1 x 10 5 cells/cm 2 in non-coated or fibronectin coated plastic dishes in 1-2% DMEM or EBM-2 supplemented with PDGF-BB(50 ng/ml, R&D) [Hellstrom, 1999;Yamashita, 2000].
  • PDGF-BB 50 ng/ml, R&D
  • RT-PCR confirmed the de novo expression of SMC specific genes, ⁇ SMA, PDGF ⁇ receptor, SM22 ⁇ , and SMI (Fig. 2d).
  • Figure 2A-D are explained in more detail as follows: a. Hoffman image (left upper) 5 days after culture with DMEM in gelatin coated glass chambers that, hBMSC formed typical vascular tube-like structures. Immunfluorscent imaging demonstrates liBMSC exhibits EC specific proteins, such as vWF, Flkl, VE-Cadherin, CD31 and UEA-1 after culturing hBMSC in EC differentiation media for 14 days. b.
  • RT-PCR analysis using EC specific gene primers, VE-Cadherin, CD34, Flk-1, Tie2 and CD31 also confirmed the differentiation of hBMSC(pre-differentiation) into EC phenotypes (post- differentiation) c.
  • hBMSC cultured in 2% DMEM containing PDGF-BB for 14 days demonstrates expression of SMC specific proteins, ⁇ -SMA and calponin by IF staining, d.
  • RT-PCR analysis shows that SMC specific genes, PDGFR- ⁇ , ⁇ -SMA, SM22 ⁇ and SMI are only expressed after induction of differentiation.
  • Thick ladder in size marker in RT-PCR(b and d) 600bp
  • hBMSC were plated at 4 x 10 4 /cm2 on plastic dishes or Poly-L-ornithine-laminin coated dishes with 100 ng/ml bFGF, 20 ng/ml EGF, and B27 supplement in DMEM/F 12 [Palmer, 1999;Mezey, 2000 ;Brazelton, 2000]. After 10-14 days, hBMSC showed morphologic and phenotypic characteristics of various neural lineage cells(Fig. 3 a).
  • Immunofluorescent cytochemistry revealed expression of phenotypic markers of astrocytes(glial acidic fibrillar protein, GFAP), oligodendrocytes(galactocerebroside, GalC), and neurons(neurofilament 200, NF200; ⁇ -tubulin III isoform, ⁇ -tubulin III) in 22 ⁇ 7 %, 15 + 6%, 57 ⁇ 9%(NF200) of hBMSC, respectively.
  • RT-PCR confirmed de novo expression of various neural lineage specific genes such as GFAP, myelin basic protein(MBP, oligodendrocyte), MAP2(microtubule-associated protein 2, neuron), GAD(glutamic acid decarboxylase, neuron), and Tau(neuron) [Sanchez-Ramos, 2000].
  • hBMSC were plated at 3-4 xlO 4 cells/cm 2 on 1% Matrigel in 2% FBS, supplemented with 10 "8 dexamethasone, 25 ng/ml HGF, 10 ng/ml FGF-4 and either 1 Omg/ml DMSO or 0.5mM sodium butyrate[Shen, 2000;Hamazaki, 2001;Oh, 2000;Schwartz, 2002].
  • 10 - 14 days of culture about 60% of hBMSC acquired epithelioid morphology.
  • RT- PCR analysis revealed de novo expression of endodermal lineage-specific genes, CK18, CK 19, ⁇ FP and albumin (Fig. 3m).
  • Phenotypic characterization of hBMSC differentiation into endodermal(epithelioid) cells IF localization of HNF 3 ⁇ (FITC) and ⁇ FP(Cy3) at day l ⁇ (g-i), and CK18(FITC) and HNF l ⁇ (Cy3) at day 14(j-l) in cultured hBMSC on Matrigel with media containing HGF, FGF-4 and DMSO. m. RT-PCR confirms de novo expression of endodermal lineage-specific genes, CK18, CK19, ⁇ FP and albumin after induced neural differentiation.
  • Example 3 Cardiomyogenic Differentiation of BMSC After Co-culture With Neonatal Rat Cardiomyocyte (NRCM) To induce cardiomyogenic differentiation, hBMSC were co-cultured with neonatal rat cardiomyocytes (NRCM). NRCM were plated at 1 x 10 5 cells/cm 2 and cultured in DMEM (low glucose) containing 10% fetal calf serum (FCS). On day 3, hBMSC labeled with Dil were added to the cultured NRCM at a 1 :4 ratio [Condorelli, 2001] and cultured up to 2 wks. After fixation and staining with antibodies against cardiac specific markers, immunofluorescent images were obtained. Dil-labeled hBMSC(Fig.
