WO2011073521A1 - Méthodes pour l'enrichissement de cellules progénitrices endothéliales adultes et leurs utilisations - Google Patents

Méthodes pour l'enrichissement de cellules progénitrices endothéliales adultes et leurs utilisations Download PDF

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WO2011073521A1
WO2011073521A1 PCT/FI2010/051036 FI2010051036W WO2011073521A1 WO 2011073521 A1 WO2011073521 A1 WO 2011073521A1 FI 2010051036 W FI2010051036 W FI 2010051036W WO 2011073521 A1 WO2011073521 A1 WO 2011073521A1
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cell
cells
adult
derived
endothelial progenitor
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Petri Salven
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Petri Salven
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/069—Vascular Endothelial cells
    • C12N5/0692—Stem cells; Progenitor cells; Precursor cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2884—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD44
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere

Definitions

  • the present invention relates to stem cells, in particular methods for identifying and enriching for adult-derived endothelial progenitor cells, the use of adult-derived endothelial progenitor cells in regenerative medicine, and targeting adult-derived endothelial progenitor cells to prevent angiogenesis.
  • vascular system The development of the vascular system is an essential event during the embryonic development of many animal species. During this process, local mesodermal precursors differentiate into vascular and endothelial cells (ECs) to form a primary vascular plexus, a process referred to as vasculogenesis (Timmermans et al, J. Cell. Mol. Med. (2009) 13(1): 87-102).
  • ECs vascular and endothelial cells
  • hESC adult-derived endothelial progenitor cell
  • hEPC adult-derived endothelial progenitor cell
  • the unique combination of cell-surface markers also includes CD44 and Sca-1.
  • the present invention relates to the discovery of enhanced methods for selectively isolating adult-derived endothelial progenitor cells.
  • Described herein are methods that comprise isolating and enriching adult-derived endothelial progenitor cells from adult-derived cell samples, where such adult-derived endothelial progenitor cells are identified by being positive for the cell-surface markers CD31
  • the methods described herein are also useful for the isolation of endothelial progenitor cells from solid tissue, such as the lung, and from vascular endothelium, such as lung vascular endothelium.
  • the methods can further comprise in vitro culturing following the selective isolation using, for example, a low-cell density adherent semi- so lid matrix colony assay.
  • an isolated cell population comprising adult vascular endothelial progenitor cells, where the endothelial progenitor cells are positive for the cell surface markers CD31 (PECAM-1), CD 105 (endoglin), CD117(c-kit), CD44 and Sca-1, and are negative for at least one cell- surface marker from the group comprising CD2, CD3, CD14, CD16, CD19, CD56, and CD235a.
  • a cell population can be a naturally isolated cell population or a genetically modified cell population.
  • a pharmaceutical composition comprising an isolated adult-derived vascular endothelial progenitor cell population, where the cells of the endothelial progenitor cell population are positive for the cell surface markers CD31 (PECAM- 1 ), CD 105 (endoglin), CD 117(c-kit), CD44 and Sca-1 , and are negative for at least one cell-surface marker from the group comprising CD2, CD3, CD 14, CD 16, CD 19, CD56, and CD235a.
  • CD31 PECAM- 1
  • CD 105 encodedoglin
  • CD 117(c-kit) CD44 and Sca-1
  • an isolated cell population comprising adult vascular endothelial progenitor cells, where the endothelial progenitor cells are positive for the cell surface markers CD31 (PECAM-1), CD 105 (endoglin), CD117(c-kit) and Sca-1, and are negative for at least one cell- surface marker from the group comprising CD2, CD3, CD14, CD16, CD19, CD56, and CD235a.
  • a cell population can be a naturally isolated cell population or a genetically modified cell population.
  • a pharmaceutical composition comprising an isolated adult-derived vascular endothelial progenitor cell population, where the cells of the endothelial progenitor cell population are positive for the cell surface markers CD31 (PECAM-1), CD 105 (endoglin), CD117(c-kit) and Sca-1, and are negative for at least one cell-surface marker from the group comprising CD2, CD3, CD14, CD16, CD19, CD56, and CD235a.
  • CD31 PECAM-1
  • CD 105 encodedoglin
  • CD117(c-kit) CD117(c-kit) and Sca-1
  • neoangiogenesis and restoration of tissue vascularization are required, such as in regenerative medicine following ischemic events and for wound healing, and for use in tissue engineering.
  • vascular endothelial progenitor cells comprising adult vascular endothelial progenitor cells, where the endothelial progenitor cells are positive for the cell surface markers CD31 (PECAM-1), CD 105 (endoglin), CD117(c-kit), CD44, and Sca-1, and are negative for at least one cell- surface marker from the group comprising CD2, CD3, CD14, CD16, CD19, CD56, and CD235a.
  • a cell population can be a naturally isolated cell population or a genetically modified cell population.
  • a pharmaceutical composition for use in inducing vascularization comprises isolated adult vascular endothelial progenitor cells, where the endothelial progenitor cells are positive for cell surface markers CD31 (PECAM-1), CD 105 (endoglin), CD117(c-kit), CD44 and Sca-1, and are negative for at least cell- surface marker from the group CD2, CD3, CD 14, CD 16, CD 19, CD56, and CD235a.
  • the isolated adult vascular endothelial progenitor cells can be autologous or allogenic cells. Also described herein are uses of such isolated adult vascular endothelial progenitor cells in inducing vascularization in tissues.
  • the ability to specifically identify endothelial stem cell-enriched cell population is useful in targeting and blocking angiogenesis, such as in tumors.
  • methods for blocking angiogenesis comprising targeting an angiogenesis inhibitor to a cell that is positive for CD31 (PECAM-1), CD 105 (endoglin), CDl 17(c-kit), CD44 and Sca-1, and that is negative for at least one of the cell- surface markers comprising CD2, CD3, CD14, CD16, CD19, CD56, and CD235a.
  • Such targeting can be performed using an antibody or a mixture of antibodies specific for CD31 (PECAM-1), CD105 (endoglin), CDl 17(c-kit), CD44 and Sca-1.
  • Figure 2 shows that blood vessel walls contains CD117 + EC
  • FIG. 1 shows a comparison of the distribution of CFCs within Lin " CD31 + CD105 + ECs that were divided into fractions enriched or depleted for Sca-1, CD117, or CD44 in a single FACS sort. The highest proportion of CFUs were found in the Lin " CD31 + CD105 + Scal + CD117 + CD44 +
  • FIG. 3A shows a flow diagram of the FACS sorting procedure used to obtain Lin " CD31 CD 105 Seal CDl 17 cells. Prior to transplantation, a single colony originating from a single GFP -tagged Lin ⁇ CD31 CD105 Scal + CDl 17 + colony forming cell was expanded for twelve days in adherent culture to amplify the cell number.
  • Figure 3B shows that functional, perfused GFP + blood vessels generated by the transplanted descendants of a single Lin ⁇ CD31 CD105 + Scal + CD117 + cell (D14 after transplantation). The mouse was perfused with fluorescent 0.2 ⁇ microspheres (red) to visualize functional blood vessels.
  • FIG. 3C shows the evaluation of self-renewal capacity, as defining characteristic of stem cells, by inoculating mice with B16 melanomas (2 million cells per mice) together with 15 CFUs of GFP-tagged isolated CD31 + CD105 + ECs. After two weeks of tumor growth, repeated isolations and serial transplantations of lineage depleted single cell suspensions containing the GFP + tagged ECs and the B16 cells. The figure shows GFP + blood vessels in the quaternary transplant.
  • the invention provides compositions of, and methods for isolating, adult-derived endothelial progenitor cells for use in a variety of therapeutic applications in regenerative medicine, where neoangiogenesis and restoration of tissue vascularization, i.e., vascular regeneration, are required, including, but not limited to, following ischemic events, wound healing, and for use in tissue engineering.
  • the invention provides methods for targeting and blocking tumor angiogenesis based on the ability to specifically identify adult-derived endothelial progenitor cell populations.
  • Described herein are methods for isolating and enriching for adult-derived endothelial progenitor cells (EPCs) from an adult-derived biological sample, such as a cell sample.
  • EPCs adult-derived endothelial progenitor cells
  • Such methods comprise selecting from an adult-derived cell sample those cells that are positive for cell- surface markers comprising CD31 (PECAM-1), CD 105 (endoglin) and CD117(c-kit), thereby enriching the adult-derived cell sample for EPCs.
  • the invention provides methods for isolating and enriching for adult-derived endothelial progenitor cells.
  • the methods comprise using the specific and novel combination of the cell-surface molecules, CD31 (PECAM-1), CD 105 (endoglin), and CD117 (c-kit), to enrich for adult-derived endothelial progenitor cells (EPCs), such as human adult-derived EPCs, from a variety of sources and biological samples, including differentiated tissues.
  • EPCs adult-derived endothelial progenitor cells
  • CD31 refers to the cell-surface adhesion molecule, also known as
  • PECAM-1 that is expressed in large amounts on endothelial cells at intercellular junctions, on T cell subsets, and to a lesser extent, on platelets and most leukocytes.
  • Expression of CD31 is required for the transendothelial migration of leukocytes through the intercellular spaces between vascular endothelial cells.
  • CD 105 also known as “endoglin,” refers to the type I membrane glycoprotein located on cell surfaces that is part of the TGF- ⁇ receptor complex.
  • the protein consists of a homodimer of 180 kDA with disulfide links. It has been found on endothelial cells, activated macrophages, fibroblasts, and smooth muscle cells.
  • CD 105 has been found to be part of the TGF- ⁇ receptor complex, it may be involved in the binding of TGF- ⁇ , TGF- ⁇ 3, activin-A, BMP-2, and BMP-7.
  • CDl 17 refers to the transmembrane cytokine receptor expressed on the surface of hematopoietic stem cells, as well as other cell types, also known as C-kit receptor.
  • c-Kit is a proto-oncogene that is a member of the receptor tyrosine kinase family and, more specifically, is closely related to the platelet derived growth factor receptor (PDGFR).
  • PDGFR platelet derived growth factor receptor
  • c-Kit is the normal cellular homolog of the HZ4-feline sarcoma virus transforming gene (v-Kit). c-Kit regulates a variety of biological responses including chemotaxis, cell proliferation, apoptosis, and adhesion.