  • FIG. 4b, f, j exhibit red fluorescence from the Dil label and cells positive for CMC specific proteins, such as cardiac troponin I(cTnl), atrial natriuretic peptide(ANP), ⁇ -myosin heavy chain ( ⁇ -MHC) appear green (Fig. 4c, g, k).
  • CMC specific proteins such as cardiac troponin I(cTnl), atrial natriuretic peptide(ANP), ⁇ -myosin heavy chain ( ⁇ -MHC) appear green (Fig. 4c, g, k).
  • the merged images illustrate a fraction of Dil-labeled hBMSC which also stained positive for the CMC specific proteins indicating that a subpopulation of hBMSC were displaying features of CMC phenotype in co-culture (Fig. 4).
  • mRNA expression of cardiac transcription factors was evaluated by RT-PCR (Fig. 4m).
  • GATA-4 and Nkx2.5 were not expressed in hBMSC(left lanes) or NRCM(middle lanes) cultures.
  • Co-culture (right lanes) of hBMSC and NRCM only induced de novo expression of GATA4 and Nkx2.5.
  • GATA-4 is a cardiac specific transcription factor which is known to activate the promoters of several cardiac genes, such as myosin light chain, TnT, Tnl and ⁇ -MHC, and ANP.
  • Nkx2.5 is a transcription factor restricted to the initial phases of CMC differentiation. [Srivastava, 2000;Bruneau, 2002] Accordingly, these findings suggest that hBMSC have entered a differentiation pathway toward CMC phenotype.
  • CFDA- SE(carboxyfluorescein diacetate succiniimidyl ester)-labeled NRCM and Dil-labeled hBMSC was used for co-culture since neither of these chemical dyes is transferred to adjacent cells. Seven days after co-culture , cultured cells were fixed and stained with cTnl followed by AMCA-conjugated secondary antibody(blue fluorescence) (Fig. 4p). To avoid the overlap of cells, we selected areas of single cell layers for imaging analysis.
  • rat aortic endothelial cells RAECs
  • RVSMCs rat vascular smooth muscle cells
  • FIGS. 4A-M are explained more as follows, a-m.
  • Co-culture of hBMSC with NRCM for investigation of cardiomyogenic differentiation Primary NRCM from F344 rats were isolated and cultured in DMEM. On day 4, Dil-labeled hBMSC were added to the cultured NRCM at a 1:4 ratio and cultured up to 2 wks.
  • IF images show co- cultured Dil-labeled hBMSC(b,f, j) as red and NRCM stained with CMC specific proteins, cTnl (c), ANP(g) and ⁇ -MHC(k) as green.
  • Double fluorescence positive cells in the merged images indicate that a subpopulation of hBMSC exhibit CMC phenotypes(arrows). Note that a few hBMSC remain non-transdifferentiated (arrowheads in b, d, j, 1). DAPI nuclear counter-staining(a, e, i) shows no overlap of nuclei suggesting this apparent trans-differentiation does not result from a mere overlap of hBMSC and NRCMs.
  • mRNA expression of cardiac transcription factors was evaluated by RT-PCR (m). Before co-culture, cardiac transcription factors, GATA4 and Nkx2.5 were not expressed in hBMSC(lane 1) and NRCM(lane 2) cultures.
  • Co-culture (lane 3) induced de novo expression of GATA4 and Nkx2.5.
  • n-q Co-culture of pre-labeled NRCM and hBMSC for investigation of cell fusion. To determine whether fusion of both cells may underlie the change into CMC phenotypes, we co-culture d NRCMs labeled with green fluorescence dye, DFCA-SE (n) and Dil- labeled hBMSC (o). Seven days after co-culture, cells were stained with cTnl followed by AMCA-conjugated secondary antibody(blue fluorescence)(p). h the IF images(n- q), triple color positive cells(arrow) are fusion cells expressing cTnl protein.
  • a hBMSC which has transdifferentiated into CMC lineage is illustrated by red and blue fluorescence positive but green negative (arrowhead), r-x.