  • c-Kit is also identical with the product of the W locus in mice and, as such, is integral to the development of mast cells and hematopoiesis.
  • the ligand for the c-Kit receptor (KL) has been identified and is encoded at the murine steel (SI) locus.
  • Kit is the human homolog of the proto-oncogene c- Kit. In various cancers, it has been found that mutations in Kit are integral for tumor growth and progression.
  • CDl 17 is often used to identify certain types of hematopoietic (blood) progenitors in the bone marrow.
  • CDl 17 is also used to identify the earliest thymocyte progenitors in the thymus, specifically early T lineage progenitors (ETP/DN1) and DN2 thymocytes, which express high levels of CDl 17. Additionally mast cells, melanocytes in the skin, and interstitial cells of Cajal in the digestive tract have been shown to express CDl 17. CDl 17 is also a marker for mouse prostate stem cells. CDl 17 is the receptor for the cytokine stem cell factor (SCF), also known as "steel factor” or "c-kit ligand". SCF exists in two forms, cell surface bound SCF and soluble (or free) SCF.
  • SCF cytokine stem cell factor
  • a method to isolate a substantially pure population of adult- derived endothelial progenitor cells where a combination of agents specific for the group of cell-surface markers comprising CD31 (PECAM-1), CD 105 (endoglin), and CDl 17 (c-kit) are contacted with a biological sample to isolate a substantially pure population of adult-derived EPCs from the biological sample.
  • the group of cell- surface markers consists essentially of CD31, CD 105, and CDl 17.
  • the group of cell-surface markers consists of CD31 , CD 105, and CDl 17.
  • the substantially pure population of EPCs is positive for the expression of CD31, CD105, and CDl 17.
  • the method further comprises the use of additional cell-surface markers, such as CD44, Seal (Ly6A/E), and lineage markers to isolate the substantially pure population of adult-derived EPCs from the biological sample.
  • additional cell-surface markers such as CD44, Seal (Ly6A/E), and lineage markers to isolate the substantially pure population of adult-derived EPCs from the biological sample.
  • CD44 refers to a cell-surface glycoprotein involved in cell-cell interactions, cell adhesion and migration.
  • CD44 is a receptor for hyaluronic acid and can also interact with other ligands, such as osteopontin, collagens, and matrix metalloproteinases (MMPs).
  • MMPs matrix metalloproteinases
  • a specialized sialofucosylated glycoform of CD44 called HCELL is found natively on human hematopoietic stem cells, and is a highly potent E- selectin and L-selectin ligand.
  • HCELL functions as a "bone homing receptor", directing migration of human hematopoietic stem cells and mesenchymal stem cells to bone marrow.
  • CD44 participates in a wide variety of cellular functions including lymphocyte activation, recirculation and homing, hematopoiesis, and tumor metastasis.
  • Sca-1 refers to anl8 kDa member of the Ly-6 family of
  • Sca-1 GPI-linked surface proteins found in mice, and is expressed by mouse hematopoietic stem cells, myeloid population and peripheral T and B cells. Sca-1 is expressed at high levels upon activation regardless of the Ly-6 haplotype. The Ly-6 family is involved in regulation and function of T cell activation. Sca-1 serves as a major phenotypic marker for mouse hematopoietic progenitor/stem cell subset.
  • Lineage cell-markers that are not expressed by endothelial progenitor cells contemplated for use in the invention include, but are not limited to, CD 13 and CD33 (expressed on myeloid cells); CD71 (expressed on erythroid cells); CD19 and B220 (expressed on B cells), CD61 (expressed on human megakaryocyte cells); Mac-1 (CDl lb/CD 18) (expressed on monocytes); Gr-1 (expressed on granulocytes); Terl 19 (expressed on erythroid cells); and I17Ra, CD2, CD3, CD4, CD5, CD8 (expressed on T cells); CD14, CD56, and CD235a.
  • the lineage markers used may be dependent on the species from which the adult-derived endothelial progenitor cells are being isolated, as determined by one of skill in the art.
  • the combination of lineage markers can comprise CD2, CD3, CD16, CD19, CD56, and CD235a.
  • the combination of lineage markers can comprise CD5, CD45R (B220), CDl lb, Gr-1 (Ly-6G/C), 7-4, and Ter-119.
  • the group of cell-surface markers comprises CD31, CD105, CD117, and CD44.
  • the group of cell- surface markers comprises CD31, CD 105, and CDl 17.
  • the group of cell-surface markers consists essentially of CD31, CD105, CDl 17, and CD44.
  • the group of cell-surface markers consists of CD31, CD105, CDl 17, and CD44.
  • the substantially pure population of adult-derived EPCs is positive for the expression of CD31, CD105, CDl 17, and CD44.
  • the group of cell-surface markers comprises CD31, CD 105, CDl 17, CD44, and one or more lineage markers.
  • the group of cell-surface markers consists essentially of CD31, CD 105, CDl 17, CD44, and one or more lineage markers.
  • the group of cell-surface markers consists of CD31, CD 105, CDl 17, CD44, and one or more lineage markers.
  • the substantially pure population of adult-derived EPCs is positive for the expression of CD31, CD105, CDl 17, and CD44, and negative for the expression of the one or more lineage markers.
  • the substantially pure population of adult-derived EPCs is positive for the expression of CD31 , CD 105and CD 117, and negative for the expression of the one or more lineage markers.
  • the one or more lineage markers comprise the group CD2, CD3, CD14, CD16, CD19, CD56, and CD235a.
  • the group of cell-surface markers comprises CD31, CD 105, CDl 17, CD44, Sca-1, and one or more lineage markers. In some embodiments of this aspect, the group of cell- surface markers comprises CD31, CD 105, CDl 17 and Sca-1, and one or more lineage markers. In some embodiments of this aspect, the group of cell-surface markers consists essentially of CD31, CD105, CDl 17, CD44,
  • the invention provides a method to isolate an enriched population of adult-derived endothelial progenitor cells, where a combination of agents specific for the group of cell- surface markers comprising CD31 (PECAM-1), CD 105 (endoglin), and CDl 17 (c-kit) are contacted with an adult-derived biological sample to isolate an enriched population of adult-derived EPCs from the biological sample.
  • the group of cell-surface markers consists essentially of CD31, CD 105, and CDl 17.
  • the group of cell- surface markers consists of CD31, CD 105, and CDl 17.
  • the enriched population of adult-derived EPCs is positive for the expression of CD31, CD105, and CDl 17.
  • the method further comprises the use of additional cell-surface markers, such as Seal (Ly6A/E), lineage markers, and CD44, to isolate the enriched population of adult-derived EPCs from the biological sample.
  • additional cell-surface markers such as Seal (Ly6A/E), lineage markers, and CD44
  • the group of cell- surface markers comprises CD31, CD105, CDl 17, and CD44. In some embodiments of this aspect, the group of cell-surface markers consists essentially of CD31, CD105, CDl 17, and CD44. In some embodiments of this aspect, the group of cell-surface markers consists of CD31 , CD105, CDl 17, and CD44. In some embodiments of this aspect and all such aspects described herein, the enriched population of adult-derived EPCs is positive for the expression of CD31, CD105, CDl 17, and CD44.
  • the group of cell- surface markers comprises CD31, CD 105, CDl 17, CD44, Sca-1, and one or more lineage markers In some embodiments of this aspect, the group of cell-surface markers consists essentially of CD31, CD105, CDl 17, CD44, Sca-1, and one or more lineage markers. In some embodiments of this aspect, the group of cell-surface markers consists of CD31, CD 105, CDl 17, CD44, Sca-1, and one or more lineage markers. In some embodiments of this aspect and all such aspects described herein, the enriched population of adult-derived EPCs is positive for the expression of CD31, CD 105, CDl 17, CD44, and Sca-1 and negative for the expression of the one or more lineage markers. In some embodiments, the one or more lineage markers comprise the group CD2, CD3, CD14, CD16, CD19, CD56, and CD235a.
  • an "adult-derived endothelial progenitor cell” refers to a population of adult immature progenitor cells that are capable of mediating post-natal vasculogenic activity, including the formation of new endothelial cells and blood vessels in vivo.
  • An adult-derived endothelial progenitor cell is a cell that can be clonally and serially replated in culture and will give rise to endothelium either by differentiation in vitro or direct incorporation into the vessel wall in vivo. Exemplary methods to test whether an isolated population is an EPC population are described herein.
  • isolated refers to a process whereby a cell or population of cells is removed from a subject or sample in which it was originally found, or a descendant of such a cell or cells.
  • isolated population refers to a population of cells that has been removed and separated from a biological sample, or a mixed or heterogeneous population of cells found in such a sample.
  • a mixed population includes, for example, a population of peripheral blood mononuclear cells obtained from isolated blood, or a cell suspension of a tissue sample.
  • an isolated population is a substantially pure population of cells as compared to the heterogeneous population from which the cells were isolated or enriched from.
  • the isolated population is an isolated population of endothelial progenitor cells.
  • the isolated population comprises a substantially pure population of endothelial progenitor cells as compared to a
  • substantially pure refers to a population of cells that is at least about 75%, at least about 80%, at least about 85%o, at least about 90%>, at least about 95%, at least about 98%>, or at least about 99% pure, with respect to the cells making up a total cell population.
  • the terms "substantially pure” or “essentially purified”, with regard to a population of endothelial progenitor cells isolated using the methods as disclosed herein, refers to a population of adult-derived EPCs that contain fewer than about 25%, fewer than about 20%, fewer than about 15%), fewer than about 10%>, fewer than about 9%, fewer than about 8%, fewer than about 7%), fewer than about 6%, fewer than about 5%, fewer than about 4%, fewer than about 4%, fewer than about 3%, fewer than about 2%, fewer than about 1%, or less than 1%, of cells that are not adult-derived EPCs as defined by the terms herein.
  • a marker may consist of any molecule found in a cell including, but not limited to, proteins (peptides and polypeptides), lipids, polysaccharides, nucleic acids and steroids.
  • morphological characteristics or traits include, but are not limited to, shape, size, and nuclear to cytoplasmic ratio.
  • functional characteristics or traits include, but are not limited to, the ability to adhere to particular substrates, ability to incorporate or exclude particular dyes, ability to migrate under particular conditions, and the ability to differentiate along particular lineages. Markers may be detected by any method available to one of skill in the art.
  • a "cell-surface marker” refers to any molecule that is expressed on the surface of a cell.
  • Cell-surface expression usually requires that a molecule possesses a transmembrane domain.
  • Some molecules that are normally not found on the cell-surface can be engineered by recombinant techniques to be expressed on the surface of a cell.
  • Many naturally occurring cell-surface markers are termed "CD” or “cluster of differentiation” molecules.
  • Cell-surface markers often provide antigenic determinants to which antibodies can bind to.
  • Cell-surface markers of particular relevance to the methods described herein include CD31, CD 105, CDl 17, CD44, and Sca-1.
  • a cell can be designated “positive” or “negative” for any of the cell-surface markers described herein, and both such designations are useful for the practice of the methods described herein.
  • a cell is considered “positive” for a cell-surface marker if it expresses the marker on its cell-surface in amounts sufficient to be detected using methods known to those of skill in the art, such as contacting a cell with an antibody that binds specifically to that marker, and subsequently performing flow cytometric analysis of such a contacted cell to determine whether the antibody is bound the cell. It is to be understood that while a cell may express messenger R A for a cell-surface marker, in order to be considered positive for the methods described herein, the cell must express it on its surface.
  • a cell is considered “negative” for a cell-surface marker if it doe not express the marker on its cell-surface in amounts sufficient to be detected using methods known to those of skill in the art, such as contacting a cell with an antibody that binds specifically to that marker and subsequently performing flow cytometric analysis of such a contacted cell to determine whether the antibody is bound the cell.
  • the agents can all comprise the same label or tag, such as fluorescent tag, and thus all cells positive for that label or tag can be excluded or removed in the methods to isolate endothelial progenitor cells described herein, so that the remaining adult-derived endothelial cells are "negative” for the one or more lineage markers used.
  • an "agent specific for a cell-surface marker” refers to an agent that can selectively react with or bind to that cell-surface marker, but has little or no detectable reactivity to another cell-surface marker or antigen.
  • an agent specific for CD117 will not be specific for CD 105.
  • agents specific for cell-surface markers recognize unique structural features of the markers.
  • an agent specific for a cell-surface marker binds to the cell-surface marker, but does not cause initiation of downstream signaling events mediated by that cell- surface marker, for example, a non-activating antibody.
  • Agents specific for cell-surface molecules include, but are not limited to, antibodies or antigen-binding fragments thereof, natural or recombinant ligands, small molecules; nucleic acid sequence and nucleic acid analogues; intrabodies; aptamers; and other proteins or peptides.
  • the preferred agents specific for cell-surface markers are antibody agents that specifically bind the cell-surface markers, and can include polyclonal and monoclonal antibodies, and antigen-binding derivatives or fragments thereof.
  • Well-known antigen binding fragments include, for example, single domain antibodies (dAbs; which consist essentially of single VL or VH antibody domains), Fv fragment, including single chain Fv fragment (scFv), Fab fragment, and F(ab')2 fragment. Methods for the construction of such antibody molecules are well known in the art.
  • Non-limiting examples of fluorescent labels or tags for labeling the antibodies for use in the methods of invention include Hydroxycoumarin, Succinimidyl ester, Aminocoumarin, Succinimidyl ester,
  • a common variation of flow cytometric techniques is to physically sort particles based on their properties, so as to purify populations of interest, using
  • fluorescence-activated cell sorting refers to a flow cytometric method for sorting a heterogeneous mixture of cells from a biological sample into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell and provides fast, objective and quantitative recording of fluorescent signals from individual cells as well as physical separation of cells of particular interest.
  • the cell suspension is entrained in the center of a narrow, rapidly flowing stream of liquid. The flow is arranged so that there is a large separation between cells relative to their diameter.
  • a vibrating mechanism causes the stream of cells to break into individual droplets. The system is adjusted so that there is a low probability of more than one cell per droplet.
  • the flow passes through a fluorescence measuring station where the fluorescent character of interest of each cell is measured.
  • An electrical charging ring is placed just at the point where the stream breaks into droplets.
  • a charge is placed on the ring based on the immediately-prior fluorescence intensity measurement, and the opposite charge is trapped on the droplet as it breaks from the stream.
  • the charged droplets then fall through an electrostatic deflection system that diverts droplets into containers based upon their charge. In some flow cytometric systems, the charge is applied directly to the stream, and the droplet breaking off retains charge of the same sign as the stream. The stream is then returned to neutral after the droplet breaks off.
  • fluorescence- activated cell sorting can be used in and with the methods, assays, and kits described herein to isolate and enrich for populations of adult-derived endothelial progenitor cells.
  • isolation and enrichment for populations of endothelial progenitor cells can be performed using bead based sorting mechanisms, such as magnetic beads.
  • the sample of cells to be isolated or enriched is contacted with magnetic beads coated with antibodies against one or more specific cell- surface antigens, such as CD31, CD 105, CD117, CD44, and Sca-1. This causes the cells expressing this antigen to attach to the magnetic beads.
  • the contacted cell solution is transferred to a strong magnetic field, such as a column or rack having a magnet.
  • beads can be coated with antibodies by a skilled artisan using standard techniques known in the art, such as commercial bead conjugation kits.
  • a negative selection step is performed to remove cells expressing one or more lineage markers, followed by fluorescence activated cell sorting to positively select adult-derived endothelial progenitor cells expressing CD31 , CD105, CD117, CD44, and Sca-1.
  • a cell sample is first contacted with labeled antibodies specific for cell- surface markers of interest, such as CD2, CD3, CD 14, CD 16, CD 19, CD56, and CD235a and the sample is then contacted with beads that are specific for the labels of the antibodies, and the cells expressing any of the markers CD2, CD3, CD14, CD16, CD19, CD56, and CD235a are removed using immunomagnetic lineage depletion.
  • populations of adult-derived EPCs isolated or enriched using a combination of agents specific for the cell- surface markers CD31, CD 105, CD117, CD44, and Sca-1 are used in colony- forming assays to expand and further characterize the adult-derived EPCs.
  • the isolated adult-derived EPCs isolated or enriched using a combination of agents specific for the cell- surface markers CD31, CD 105, CD117, CD44, and Sca-1 are used in colony- forming assays to expand and further characterize the adult-derived EPCs.
  • the isolated adult-derived EPCs isolated or enriched using a combination of agents specific for the cell- surface markers CD31, CD 105, CD117, CD44, and Sca-1 are used in colony- forming assays to expand and further characterize the adult-derived EPCs.
  • the isolated adult-derived EPCs isolated or enriched using a combination of agents specific for the cell- surface markers CD31, CD 105, CD117, CD44, and Sca-1 are used in colony
  • EPCs are expanded in culture using methods known to one of skill in the art prior to use in a subject in need, for example. Such expansion methods can comprise the use of low- density adherent semi- so lid matrix colony assays as described herein in the Example section. Isolated adult-derived EPCs can be cultured under primary culture conditions resulting in the outgrowth of EPC-derived discrete, adherent colonies by day 5 to day 14 termed herein as "colony- forming unit-ECs" (CFU-ECs). Conditions for such cultures can be found in the Example section and Current Protocols in Stem Cell Biology, 2008, 2C 1.1 - 2C1.27.
  • CFU-ECs display phenotypic and functional characteristics of adult endothelial cells, including expression of cell surface markers, CD31, CD 105, CD 144, CD 146, vWF, and KDR, uptake of AcLDL (acetylated low-density lipoprotein), upregulate VCAM-1, and form capillary- like tubes when plated on Matrigel (Lin et al., 2000; Gulati et al., 2003; Hur et al., 2004; Ingram et al., 2004; Yoder et al., 2007).
  • the individual endothelial cell colonies or CFU-ECs thus formed can be clonally isolated and serially subcultured or replated, using techniques known to the skilled artisan.
  • the use of a combination of agents specific for the cell- surface markers CD31, CD 105, CD 117, CD44, and Sca-1 for the isolation or enrichment of adult-derived EPCs results in an increased yield of CFU-ECs when compared to a method of isolation or enrichment of EPCs using a combination of agents specific only for the cell- surface markers CD31 and CD 105.
  • the increased yield is at least 5 fold greater.
  • CFU-ECs obtained using a combination of agents specific for the cell- surface markers CD31, CD 105, CD 117, CD44, and Sca-1 for the isolation or enrichment of adult-derived EPCs have an increased ability to form new colonies when serially subcultured, when compared to CFU-EPCs obtained using a method of isolation or enrichment of adult-derived EPCs using a combination of agents specific only for the cell-surface markers CD31 and CD 105.
  • the increased ability to form new colonies is at least 6 fold greater.
  • the terms “increased,” “increase,” or “enhance” are all used herein to generally mean an increase by a statically significant amount; for the avoidance of any doubt, the terms “increased,” “increase,” or “enhance” mean an increase, as compared to a reference level, of at least about 10%, of at least about 15%, of at least about 20%, of at least about 25%, of at least about 30%, of at least about 35%, of at least about 40%, of at least about 45%, of at least about 50%, of at least about 55%, of at least about 6o%, of at least about 65%>, of at least about 70%>, of at least about 75%>, of at least about 80%>, of at least about 85%>, of at least about 90%>, of at least about 95%>, or up to and including a 100%, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold, at least about a 6-fold,
  • the CFU-ECs generated using the EPCs isolated by the methods described herein can be tested for the expression of specific cell-surface markers including CD31, CD105, CD144, CD146, vWF, and KDR, and the lack of expression of the hematopoietic cell specific surface antigen CD45 or
  • the CFU-ECs generated using the EPCs isolated by the methods described herein are tested for the ability to uptake AcLDL (acetylated low- density lipoprotein).
  • AcLDL acetylated low- density lipoprotein
  • fluorescently labeled AcLDL is contacted with a CFU-EC population and the ability of the CFU-EC population to uptake the fluorescently labeled AcLDL assayed using a flow cytometer.
  • isolated adult-derived vascular endothelial progenitor cells that are positive for the cell-surface markers CD31, CD 105, CD117, CD44, and Sca-1, and negative for one or more lineage cell-surface markers, such as CD2, CD3, CD14, CD16, CD19, CD56, and CD235a.