  • CDFA-SE-labeled rat aortic endothelial cells(RAECs) or rat vascular smooth muscle cells(RVSMCs) were co-cultured with Dil-labeled hBMSC at a 4:1 ratio and cultured for 7 days. After fixation, cells were stained with VE-Cadherin or ⁇ - SMA followed by AMCA-conjugated secondary antibody(blue fluorescence).
  • Arrow in r-u indicates fusion between RAECs(green) and hBMSC(red) which expresses VE- Cadherin. Arrowheads in r-u illustrate ECs differentiated from hBMSC. Arrow in v-x indicates fusion between RVSMCs(green) and hBMSC(red).
  • Example 4 Regenerative Effect On Acute Ischemic Myocardium
  • hBMSC hBMSC
  • shortly after coronary ligation in nude rats 8 x 10 5 hBMSC were transplanted in the wall of peri-infarct area.
  • the same number of human total bone marrow cells (TBMC) and the same volume of PBS were used as controls.
  • 14 rats receiving hBMSC survived while 12 rats survived in TBMC and PBS groups during the study period of 4 wks.
  • echocardiography was performed before and 4 wks after the surgery.
  • Baseline left ventricular end-diastolic and end-systolic dimensions (LVEDD, LVESD) fractional shortening(FS) and wall motion score index(WMSI) were similar among rats receiving hBMSC, TBMC or PBS(data not shown).
  • Echocardiography performed 4 wks after treatment revealed that, both LVEDD and LVESD were significantly smaller (LVEDD, P ⁇ 0.05 versus TBMC and PBS, respectively; LVESD, P ⁇ 0.01 versus TBM and PBS, respectively) (Fig. 5a,b) and thus FS was significantly greater (P ⁇ 0.01 versus TBMC and PBS, respectively) (Fig. 5c) in rats receiving hBMSC compared with those treated with TBMC or PBS.
  • Wall motion score index(WMSI) which represents the extent of regional wall motion abnormalities, was significantly better (P ⁇ 0.01 versus TBMC and PBS, respectively) (Fig. 5d). LVEDD, LVESD, FS, and WMSI were not different between TBM and PBS treated rats. Of note, the incidence of dyskinesia was 14% vs. 58% and 67% in hBMSC vs. TBMC and PBS treated rats (P ⁇ 0.05).
  • FIG. 5A-D is explained more fully as follows, a-d. Echocardiographic parameters 4 wks after MI and cell transplantation show smaller LVEDD, LVESD and better FS and WMSI in the hBMSC transplanted rats compared to the TBMC and PBS treated rats, indicating improved cardiac function.
  • WMSI Wall motion score index, e, f. Invasive hemodynamic measurements using Millar catheter 4 wks after the hBMSC transplantation.
  • LV systolic pressure (e) and +dP/dt and -dP/dt were significantly augmented in the hBMSC transplanted rats compared to the control groups'.
  • Example 5 Engraftment and multi-lineage trans-differentiation of transplanted hBMSC in vivo To determine the extent and magnitude of transplanted hBMSC engraftment, 4 wk-myocardial sections from frozen samples were examined. Under fluorescent microscopy, engraftment of numerous Dil-labeled BMSCs in the peri-infarct and infarct region was observed (Fig. 6a,b). In contrast, the TBMC transplanted heart shows smaller numbers of scattered Dil-labeled cells, which are mostly located within the infarct region (Fig. 5 c, d).
  • hnmunophenotypic characterization revealed that engrafted Dil-labeled hBMSC(red) were stained positive for CMC specific proteins, cTnl and ANP(green)( ⁇ -MHC data not shown) and formed a mass of morphologically mature appearing CMCs, indistinguishable from host CMCs in the periinfarct and infarct area (Fig 6. e-1), indicative of CMC differentiation.
  • Differentiation of hBMSC into ECs and SMCs was also confirmed by the co-localization of transplanted hBMSC with ILB4 positive vascular ECs and ⁇ -SMA positive SMCs, suggestive of differentiation into EC and SMC phenotypes(Fig 6. m-t).
  • CMC differentiation only in hBMSC transplanted rats Differentiation into CMC, defined by morphologic similarity and CMC specific protein expression(cTnl), was observed in 12 out of 14 surviving hBMSC transplanted rats(85%) could be detected. An organized mass of regenerating CMCs similar to normal host myocardium as seen in Fig 6h and Fig 8c was found in 9 out of 14 rats(64%) and only scattered cellular differentiation was observed in 3 other rats (21%). However, CMC specific protein expression was observed in all hBMSC treated rats. EC and SMC differentiation defined by morphology and marker positivity was found in all rats examined.