  • Such isolated adult-derived vascular endothelial progenitor cells can be genetically modified to express one or more therapeutic agents, for example, an anti-inflammatory agents.
  • compositions comprising isolated adult-derived vascular endothelial progenitor cells that are positive for the cell-surface markers CD31, CD 105, CD117, CD44, and Sca-1, and negative for one or more lineage cell-surface markers.
  • isolated adult-derived vascular endothelial progenitor cells and pharmaceutical compositions comprising such cells can be used, for example, in subjects in need of vascularization or vascular regeneration.
  • isolated adult-derived vascular endothelial progenitor cells that are positive for the cell-surface markers CD31 , CD 105, CD117, CD44, and Sca-1, and negative for one or more lineage cell-surface markers, such as CD2, CD3, CD14, CD16, CD19, CD56, and CD235a in promoting vascular regeneration or vascularization in a subject or tissue in need thereof.
  • the isolated adult-derived vascular endothelial progenitor cells are autologous to the subject in need, while in other embodiments, the cells are allogeneic to the subject in need. [0068]
  • methods for treating a subject in need of vascular regeneration are provided herein.
  • a method of promoting vascular regeneration in a subject in need thereof comprising administering to a subject an effective amount of adult-derived endothelial progenitor cells isolated from a biological sample that are positive for the group of cell- surface markers comprising CD31, CD 105, and CDl 17, to promote vascular regeneration in the subject.
  • a method of promoting vascularization in a tissue comprising administering to a tissue in need of vascularization an effective amount of adult-derived endothelial progenitor cells isolated from a biological sample that are positive for the group of cell-surface markers comprising CD31, CD105, and CDl 17, to promote vascular regeneration in the subject.
  • the method further comprises the use of additional cell-surface markers, such as CD44, Seal (Ly6A/E), and lineage markers, to isolate the adult-derived EPCs from the biological sample for use in the subject or tissue in need.
  • additional cell-surface markers such as CD44, Seal (Ly6A/E), and lineage markers
  • the group of cell-surface markers consists essentially of CD31, CD105, CDl 17, CD44, and one or more lineage markers. In some embodiments of these aspects, the group of cell- surface markers consists of CD31, CD 105, CDl 17, CD44, and one or more lineage markers. In some embodiments of these aspects and all such aspects described herein, the EPCs are positive for the expression of CD31, CD105, CDl 17, and CD44, and negative for the expression of the one or more lineage markers, such as CD2, CD3, CD14, CD16, CD19, CD56, and CD235a.
  • the group of cell-surface markers comprises CD31, CD 105, CDl 17, CD44, Sca-1, and one or more lineage markers. In some embodiments of these aspects, the group of cell-surface markers consists essentially of CD31, CD 105, CDl 17, CD44, Sca-1, and one or more lineage markers. In some embodiments of these aspects, the group of cell- surface markers consists of CD31, CD 105, CDl 17, CD44, Sca-1, and one or more lineage markers.
  • the EPCs are positive for the expression of CD31, CD 105, CDl 17, CD44, and Sca-1, and negative for the expression of the one or more lineage markers, such as CD2, CD3, CD14, CD16, CD19, CD56, and CD235a.
  • the method further comprises expanding the isolated adult-derived endothelial progenitor cells in vitro in order to increase or expand the number of endothelial cell progenitors for use in the methods described herein.
  • the isolated endothelial progenitor cells can be expanded to proliferate to form CFU-ECs, using the methods described herein or known to one of skill in the art.
  • Such expansion can comprise, for example, the use of a low-cell density adherent semi- so lid matrix colony assay, as described in the Examples section herein.
  • the method comprises administering an effective amount of the expanded population of endothelial progenitor cells to the subject in need thereof.
  • the expression of any of the cell-surface markers described herein on the CFU-ECs can be determined prior to the administration to the subject in need.
  • the populations of substantially pure and enriched endothelial progenitor cells that can be obtained using the methods described herein can be used to promote vascular regeneration for the treatment of a variety of conditions.
  • vascular regeneration refers to the formation of new blood vessels or the replacement of damaged blood vessels (e.g., capillaries) after injuries or traumas, as described herein, including but not limited to, cardiac disease, or ischemia.
  • Angiogenesis is a term that can be used interchangeably to describe such phenomena.
  • the subject in need of vascular regeneration has had or is having ischemia or ischemic damage.
  • ischemia is a restriction in blood supply, with resultant damage or dysfunction of tissue. Ischemia is also described herein as an inadequate flow of blood to a part of the body, caused by constriction or blockage of the blood vessels supplying it.
  • Ischemia is a feature of heart diseases, transient ischemic attacks, cerebrovascular accidents, ruptured arteriovenous malformations, and peripheral artery occlusive disease.
  • the heart, the kidneys, and the brain are among the organs that are the most sensitive to ischemic damage.
  • Ischemia in brain tissue for example due to stroke or head injury, causes a process called the ischemic cascade to be unleashed, in which proteolytic enzymes, reactive oxygen species, and other harmful chemicals damage and may ultimately kill brain tissue.
  • heart disease includes cardiomyopathy, hypertrophic cardiomyopathy, dilated cardiomyopathy, atherosclerosis, coronary artery disease, ischemic heart disease, myocarditis, viral infection, wounds, hypertensive heart disease, valvular disease, congenital heart disease, myocardial infarction, congestive heart failure, arrhythmias, etc.
  • the methods described herein are particularly useful in treating diseases of the heart involving damage to cardiac tissue due to restricted blood flow, i.e., cardiac ischemia.
  • Non-limiting examples of adverse effects of myocardial ischemia and myocardial ischemic damage include: myocyte damage (e.g., myocyte cell loss, myocyte hypertrophy, myocyte cellular hyperplasia), angina (e.g., stable angina, variant angina, unstable angina, sudden cardiac death), myocardial infarction, and congestive heart failure. Damage due to myocardial ischemia may be acute or chronic, and consequences may include scar formation, cardiac remodeling, cardiac hypertrophy, wall thinning, dilatation, and associated functional changes.
  • a subject in need of vascular regeneration has a wound.
  • Endothelial progenitor cells are useful in wound healing as they can increase vascularization, increase cell migration to the site of injury, decrease the amount of scarring, and increase tissue regeneration.
  • a wound refers to a lesion caused by an injury or damage, usually restricted to those with disruption of the normal continuity of structures, such as the epidermal or dermal layers of the skin, continuity of the vasculature, or any damage to a layer of a tissue, such as a corneal epithelial layer.
  • a wound can be an acute wound or a chronic wound.
  • acute wounds are those wounds that heal promptly, within 30 days in a normal subject.
  • Non-limiting examples of acute wounds that can be treated with the endothelial progenitor cell isolated using the methods described herein include abrasions, avulsions, contusions, crush wounds, cuts, lacerations, projectile wounds and puncture wounds.
  • Chronic wounds as defined herein, are those wounds that take longer than 30 days to heal, and include, but are not limited to, diabetic skin sores, pressure sores, surgical wounds, spinal injury wounds, burns, chemical-induced wounds and wounds due to blood vessel disorders.
  • a "skin wound” is defined herein as a break in the continuity of skin tissue that is caused by direct injury to the skin.
  • Several classes including punctures, incisions, excisions, lacerations, abrasions, atrophic skin, or necrotic wounds and burns generally characterize skin wounds. The methods described herein are useful for enhancing the healing of all wounds of the skin, including wounds in diabetics, normal patients and surgical patients.
  • a "tissue wound” as used herein is a wound to an internal organ, such as a blood vessel, intestine, colon, etc.
  • the endothelial progenitor cell isolated using the methods described herein are useful for enhancing the wound healing process in tissue wounds, whether they arise naturally or as the result of surgery. For instance, during the repair of arteries, the vessels need to be sealed and wound healing must be promoted as quickly as possible.
  • the substantially pure and enriched endothelial progenitor cells isolated using the methods described herein can accelerate such processes.
  • the endothelial progenitor cells used in the methods described herein can be obtained from any biological source or sample.
  • the cells are typically isolated from a sample from the subject in need of treatment so that there are no rejection issues (i.e., autologous transplantation).
  • the cells may be isolated from the peripheral blood of the subject, from the bone marrow, from a tissue sample, etc.
  • the cells may be isolated from a biological source or sample from a relative, an MHC-matched donor, a donor of the same blood type, or any donor of the same species.
  • cross-species cells are used (i.e., xenogeneic transplantation).
  • the methods for promoting vascular regeneration through the administration of endothelial progenitor cells can further comprise the administration of one or more therapeutic agents.
  • the therapeutic agent may be a protein, a peptide, a polynucleotide, an aptamer, a virus, a small molecule, a chemical compound, a cell, etc.
  • combinations of the above pro-angiogenic factors are used.
  • Derivatives or modified versions of these pro-angiogenic factors are also useful in the invention. These modified versions are typically 75%, 80%, 90%, 95%, 98%, 99%, or 100% identical to the wild protein or peptide. In certain embodiments, these modified versions show at least 50%>, 75%>, 80%>, or 90%> overall identity and share recognized or conserved sequence elements. Modified versions, fusions, or derivatives also include forms in which at least conserved or characteristic sequence elements have been placed in non- natural environments. In certain embodiments, the modified versions or derivatives have enough of the sequence of a pro-angiogenic factor to have the substantially the same activity as the naturally occurring factor. Any other pro-angiogenic factors known or discovered in the future may be used in the methods described herein.
  • treatment includes not just the improvement of symptoms or markers, but also a cessation of at least slowing of progress or worsening of symptoms that would be expected in absence of treatment.
  • Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • a reduction in a biochemical marker of cardiac dysfunction of myocardial infarction for example a reduction in, as an illustrative example only, at least one of the following biomarkers as disclosed in U.S Patent Application 2005/0250156, which is incorporated herein by reference, include for example, protein biomarkers in the blood such as; troponin I and T (Tnl/TnT), creatine kinase-MB isoform (CK0MB), myoglobin (MYO), hsCRP, H-FABP, MPO, BNP, p-selectin, sCD40L, GPIIb/IIIa, PTF 1.2, DD, TAT, BTG, PF4, PECAM-1, TPP, IL-6, IL-18, PIGF, PaPP-A, glutathione peroxidase, plasma thioredoxin, cyctatin C, and serum deoxyribonuclease I and ATP/
  • the efficacy of the methods described herein can be judged using an experimental animal model of cardiac dysfunction, e.g., mice or rats, or for example, induction of myocardial infarction in animal models, or an animal model which has been genetically modified to develop cardiac abnormalities.
  • An effective amount can be assessed in an animal models of ischemia/reperfusion injury when adult endothelial progenitor cells are administered just before reperfusion, such as disclosed in Smits et al, J Pharmacol Exp Ther 1998;286:611-618 ; McVey et al., ,J Cardiovasc Pharmacol
  • the endothelial progenitor cells are administered to the site of cardiac tissue damaged following a heart attack.
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrasternal injection and infusion.
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous
  • systemic administration means the administration of pharmaceutical compositions other material other than directly into the diseased tissue, such as cardiac tissue, such that it enters the subjects system and, thus, is subject to metabolism and other like processes.
  • administration of the adult endothelial progenitor cells of the invention can include formulation into
  • phrases "pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in maintaining the activity of or carrying or transporting the subject adult human endothelial progenitor cells from one organ, or portion of the body, to another organ, or portion of the body.
  • each carrier must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation.
  • Described herein are methods for targeting adult-derived endothelial progenitor cells for the inhibition or amelioration of angiogenesis-dependent diseases or disorders.
  • angiogenic vasculature such as those found in tumors, the numbers and percentages of adult-derived endothelial progenitor cells expressing the cell-surface markers CD31, CD 105, CD 117, CD44, and Sca-1 are greatly increased, relative to quiescent vasculature.
  • the methods described herein comprise targeting an adult-derived endothelial progenitor cell using one or more antibodies, or a mixture of antibodies, specific for the cell-surface markers CD31, CD 105, CDl 17, CD44, and Sca-1, to block angiogenesis in a subject or tissue in need.
  • the antibody or antibodies can further comprise a tag, such as a toxin, that can be used to lyse or destroy the cell expressing the combination of cell-surface markers.
  • angiogenesis refers to the sprouting of new blood vessels from pre-existing blood vessels or through the proliferation of adult-derived endothelial progenitor cells, as described herein, characterized by endothelial cell proliferation and migration triggered by pro-angiogenic factors.
  • Angiogenesis can be a good and necessary process, for example, in wound healing, or it can be an aberrant and undesired process with detrimental consequences, such as the growth of solid tumors and metastasis, and hemangiomas. Aberrant angiogenesis can lead to certain pathological conditions such as death, blindness, and disfigurement.
  • angiogenesis-dependent disease or disorder refers to diseases or disorders that are dependent on a rich blood supply and blood vessel proliferation for the diseases' pathological progression (e.g. metastatic tumors) or diseases or disorders that are the direct result of aberrant blood vessel proliferation (e.g. diabetic retinopathy and hemangiomas).
  • Examples include abnormal vascular proliferation, ascites formation, psoriasis, age-related macular degeneration, thyroid hyperplasia, preeclampsia, rheumatoid arthritis and osteoarthritis, Alzheimer's disease, obesity, pleural effusion, atherosclerosis, endometriosis, diabetic/other retinopathies, ocular neovascularizations such as neovascular glaucoma and corneal neovascularization.
  • angiogenesis inhibitors, drugs, or treatments are available for use in the methods described herein, and are known to one of skill in the art.
  • the angiogenesis inhibitors for use in the methods described herein include but are not limited to monoclonal antibody therapies directed against specific pro-angiogenic growth factors and/or their receptors. Examples of these are: bevacizumab (Avastin®), cetuximab (Erbitux®), panitumumab (VectibixTM), and trastuzumab (Herceptin®).
  • the angiogenesis inhibitors for use in the methods described herein include but are not limited to small molecule tyrosine kinase inhibitors (TKIs) of multiple pro-angiogenic growth factor receptors.
  • TKIs small molecule tyrosine kinase inhibitors
  • the three TKIs that are currently approved as anti-cancer therapies are erlotinib (Tarceva®), sorafenib
  • VEGF/VPF protein As the VEGF/VPF protein interacts with the VEGFRs, inhibition of either the ligand VEGF, e.g. by reducing the amount that is available to interact with the receptor; or inhibition of the receptor's intrinsic tyrosine kinase activity, blocks the function of this pathway. This pathway controls endothelial cell growth, as well as permeability, and these functions are mediated through the VEGFRs.
  • VEGF inhibitors include for example, AVASTIN® (bevacizumab), an anti-VEGF monoclonal antibody of Genentech, Inc. of South San Francisco, CA, VEGF Trap (Regeneron / Aventis).
  • Additional VEGF inhibitors include CP-547,632 (3-(4-Bromo-2,6-difiuoro- benzyloxy)-5-[3-(4-pyrrolidin 1-yl- butyl)-ureido]-isothiazole-4- carboxylic acid amide hydrochloride; Pfizer Inc. , NY), AG13736, AG28262 (Pfizer Inc.), SU5416, SU11248, & SU6668 (formerly Sugen Inc., now Pfizer, New York, New York), ZD-6474
  • vatalanib also known as PTK-787, ZK-222584: Novartis & Schering: AG
  • MACUGEN® pegaptanib octasodium, NX-1838, EYE-001, Pfizer
  • IM862 glufanide disodium, Cytran Inc. of Kirkland, Washington, USA
  • VEGFR2-selective monoclonal antibody DC 101 ImClone Systems, Inc.
  • angiozyme a synthetic ribozyme from Ribozyme (Boulder, Colorado) and Chiron
  • VEGF inhibitors are also disclosed in US Patent No. 6,534,524 and
  • VEGF inhibitors are described in, for example in WO 99/24440 (published May 20, 1999), International Application PCT/IB99/00797 (filed May 3, 1999), in WO 95/21613 (published August 17, 1995), WO 99/61422 (published December 2, 1999), U.S. Pat. Publ. No. 20060094032 "siRNA agents targeting VEGF", U.S. Patent 6, 534,524 (discloses AG13736), U.S. Patent 5,834,504 (issued November 10, 1998), WO 98/50356 (published November 12, 1998), U.S. Patent 5, 883,113 (issued March 16, 1999), U.S.
  • Patent 5, 886,020 (issued March 23, 1999), U.S. Patent 5,792,783 (issued August 11, 1998), U.S. Patent No. US 6,653,308 (issued November 25, 2003), WO 99/10349 (published March 4, 1999), WO 97/32856 (published September 12, 1997), WO 97/22596 (published June 26, 1997), WO 98/54093 (published December 3, 1998), WO 98/02438 (published January 22, 1998), WO 99/16755 (published April 8, 1999), and WO 98/02437 (published January 22, 1998), WO 01/02369 (published January 11, 2001); U.S. Provisional Application No. 60/491,771 piled July 31, 2003); U.S. Provisional Application No. 60/460,695 (filed April 3, 2003); and WO
  • the angiogenesis inhibitors for use in the methods described herein include anti-angiogenic factors such as alpha-2 antiplasmin (fragment), angiostatin (plasminogen fragment), antiangiogenic antithrombin III, cartilage-derived inhibitor (CDI), CD59 complement fragment, endostatin (collagen XVIII fragment), fibronectin fragment, gro-beta ( a C-X-C chemokine), heparinases heparin hexasaccharide fragment, human chorionic gonadotropin (hCG), interferon alpha/beta/gamma, interferon inducible protein (IP- 10), interleukin-12, kringle 5 (plasminogen fragment), beta- thrombo globulin, EGF (fragment), VEGF inhibitor, endostatin, fibronection (45 kD fragment), high molecular weight kininogen (domain 5), NK1, NK2, NK3 fragments of HGF,
  • anti-angiogenic factors such
  • thalidomide TNP470, the bisphosphonate family such as amino-bisphosphonate zoledronic acid, bombesin/gastrin-releasing peptide (GRP) antagonists such as RC-3095 and RC-3940-II (Bajol AM, et. al, British Journal of Cancer (2004) 90, 245-252), anti- VEGF peptide RRKRRR (dRK6) (Seung-Ah Yoo, J.Immuno, 2005, 174: 5846-5855).
  • GFP bombesin/gastrin-releasing peptide
  • the methods described herein include the use of more than one anti-angiogenic factor or angiogenesis inhibitor.
  • the therapy can also be administered in conjunction with other anti-cancer treatment such as biological, chemotherapy and radiotherapy.
  • Biological therapies use the body's immune system, either directly or indirectly, to fight cancer or to lessen the side effects that may be caused by some cancer treatments.
  • Immune response modifying therapies such as the administration of interferons, interleukins, colony-stimulating factors, monoclonal antibodies, vaccines, gene therapy, and nonspecific immunomodulating agents are also envisioned as anti-cancer therapies.
  • the methods described herein are directed at angiogenic diseases and disorders.
  • angiogenesis occurs at inappropriate locations, is aberrant, and/or uncontrolled and results in undesirable effects, then that angiogenesis is considered pathological.
  • the pathological angiogenic diseases and disorders include but are not limited to cancer, ascites formation, psoriasis, age-related macular degeneration, thyroid hyperplasia, preeclampsia, rheumatoid arthritis and osteoarthritis, Alzheimer's disease, obesity, pleura effusion, atherosclerosis, endometriosis, diabetic/other
  • the pathological angiogenic disease or disorder is cancer, where the rapidly dividing neoplastic cancer cells require an efficient blood supply to sustain their continual growth of the tumor.
  • cancer refers to any of various malignant neoplasms characterized by the proliferation of anaplastic cells that tend to invade surrounding tissue and metastasize to new body sites and also refers to the pathological condition characterized by such malignant neoplastic growths.
  • the blood vessels provide conduits to metastasize and spread elsewhere in the body.
  • the cancer cells Upon arrival at the metastatic site, the cancer cells then work on establishing a new blood supply network. Inhibition of angiogenesis at the primary tumor site and secondary tumor site serve to prevent and limit the progression of the disease.