  • transplanted hBMSC were robustly engrafted in the infarct/ischemic myocardium, survived for prolonged periods of time and resulted in de novo myogenesis and vasculogenesis.
  • pathologic investigation revealed no teratoma, angiomatosis or bone formation in hBMSC transplanted hearts.
  • Figure 6A-D are explained in more detail as follows. Engraftment of Dil- labeled hBMSC and TBMC into infarcted myocardium. Numerous hBMSC(red fluorescence)(a) are engrafted into infarct and peri-infarct region of myocardium at 4 wks after transplantation. In contrast, considerably fewer TBMCs(red fluorescence) are seen, mostly within the infarct area(c). (b) and (d) are the Hoffman images of (a) and (c) showing the localization of engrafted cells, e-1. Immunophenotypic characterization of differentiated hBMSC into CMCs.
  • Example 6 Transplanted hBMSC Augmented Proliferation and Survival of Host Myocardium Since the profound physiologic effect of hBMSC transplantation may not solely be explained in some of the rats by the extent of multilineage differentiation in situ, we next sought to elucidate whether transplanted hBMSC affected proliferation and survival of host myocardial cells.
  • mini-osmotic pumps were implanted for delivering 5-Bromo-2'- deoxyuridine(BrdU) constantly for 4 wks to label all cells that entered the S phase of the cell cycle, hi sections from hBMSC transplanted hearts, numerous proliferating cells were observed both in host myocardial cells and transplanted cells(Fig. 7a-d). In contrast, considerably fewer BrdU-positive cells were observed in TBMC or PBS injected hearts(Fig. 7e,f).
  • a BrdU index the percentage of the BrdU positive nuclei to total number of nuclei counted in each section was significantly higher in hBMSC transplanted hearts than controls (hBMSC vs. TBMC, PBS; 25.8 + 5.2% vs. 11.2 ⁇ 3.1% , 9.5 + 2.8 %, P ⁇ 0.001).
  • Double IF histochemistry using mAb against BrdU and either ILB4 or cTnl revealed that CMCs and ECs were positive for BrdU(Fig. 7g-n).
  • the BrdU indices of ECs and CMCs were again higher in hBMSC transplanted hearts(vs. TBMC, PBS; ECs, 8.3 + 2.7 vs.
  • the apoptotic index the percentage of TUNEL(+) nuclei to total number of nuclei counted in each section, was 3 times lower in hBMSC transplanted hearts (apoptotic index vs. TBMC, PBS, 2.1 + 0.8 vs. 6.6 + 1.2, 7.3 ⁇ 1.1, P ⁇ 0.01)(Fig. 7o-q). These differences were particularly evident within the periinfarct area.
  • Figure 7A-U is explained more fully as follows, a-h. Identification of prohferative cells by BrdU imunohistochemistry. In sections from hBMSC transplanted heart (a-d), numerous BrdU positive cells(green dot) were observed (c) both in host myocardial cells and transplanted cells. In contrast, fewer BrdU positive cells were observed in TBMC(e) or PBS(f) injected rat hearts, g-j. Concomitant staining with antibody against BrdU(h) and ILB4(i) demonstrates the presence of BrdU positive cells(arrows) in capillary ECs of a hBMSC transplanted heart, k-o.
  • Concomitant staining with antibody against BrdU(l) and cTnI(m) shows the presence of BrdU positive cells(arrows) in mature CMCs(n) of a hBMSC transplanted heart, m- o.
  • a representative image obtained after staining with mAb against ⁇ -sarcomeric actinin(red) and TUNEL(green) demonstrates CMC apoptosis(arrows) from a PBS injected heart, t-u.
  • a representative image obtained after staining with ILB4(red) and TUNEL(green) demonstrates EC apoptosis(arrows) from a PBS injected heart.
  • Example 7 Paracrine effect of transplanted hBMSC: upregulation of angiogenic cytokines and cardiac transcription factors
  • angiogenic cytokines such as VEGF- A, angiopoietin-l(Ang-l) and 2(Ang-2), hepatocyte growth factor(HGF), basic fibroblast growth factor(bFGF), platelet-derived growth factor- B(PDGF-B), transforming growth factor(TGF)- ⁇ and cardiac transcription factors such as Nkx2.5, GATA-4, MEF2c by semi-quantitative RT-PCR using harvested cardiac samples at 14 and 28 days after hBMSC or PBS treatment was evaluated.
  • VEGF- A angiopoietin-l(Ang-l) and 2(Ang-2
  • HGF hepatocyte growth factor
  • bFGF basic fibroblast growth factor
  • PDGF-B platelet-derived growth factor- B
  • TGF transforming growth factor
  • transplanted hBMSC could enhance myogenesis by augmenting host CMC differentiation and/or directly differentiating into new CMCs.
  • upregulation of multiple factors can, in part, explain the prohferative effect of transplanted hBMSC on host ECs and CMCs.
  • Example 8 hBMSC Transplantation Increased Capillary and CMC density and Decreased Myocardial Fibrosis.
  • capillary and CMC density were quantified after CD31 and HE staining, respectively and the percent circumferential fibrosis area was measured.
  • Capillary density was 2 and 2.3 fold higher in the hBMSC transplanted rats than in the TBMC and PBS injected rats(P ⁇ 0.01)(Fig. 9 a-d).
  • CMC density was also 2.4 and 2.8 fold higher in the hBMSC transplanted rats than in the TBMC and PBS injected rats(P ⁇ 0.01)(Fig. 9d-g).
  • the cells were grown in complete DMEM with low(lg) glucose containing 17% of FBS (Biowhittaker; lot selected for promoting expansion of marrow cells), 100 U/ml penicillin, and 100 ⁇ g/ml streptomycin and 2mM glutamate at 37°C and 5% CO 2 for 4-6 days, the medium was replaced with fresh complete medium, and the adherent cells were grown to 60% confluency.
  • the cells were reseeded in complete medium into 25cm 2 tissue culture flask(T25) at a density of 1 x 10 4 cells/cm 2 . After the cells were 60% confluent, the cells were serially reseeded into T75 and T175 flasks at the same density.
  • Fluorescent-Activated Cell Sorting Fluorescent-activated Cell Sorting (FACS) analysis of hBMSC was performed on cultured hBMSC. Cells from at least 3 different clones of pre- and post-clonal isolation were used, and for clonal lines, two sets of cells at 5 PDs and 120 PDs were used. The procedure of FACS staining was described previously ⁇ Kalka, 2000 #11 ⁇ .
  • Quantitative FACS was performed on a FACStar flow cytometer(B-D).
  • Antibodies used for FACS analysis were FITC- or Phycoerythrin(PE)-conjugated antibodies against CD4, CD8, CDllb(Mac-l), CD13, CD14, CD15, CD29, CD30, CD31, CD34, CD44, CD71, CD73, , CD90(Thyl), CD117(c-kit), CD146, CD166, HLA-DR, HLA-ABC, ⁇ 2-microglobulin from Beckton Dickson(BD), CD133(AC133) from Miltenyl Biotech(Auburn, CA, USA), and CD105(Endoglin) from Ancell(Bayport, MN, USA), and unconjugated antibodies against Oct4 from Santa-Cruz.
  • DNA ploidy analysis DNA contents per cells were determined by pre-treating cells with Ribonuclease(100 ⁇ g/ml) and staining with propidium iodide(50 ⁇ g/ml) and subsequent FACS analysis.
  • telomere length assay We used TeloTAGGG Telomere Length Assay kit(Roche, Indianapolis, IN) for determination of telomere length of hBMSC at 5 PDs and 120 PDs from 3 different clones. Briefly, after isolation(l ⁇ g) and digestion of genomic DNA, DNA fragments were separated by gel electrophoresis and transferred to a Nylon membrane by Southern blotting. The blotted DNA fragments are hybridized to a digoxigenin (DIG)- labeled probe specific for telomeric restriction fragment(TRF) and incubated with a DIG-specific ab covalently coupled to alkaline phosphate.
  • DIG digoxigenin
  • telomere probe is visualized by virtue of alkaline phosphatase metabolizing CDP-Star, a highly sensitive chemiluminescence substrate.
  • the average TRF length was determined by comparing the signals relative to a molecular weight standard.
  • hBMSC 5 x 10 cells/cm in either 0.1 % gelatin or vitronectm coated glass chamber slides in DMEM or EBM-2(Clonetics) with 2-5% FBS, 10 "8 dexamethasone and 10 ng/ml VEGF(R&D, Minneapolis, MN) and cultured up to 14 days.