  • carcinomas and sarcomas such as those found in the anus, bladder, bile duct, bone, brain, breast, cervix, colon/rectum,
  • endometrium esophagus, eye, gallbladder, head and neck, liver, kidney, larynx, lung, mediastinum (chest), mouth, ovaries, pancreas, penis, prostate, skin, small intestine, stomach, spinal marrow, tailbone, testicles, thyroid and uterus.
  • carcinomas include papilloma/carcinoma, choriocarcinoma, endodermal sinus tumor, teratoma, adenoma/adenocarcinoma, melanoma, fibroma, lipoma, leiomyoma, rhabdomyoma, mesothelioma, angioma, osteoma, chondroma, glioma, lymphoma/leukemia, squamous cell carcinoma, small cell carcinoma, large cell undifferentiated carcinomas, basal cell carcinoma and sino nasal undifferentiated carcinoma.
  • sarcomas include soft tissue sarcoma such as alveolar soft part sarcoma, angiosarcoma, dermato fibrosarcoma, desmoid tumor, desmoplastic small round cell tumor, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, hemangiopericytoma, hemangiosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphosarcoma, malignant fibrous histiocytoma, neurofibrosarcoma, rhabdomyosarcoma, synovial sarcoma, and Askin's tumor, Ewing's sarcoma (primitive neuroectodermal tumor), malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, and chondrosarcoma. Abnormal build up and growth
  • RA rheumatoid arthritis
  • RA rheumatoid arthritis
  • synovial tissue swelling characterized by synovial tissue swelling, leukocyte ingress and angiogenesis, or new blood vessel growth.
  • RA rheumatoid arthritis
  • Angiogenesis is now recognized as a key event in the formation and maintenance of the pannus in RA (Paleolog, E. M., Arthritis Res. 2002;4 Suppl 3:S81-90; Afuwape AO, Histol Histopathol. 2002;17(3):961-72).
  • RA rheumatoid arthritis
  • VEGF vascular permeability factor
  • RRKRRR anti-VEGF hexapeptide RRKRRR (dRK6) can suppress and mitigate the arthritis severity (Seung-Ah Yoo, et. al.,2005, supra). Accordingly, encompassed in the methods disclosed herein are subjects treated for rheumatoid arthritis. [0108] In some embodiments, the methods described herein are used in blocking pathological angiogenesis that occurs in Alzheimer's disease. Alzheimer's disease (AD) is the most common cause of dementia worldwide.
  • AD Alzheimer's disease
  • statins statins
  • histamine H2 -receptor blockers or calcium- channel blockers
  • AD angiogenesis in the brain vasculature can play an important role in AD.
  • the brain endothelium secretes the precursor substrate for the beta-amyloid plaque and a neurotoxic peptide that selectively kills cortical neurons.
  • amyloid deposition in the vasculature leads to endothelial cell apoptosis and endothelial cell activation which leads to neovascularization.
  • Vessel formation could be blocked by the VEGF antagonist SU 4312 as well as by statins, indicating that anti- angiogenesis strategies can interfere with endothelial cell activation in AD (Schultheiss C, el. al, 2006; Grammas P., et. al., 1999) and can be used for preventing and/or treating AD. Accordingly, encompassed in the methods disclosed herein are subjects treated for
  • the methods described herein are used in blocking pathological angiogenesis that occurs in endometriosis. Excessive endometrial
  • Differentiation is a developmental process whereby cells assume a specialized phenotype, e.g., acquire one or more characteristics or functions distinct from other cell types.
  • the differentiated phenotype refers to a cell phenotype that is at the mature endpoint in some developmental pathway (a so called terminally differentiated cell).
  • terminally differentiated cell In many, but not all tissues, the process of differentiation is coupled with exit from the cell cycle. In these cases, the terminally differentiated cells lose or greatly restrict their capacity to proliferate.
  • the terms “differentiation” or “differentiated” refer to cells that are more specialized in their fate or function than at a previous point in their development, and includes both cells that are terminally differentiated and cells that, although not terminally differentiated, are more specialized than at a previous point in their development.
  • the development of a cell from an uncommitted cell (for example, a stem cell), to a cell with an increasing degree of commitment to a particular differentiated cell type, and finally to a terminally differentiated cell is known as progressive differentiation or progressive commitment.
  • a cell that is "differentiated” relative to a progenitor cell has one or more phenotypic differences relative to that progenitor cell.
  • pathology refers to symptoms, for example, structural and functional changes in a cell, tissue, or organs, which contribute to a disease or disorder.
  • the pathology may be associated with a particular nucleic acid sequence, or "pathological nucleic acid” which refers to a nucleic acid sequence that contributes, wholly or in part to the pathology, as an example, the pathological nucleic acid may be a nucleic acid sequence encoding a gene with a particular pathology causing or pathology-associated mutation or polymorphism.
  • the pathology may be associated with the expression of a pathological protein or pathological polypeptide that contributes, wholly or in part to the pathology associated with a particular disease or disorder.
  • CD 117 " expressing ECs from the blood vessel wall endothelium comprise a subpopulation of EC progenitors with a very high proliferative potential that are able to produce discrete EC colonies in low-cell density adherent cultures.
  • CD31 CD105 Scal + CDl 17 + can produce several hundreds of thousands of endothelial daughter cells and generate a functional blood vessel after transplantation in vivo.
  • CD117 + and CD44 + ECs constituted the majority of all ECs, with long segments of vasculature being composed of CD117 + CD44 + ECs.
  • Sections (5-10 ⁇ ) were cut and antigen retrieval was performed using 2M HC1 for 15min at room temperature followed by neutralization in 0.1 M sodium borate for lOmin. Rinsing 3 times with PBS for 5min was followed by blocking with 5% goat serum for 30 min at room temperature. Subsequently, the sections were incubated with FITC-labeled rat anti-BrdU mAb to BrdU (Abeam) at 1 :50) and rabbit polyclonal anti-vWF at 1 :700 overnight at 4°C. Negative controls were performed by omitting the primary antibodies or using irrelevant controls of the same isotype.
  • Mouse lung endothelial cells were isolated from lungs dissected from adult wild type C57BL/6 or GFP-tagged transgenic C57BL/6-Tg(ACTB-EGFP)10sb/J mice. Mice were anesthetized with Rompun vet (Bayer) and Ketaminol vet (intervet). The chest was opened through a midline sternotomy. The left ventricle was identified and the ventricular cavity was entered through the apex with a 27-gauge needle. The right ventricle was identified and an incision was made in the free wall to exsanguinate the animal and to allow the excess perfusate to exit the vascular space.
  • Scoring of colonies was performed with an inverted microscope. Colonies containing 15 or more cells on day 7 were counted. In preliminary experiments the isolated cell populations were plated at various plating densities, and an ideal plating density for each subpopulation was determined.
  • the B 16-F 1 melanoma cell line was maintained in DMEM supplemented with 2 mM L-glutamine, Pen/Strep (lOOU/lOOg/ml), and 10%> fetal bovine serum (PromoCell).
  • the mice were injected in the ear with B16 cells (2> ⁇ 10 6 cells in 30 ⁇ ).
  • B16 cells (2> ⁇ 10 6 cells in 30 ⁇ ).
  • GFP+ isolated mouse lung endothelial cells were mixed with B6 cells prior to injection. The tumors were allowed to grow for 10-20 days, the mice were killed, and the tissues were processed for analyses.
  • Matrigel plugs 400 ⁇ 1 per injection Basement Membrane Matrix; BD PharMingen
  • recombinant murine VEGF164 100 ng/ml; R&D Systems
  • the plugs were excised and processed for tissue analyses at 1-2 weeks after injection.
  • Fluorescent carboxy late-modified microspheres, 0.2 um, red fluorescent (580/605) (FluoSpheres, Molecular Probes) were used diluted 1 :6 with PBS. Mice were anesthetized with Rompun vet (Bayer) and Ketaminol vet (Iintervet).
  • Tridil nitroglycerin, Orion Oyj
  • the chest was opened through a midline sternotomy.
  • the left ventricle was identified and the ventricular cavity was entered through the apex with a 27- gauge needle.
  • the right ventricle was identified and an incision was made in the free wall to exsanguinate the animal and to allow the excess perfusate to exit the vascular space.
  • the animal was perfused with approximately 2-6 ml of PBS at approximately 10 ml/min and then with the fluorescent microspheres.
  • cryosections For immunohistochemistry of cryosections, samples were fixed for 1 h with 2% PFA and incubated in 20% sucrose/PBS overnight. After the cryopreservation, tissues were embedded in OCT compound (Tissue-Tek; Sakura Finetek Europe) and frozen at -70°C. Sections (8-80 ⁇ ) were stained with the primary antibodies overnight at 4°C and subsequently detected with fluorochrome-conjugated secondary antibodies for 30 min at room temperature. Finally, the sections were mounted with antifading medium (Vectashield).
  • E-cadherin Provides a Cell-surface Marker for Further Enrichment of CD117+ Vascular Endothelial Stem Cells
  • the CD117- E-cadherin- fraction of lin-CD31+CD105+Sca-l+ ECs was greatly depleted of CFCs
  • hematopoietic stem-cell antigen Sca-1 is a member of the Ly-6 antigen family. Proc Natl Acad Sci U S A 86, 4634-8. Liu, A. Y. et al. (1997) Cell-cell interaction in prostate gene regulation and

Abstract

La présente invention a pour objet général la découverte de nouvelles méthodes améliorées pour l'isolement et l'enrichissement de populations cellulaires comprenant des cellules souches endothéliales adultes ou des cellules progénitrices endothéliales adultes, une large proportion des cellules isolées étant capable de subir une expansion clonale. En particulier, les inventeurs ont découvert que l'utilisation d'une association spécifique et unique des marqueurs de surface cellulaire enrichit significativement les cellules progénitrices endothéliales adultes. Les méthodes selon la présente invention fournissent des améliorations significatives et surprenantes de la capacité à sélectionner et à isoler les cellules progénitrices endothéliales adultes, comparées à des méthodes dans lesquelles seules les cellules positives pour les marqueurs CD31 et CD 105 sont utilisées. En outre, l'invention concerne des méthodes pour la culture ex vivo et l'expansion de cellules endothéliales clonales issues d'une cellule progénitrice endothéliale unique et l'utilisation de telles populations cellulaires.