  • hBMSC were replated at 1 x 10 5 cells/cm 2 in non-coated or fibronectin coated culture dishes in 1-2% DMEM or EBM-2 supplemented with PDGF-BB(50 ng/ml, R&D) [Hellstrom, 1999 #756;Yamashita, 2000 #757] and cultured for 14 days.
  • hBMSC were plated at 4 x 10 4 /cm 2 on Poly-L-ornithin-laminin coated dishes(B-D) or plastic dishes with 100 ng/ml bFGF and 20 ng/ml EGF, B27 supplement in DMEM/F 12 [Palmer, 1999 #764;Mezey, 2000 #750;Brazelton, 2000 #749] and cultured up to 14 days.
  • hBMSC were plated at 3-5 xlO 4 cells/cm 2 on 1% matrigel(BD) in 2% FBS, supplemented with 10 "8 dexamethasone, 25 ng/ml HGF, 10 ng/ml FGF-4 and cultured for 7 days, and thereafter either 1 Omg/ml DMSO or 0.5mM sodium butyrate were added and cultured for 7 more days[Shen, 2000 #761;Hamazaki, 2001 #762;Oh, 2000 #763;Schwartz, 2002 #747].
  • hBMSC were co-culture d with freshly isolated NRCM.
  • NRCM from F344 rats were isolated as described [De Luca, 2000 #759] and plated at 2 x 10 5 cells/cm 2 .
  • Dil-labeled hBMSC were added to the NRCM culture plates at a 1:4 ratio in DMEM containing 10% fetal calf serum(FCS)[Condorelli, 2001 #758] and cultured up to 10 days.
  • Rat aortic endothelial cells were cultured in EBM-2 containing EGM- 2 MV Single Quots(Cambrex).
  • Rat vascular smooth muscle cells RVSMCs were cultured in DMEM with lg/L glucose(Cellgro) containing 15% FBS, 2mM L- glutamine and antibiotics.
  • NRCMs were isolated and cultured as described above. When the cells reached 50-60% confluency(within 3 days), cells were stained with 25 ⁇ M of Vybrant CFDA SE cell tracer kit(Molecular Probes, Engene, OR) according to the manufacturer's instruction. Cells labeled with CFDA SE is shown green under the fluorescent microscopy.
  • hBMSC red fluorescence
  • SMC markers we used abs against ⁇ -smooth muscle actin(mouse mAb)(l: 300), calponin(mouse mAb)(l:250, DAKO, Carpinteria, CA, USA).
  • neural lineage markers we used abs against NF-200(mouse mAb)(l:400, Sigma), ⁇ -tubulinIII(mouse mAb)(l:100, Sigma), Gal-C(rabbit pAb)(l:100, Sigma), GFAP(goat pAb)(l:200, Santa-Cruz).
  • CMC markers abs against cTn I(two forms: mouse mAb and rabbit pAb, l:100)(Chemicon, Temecular, CA), ventricular myosin heavy chain ⁇ ( ⁇ -MHC)(mouse mAb, l:100)(Chemicon), ⁇ -sarcomeric actinin (clone EA-53, mouse mAb, 1 :200)(Sigma), ANP(rabbit pAb)(Chemicon).
  • Control mouse, rabbit or goat IgG were from Sigma. Secondary anti-mouse, rabbit or goat antibodies(AMCA, 1:150, FITC or Cy-2, 1:200; Cy-3, 1:200) were from Jackson hnmunoresearch(West Grove, PA).
  • TBMCs The preparation of TBMCs was the same as that of hBMSC before culture.
  • hBMSC and TBMCs were labeled with carbocyanine dye, Dil(1.5 ⁇ g/ml, Molecular Probes) before the cell transplantation as described before[Kalka, 2000;Kawamoto, 2001].
  • carbocyanine dye Dil(1.5 ⁇ g/ml, Molecular Probes)
  • Separate rats(n 4, each group) were implanted underneath the back skin with miniosmotic pumps (model 2ML4, Alzet, Palo Alto, CA) that infused 1.25 mg BrdU/d (Sigma)(dissolved in a 1:1 (vol/vol) mixture of dimethyl sulfoxide and 0.154 M NaCl) after the surgery and sacrificed at 4 wks ⁇ McEwan, 1998 ⁇ .
  • Acute myocardial infarction model MI was induced as described 'previously in our lab [Kawamoto, 2003;Kawamoto, 2001]. Following left thoracotomy and incision of the pericardium under artificial ventilation(model 683, Harvard Apparatus), the left anterior descending (LAD) coronary artery was ligated near its origin with 6-0 prolene suture (Ethicon).