PCT/FI2010/051036 2009-12-15 2010-12-15 Méthodes pour l'enrichissement de cellules progénitrices endothéliales adultes et leurs utilisations WO2011073521A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015119642A1 (fr) * 2014-02-10 2015-08-13 The Johns Hopkins University Amélioration du devenir endothélial de cellules souches pluripotentes humaines par une faible pression d'oxygène
CN108060116A (zh) * 2018-02-12 2018-05-22 重庆医科大学附属儿童医院 一种胎鼠内皮祖细胞的提取分离培养方法
CN113552363A (zh) * 2020-04-03 2021-10-26 中国科学院广州生物医药与健康研究院 Cd44作为造血干/祖细胞的标志物及其应用

Citations (33)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4963489A (en) 1987-04-14 1990-10-16 Marrow-Tech, Inc. Three-dimensional cell and tissue culture system
WO1995021613A1 (fr) 1994-02-09 1995-08-17 Sugen, Inc. Composes destines au traitement de troubles associes a la vasculogenese et/ou a l'angiogenese
US5559022A (en) 1992-10-09 1996-09-24 Advanced Tissue Sciences, Inc. Liver reserve cells
WO1997022596A1 (fr) 1995-12-18 1997-06-26 Zeneca Limited Derives de quinazoline
WO1997032856A1 (fr) 1996-03-05 1997-09-12 Zeneca Limited Derives de 4-anilinoquinazoline
US5672346A (en) 1992-07-27 1997-09-30 Indiana University Foundation Human stem cell compositions and methods
WO1998002438A1 (fr) 1996-07-13 1998-01-22 Glaxo Group Limited Composes heteroaromatiques bicycliques en tant qu'inhibiteurs de la proteine tyrosine kinase
WO1998002437A1 (fr) 1996-07-13 1998-01-22 Glaxo Group Limited Composes heteroaromatiques bicycliques en tant qu'inhibiteurs de la proteine tyrosine kinase
US5792783A (en) 1995-06-07 1998-08-11 Sugen, Inc. 3-heteroaryl-2-indolinone compounds for the treatment of disease
US5827735A (en) 1992-06-22 1998-10-27 Morphogen Pharmaceuticals, Inc. Pluripotent mesenchymal stem cells and methods of use thereof
WO1998050356A1 (fr) 1997-05-07 1998-11-12 Sugen, Inc. Derives de 2-indolinone utilises en tant que modulateurs de l'activite de la proteine kinase
WO1998054093A1 (fr) 1997-05-30 1998-12-03 Merck & Co., Inc. Nouveaux inhibiteurs d'angiogenese
WO1999010349A1 (fr) 1997-08-22 1999-03-04 Zeneca Limited Derives d'oxindolylquinazoline utiles comme inhibiteurs d'angiogenese
WO1999016755A1 (fr) 1997-09-26 1999-04-08 Merck & Co., Inc. Nouveaux inhibiteurs de l'angiogenese
WO1999024440A1 (fr) 1997-11-11 1999-05-20 Pfizer Products Inc. Derives de thienopyrimidine et thienopyridine utiles comme agents anticancereux
WO1999061422A1 (fr) 1998-05-29 1999-12-02 Sugen, Inc. Inhibiteurs de la proteine kinase 2-indolinone a substitution pyrrole
WO1999062890A1 (fr) 1998-06-04 1999-12-09 Pfizer Products Inc. Derives isothiazole utiles en tant qu'agents anticancereux
US6121230A (en) 1994-05-26 2000-09-19 Metris Therapeutics Limited Anti-VEGF agents in the treatment of endometriosis
WO2001002369A2 (fr) 1999-07-02 2001-01-11 Agouron Pharmaceuticals, Inc. Composes d'indazole et compositions pharmaceutiques inhibant les proteines kinases, et procedes d'utilisation de ceux-ci
WO2001095353A2 (fr) 2000-06-07 2001-12-13 Siemens Aktiengesellschaft Procede d'organisation du deroulement de processus de commutation commandes electroniquement
WO2002044158A1 (fr) 2000-11-28 2002-06-06 Pfizer Products Inc. Sels d'un isothiazole-4-carboxamide et leur utilisation comme agents anti-hyperproliferatifs
US6534524B1 (en) 1999-07-02 2003-03-18 Agouron Pharmaceuticals, Inc. Indazole compounds and pharmaceutical compositions for inhibiting protein kinases, and methods for their use
US6653308B2 (en) 2001-02-15 2003-11-25 Sugen, Inc. 3-(4-amidopyrrol-2-ylmethylidene)-2-indolinone derivatives as protein kinase inhibitors
WO2003106462A1 (fr) 2002-06-14 2003-12-24 Pfizer Inc. Derives d'amide heteroaryle benzocondense de thienopyridines utilisees en tant qu'agents therapeutiques, compositions pharmaceutiques les contenant et procedes d'utilisation associes
WO2005035738A1 (fr) * 2003-10-07 2005-04-21 Biomaster Inc. Differentiation cellulaire de cellules precurseurs issus des tissus adipeux
WO2005042730A2 (fr) * 2003-11-04 2005-05-12 Biomaster, Inc. Procede et systeme pour preparer des cellules souches a partir de tissus gras
WO2005078073A2 (fr) * 2004-02-09 2005-08-25 Indiana University Research And Technology Corporation Isolement, expansion et utilisation de progeniteurs clonogenes de cellules endotheliales
US20060094032A1 (en) 2004-03-12 2006-05-04 Fougerolles Antonin D iRNA agents targeting VEGF
WO2007102162A2 (fr) * 2006-03-08 2007-09-13 Kwalata Trading Limited Cellules souches régulatrices
US20080050347A1 (en) * 2006-08-23 2008-02-28 Ichim Thomas E Stem cell therapy for cardiac valvular dysfunction
WO2008043181A1 (fr) * 2006-10-12 2008-04-17 Mcgill University Augmentation de populations de cellules souches par la modulation de la protéine tyrosine phosphatase des lymphocytes t (tc-ptp)
WO2008083962A1 (fr) * 2007-01-09 2008-07-17 Lead Pharma Holding B.V. Obtention de nouvelles cellules progénitrices de cardiomyocytes et cardiomyocytes obtenues à partir de ces cellules
WO2008085221A2 (fr) * 2006-10-27 2008-07-17 Caritas St. Elizabeth Medical Center Of Boston, Inc. Utilisation thérapeutique de cellules exprimant cd31

Patent Citations (37)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4963489A (en) 1987-04-14 1990-10-16 Marrow-Tech, Inc. Three-dimensional cell and tissue culture system
US5827735A (en) 1992-06-22 1998-10-27 Morphogen Pharmaceuticals, Inc. Pluripotent mesenchymal stem cells and methods of use thereof
US5672346A (en) 1992-07-27 1997-09-30 Indiana University Foundation Human stem cell compositions and methods
US5559022A (en) 1992-10-09 1996-09-24 Advanced Tissue Sciences, Inc. Liver reserve cells
WO1995021613A1 (fr) 1994-02-09 1995-08-17 Sugen, Inc. Composes destines au traitement de troubles associes a la vasculogenese et/ou a l'angiogenese
US6121230A (en) 1994-05-26 2000-09-19 Metris Therapeutics Limited Anti-VEGF agents in the treatment of endometriosis
US5883113A (en) 1995-06-07 1999-03-16 Sugen, Inc. 3-(4'-Bromobenzylindenyl)-2-indolinone and analogues thereof for the treatment of disease
US5886020A (en) 1995-06-07 1999-03-23 Sugen, Inc. 3-(4'-dimethylaminobenzylidenyl)-2-indolinone and analogues thereof for the treatment of disease
US5792783A (en) 1995-06-07 1998-08-11 Sugen, Inc. 3-heteroaryl-2-indolinone compounds for the treatment of disease
US5834504A (en) 1995-06-07 1998-11-10 Sugen, Inc. 3-(2'-halobenzylidenyl)-2-indolinone compounds for the treatment of disease
WO1997022596A1 (fr) 1995-12-18 1997-06-26 Zeneca Limited Derives de quinazoline
WO1997032856A1 (fr) 1996-03-05 1997-09-12 Zeneca Limited Derives de 4-anilinoquinazoline
WO1998002437A1 (fr) 1996-07-13 1998-01-22 Glaxo Group Limited Composes heteroaromatiques bicycliques en tant qu'inhibiteurs de la proteine tyrosine kinase
WO1998002438A1 (fr) 1996-07-13 1998-01-22 Glaxo Group Limited Composes heteroaromatiques bicycliques en tant qu'inhibiteurs de la proteine tyrosine kinase
WO1998050356A1 (fr) 1997-05-07 1998-11-12 Sugen, Inc. Derives de 2-indolinone utilises en tant que modulateurs de l'activite de la proteine kinase
WO1998054093A1 (fr) 1997-05-30 1998-12-03 Merck & Co., Inc. Nouveaux inhibiteurs d'angiogenese
WO1999010349A1 (fr) 1997-08-22 1999-03-04 Zeneca Limited Derives d'oxindolylquinazoline utiles comme inhibiteurs d'angiogenese
WO1999016755A1 (fr) 1997-09-26 1999-04-08 Merck & Co., Inc. Nouveaux inhibiteurs de l'angiogenese
WO1999024440A1 (fr) 1997-11-11 1999-05-20 Pfizer Products Inc. Derives de thienopyrimidine et thienopyridine utiles comme agents anticancereux
WO1999061422A1 (fr) 1998-05-29 1999-12-02 Sugen, Inc. Inhibiteurs de la proteine kinase 2-indolinone a substitution pyrrole
WO1999062890A1 (fr) 1998-06-04 1999-12-09 Pfizer Products Inc. Derives isothiazole utiles en tant qu'agents anticancereux
US6235764B1 (en) 1998-06-04 2001-05-22 Pfizer Inc. Isothiazole derivatives useful as anticancer agents