  • a WMSI is derived by dividing the sum of wall motion scores by the number of visualized segments; it represents the extent of regional wall motion abnormalities.
  • a normal WMSI is 1.
  • 1.4 French high fidelity pressure transducer (Micro-tip catheter, Millar Instrument, Houston, TX) was introduced into the left ventricle via right carotid artery.
  • LVSP, LVEDP, the +dP/dt and -dP/dt were recorded using polygraph(Model 7P)(Grass Instrument, West Warwick, RI, USA) [Kawamoto, 2001].
  • BrdU was detected with the sheep anti-BrdU antibody(l:50; Biodesign) followed by Streptavidin-FITC(1:100; Vector). Nuclear counterstaining was performed with DAPI. BrdU positive cells were counted in the peri-infarct area where the scar tissue comprises less than 20% of the visual field (x 200 magnification).
  • In situ labeling of fragmented DNA was performed with terminal deoxynucleotidyltransferase(TdT)-mediated dUTP-biotin nick end labeling(TUNEL) method with the use of an in situ cell detection kit(Roche) as described [Fujio, 2000].
  • RT-PCR analysis was performed as described previously ⁇ Yoon, 2003 ⁇ .
  • Total RNA was extracted from cultured cells or from hearts using RNaqueous kit (Ambion, Austin, Texas, USA) according to the manufacturer's instruction.
  • One microgram of total RNA were reverse transcribed using random hexamer and Moloney murine leukemia virus reverse transcriptase(Superscript II kit, Roche).
  • the RT product was subjected to PCR using Advantage cDNA polymerase mix(Clontech) or Taq polymerase(Roche).
  • Advantage cDNA polymerase mix (Clontech) or Taq polymerase(Roche).
  • quantitative RT-PCR quantification of mRNA expression of each gene was calculated based on the GAPDH expression.
  • PCR primers and annealing temperature used is described in Table 1.
  • FIG. 10 FACS analysis using multiple surface epitopes demonstrated none to minimal expression ( ⁇ 1%) of CD117 in hBMSC.
  • Hepatocyte growth factor is a potent angiogenic factor which stimulates endothelial cell motility and growth. J Cell Biol 119, 629-41 (1992).
  • TGF-betal Transforming growth factor betal

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Abstract

L'invention concerne une cellule souche de moelle osseuse isolée (BMSC) possédant des niveaux indétectables ou faibles de marqueurs cellulaires sélectionnés, dont ceux typiques des cellules endothéliales, neuronales et des muscles lisses. L'invention porte également sur des greffons et sur des produits pharmaceutiques comprenant lesdites cellules. Des méthodes de fabrication des BMSC sont également décrites. Le tout trouve une plage étendue d'applications, dont l'utilisation dans la prévention et le traitement de maladies cardiovasculaires.
EP04796882A 2003-10-28 2004-10-28 Nouvelles cellules souches multipotentes Withdrawn EP1685237A4 (fr)

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WO2007047963A2 (fr) * 2005-10-20 2007-04-26 Caritas St. Elizabeth Medical Center Of Boston, Inc. Utilisation de cellules souches derivees de la moelle osseuse dans le traitement de l'ischemie
CN101573441A (zh) * 2005-12-08 2009-11-04 路易斯维尔大学研究基金会有限公司 非常小的胚胎样（vsel）干细胞及分离和利用其的方法
US9155762B2 (en) 2005-12-08 2015-10-13 University Of Louisville Research Foundation, Inc. Uses and isolation of stem cells from bone marrow
AU2007243221A1 (en) * 2006-04-28 2007-11-08 Tulane University Health Sciences Center Methods for treating diabetes
US20100260725A1 (en) * 2007-09-24 2010-10-14 Mohapatra Shyam S Materials and Methods for Treating Allergic and Inflammatory Conditions
WO2011053690A1 (fr) * 2009-10-30 2011-05-05 The University Of North Carolina At Chapel Hill Cellules souches pluripotentes à partir de l'arbre biliaire extrahépatique et procédés pour leur isolement
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US11072777B2 (en) 2016-03-04 2021-07-27 University Of Louisville Research Foundation, Inc. Methods and compositions for ex vivo expansion of very small embryonic-like stem cells (VSELs)
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