WO2001002369A2 (fr) 1999-07-02 2001-01-11 Agouron Pharmaceuticals, Inc. Composes d'indazole et compositions pharmaceutiques inhibant les proteines kinases, et procedes d'utilisation de ceux-ci
US6534524B1 (en) 1999-07-02 2003-03-18 Agouron Pharmaceuticals, Inc. Indazole compounds and pharmaceutical compositions for inhibiting protein kinases, and methods for their use
WO2001095353A2 (fr) 2000-06-07 2001-12-13 Siemens Aktiengesellschaft Procede d'organisation du deroulement de processus de commutation commandes electroniquement
WO2002044158A1 (fr) 2000-11-28 2002-06-06 Pfizer Products Inc. Sels d'un isothiazole-4-carboxamide et leur utilisation comme agents anti-hyperproliferatifs
US6653308B2 (en) 2001-02-15 2003-11-25 Sugen, Inc. 3-(4-amidopyrrol-2-ylmethylidene)-2-indolinone derivatives as protein kinase inhibitors
WO2003106462A1 (fr) 2002-06-14 2003-12-24 Pfizer Inc. Derives d'amide heteroaryle benzocondense de thienopyridines utilisees en tant qu'agents therapeutiques, compositions pharmaceutiques les contenant et procedes d'utilisation associes
WO2005035738A1 (fr) * 2003-10-07 2005-04-21 Biomaster Inc. Differentiation cellulaire de cellules precurseurs issus des tissus adipeux
WO2005042730A2 (fr) * 2003-11-04 2005-05-12 Biomaster, Inc. Procede et systeme pour preparer des cellules souches a partir de tissus gras
WO2005078073A2 (fr) * 2004-02-09 2005-08-25 Indiana University Research And Technology Corporation Isolement, expansion et utilisation de progeniteurs clonogenes de cellules endotheliales
US20060094032A1 (en) 2004-03-12 2006-05-04 Fougerolles Antonin D iRNA agents targeting VEGF
WO2007102162A2 (fr) * 2006-03-08 2007-09-13 Kwalata Trading Limited Cellules souches régulatrices
US20080050347A1 (en) * 2006-08-23 2008-02-28 Ichim Thomas E Stem cell therapy for cardiac valvular dysfunction
WO2008043181A1 (fr) * 2006-10-12 2008-04-17 Mcgill University Augmentation de populations de cellules souches par la modulation de la protéine tyrosine phosphatase des lymphocytes t (tc-ptp)
WO2008085221A2 (fr) * 2006-10-27 2008-07-17 Caritas St. Elizabeth Medical Center Of Boston, Inc. Utilisation thérapeutique de cellules exprimant cd31
WO2008083962A1 (fr) * 2007-01-09 2008-07-17 Lead Pharma Holding B.V. Obtention de nouvelles cellules progénitrices de cardiomyocytes et cardiomyocytes obtenues à partir de ces cellules

Non-Patent Citations (47)

* Cited by examiner, † Cited by third party
Title
"Animal Cell Culture", 2000, OXFORD UNIVERSITY PRESS
"Current Protocols in Cell Biology", 2000, JOHN WILEY & SONS
"Embryonic Stem Cells, Methods and Protocols", 2002, HUMANA PRESS
AFUWAPE AO, HISTOL HISTOPATHOL., vol. 17, no. 3, 2002, pages 961 - 72
BAJOL AM, BRITISH JOURNAL OF CANCER, vol. 90, 2004, pages 245 - 252
BARKER, N. ET AL.: "Identification of stem cells in small intestine and colon by marker gene Lgr5", NATURE, vol. 449, 2007, pages 1003 - 7
BOMPAIS, H. ET AL.: "Human endothelial cells derived from circulating progenitors display specific functional properties compared with mature vessel wall endothelial cells", BLOOD, vol. 103, 2004, pages 2577 - 84, XP002351475, DOI: doi:10.1182/blood-2003-08-2770
BUDDE ET AL., CARDIOVASC RES, vol. 47, 2000, pages 294 - 305
CARMELIET, P.: "Angiogenesis in life, disease and medicine", NATURE, vol. 438, 2005, pages 932 - 6, XP008084723, DOI: doi:10.1038/nature04478
CLARK, W. R.: "The Experimental Foundations of Modern Immunology", 1986, WILEY & SONS, INC.
COULTAS, L.; CHAWENGSAKSOPHAK, K.; ROSSANT, J.: "Endothelial cells and VEGF in vascular development", NATURE, vol. 438, 2005, pages 937 - 45, XP002383770, DOI: doi:10.1038/nature04479
CURRENT PROTOCOLS IN STEM CELL BIOLOGY, 2008, pages 2C 1.1 - 2C1.27
DOME B ET AL: "Circulating endothelial cells, bone marrow-derived endothelial progenitor cells and proangiogenic hematopoietic cells in cancer: From biology to therapy", CRITICAL REVIEWS IN ONCOLOGY / HEMATOLOGY, ELSEVIER SCIENCE IRELAND LTD., LIMERICK, IE, vol. 69, no. 2, 1 February 2009 (2009-02-01), pages 108 - 124, XP025866134, ISSN: 1040-8428, [retrieved on 20080903], DOI: DOI:10.1016/J.CRITREVONC.2008.06.009 *
DOR, Y.; BROWN, J.; MARTINEZ, 0.1.; MELTON, D. A.: "Adult pancreatic beta-cells are formed by self-duplication rather than stem-cell differentiation", NATURE, vol. 429, 2004, pages 41 - 6, XP002388399, DOI: doi:10.1038/nature02520
EBBA BRAKENHIELM, CIRCULATION RESEARCH, vol. 94, 2004, pages 1579
EICHMANN, A. ET AL.: "Vascular development: from precursor cells to branched arterial and venous networks", INT J DEV BIOL, vol. 49, 2005, pages 259 - 67
GOUMANS M J ET AL: "TGF-beta1 induces efficient differentiation of human cardiomyocyte progenitor cells into functional cardiomyocytes in vitro", STEM CELL RESEARCH,, vol. 1, no. 2, 1 June 2008 (2008-06-01), pages 138 - 149, XP022764784, ISSN: 1873-5061, [retrieved on 20080312], DOI: DOI:10.1016/J.SCR.2008.02.003 *
HEALY, DL., HUM REPROD UPDATE, vol. 4, no. 5, September 1998 (1998-09-01), pages 736 - 40
IKUTA, K.; WEISSMAN, 1. L.: "Evidence that hematopoietic stem cells express mouse c-kit but do not depend on steel factor for their generation", PROC NATL ACAD SCI U S A, vol. 89, 1992, pages 1502 - 6, XP002539252, DOI: doi:10.1073/pnas.89.4.1502
INGRAM, D. A. ET AL.: "Identification of a novel hierarchy of endothelial progenitor cells using human peripheral and umbilical cord blood", BLOOD, vol. 104, 2004, pages 2752 - 60, XP002351443, DOI: doi:10.1182/blood-2004-04-1396
INGRAM, D. A. ET AL.: "Vessel wall-derived endothelial cells rapidly proliferate because they contain a complete hierarchy of endothelial progenitor cells", BLOOD, vol. 105, 2005, pages 2783 - 6, XP002351444, DOI: doi:10.1182/blood-2004-08-3057
JACKSON ET AL., PNAS, vol. 96, no. 25, 1999, pages 14482 86
KLEIN: "Immunology", 1982, JOHN WILEY
KOCH, A. E., ANN. RHEUM. DIS., vol. 59, 2000, pages I65 - 71
LEONG, K. G.; WANG, B. E.; JOHNSON, L.; GAO, W. Q.: "Generation of a prostate from a single adult stem cell", NATURE, vol. 456, 2008, pages 804 - 8, XP002567668, DOI: doi:10.1038/nature07427
LIU, A. Y. ET AL.: "Cell-cell interaction in prostate gene regulation and cytodifferentiation", PROC NATL ACAD SCI U S A, vol. 94, 1997, pages 10705 - 10
MCVEY ET AL., J CARDIOVASC PHARMACOL, vol. 33, 1999, pages 703 - 710
PALEOLOG, E. M., ARTHRITIS RES., vol. 4, no. 3, 2002, pages 81 - 90
PERINO M G ET AL: "Cardiomyogenic stem and progenitor cell plasticity and the dissection of cardiopoiesis", JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY, ACADEMIC PRESS, GB, vol. 45, no. 4, 1 October 2008 (2008-10-01), pages 475 - 494, XP025584918, ISSN: 0022-2828, [retrieved on 20080511], DOI: DOI:10.1016/J.YJMCC.2008.05.002 *
PITTINGER ET AL., SCIENCE, vol. 284, 1999, pages 143 47
PROCKOP, SCIENCE, vol. 276, 1997, pages 71 74
ROITT, I.: "Essential Immunology", 1991, BLACKWELL SCIENTIFIC PUBLICATIONS
SATOSHI NISHIMURA, DIABETES, vol. 56, 2007, pages 1517 - 1526
SEUNG-AH YOO, J.IMMUNO, vol. 174, 2005, pages 5846 - 5855
SMITS ET AL., J PHARMACOL EXP THER, vol. 286, 1998, pages 611 - 618
STINGL, J. ET AL.: "Purification and unique properties of mammary epithelial stem cells", NATURE, vol. 439, 2006, pages 993 - 7, XP002465395
THEISE ET AL., HEPATOLOGY, vol. 31, 2000, pages 235 40
TIMMERMANS ET AL., J. CELL. MOL. MED., vol. 13, no. 1, 2009, pages 87 - 102
TIMMERMANS, F. ET AL.: "Endothelial outgrowth cells are not derived from CD133+ cells or CD45+ hematopoietic precursors", ARTERIOSCLER THROMB VASE BIOL, vol. 27, 2007, pages 1572 - 9, XP055021982, DOI: doi:10.1161/ATVBAHA.107.144972
TUMBAR, T. ET AL.: "Defining the epithelial stem cell niche in skin", SCIENCE, vol. 303, 2004, pages 359 - 63, XP002531165, DOI: doi:10.1126/science.1092436
UNTERGASSER G ET AL: "CD34<+>/CD133<-> circulating endothelial precursor cells (CEP): Characterization, senescence and in vivo application", EXPERIMENTAL GERONTOLOGY, ELSEVIER SCIENCE, OXFORD, GB, vol. 41, no. 6, 1 June 2006 (2006-06-01), pages 600 - 608, XP025083299, ISSN: 0531-5565, [retrieved on 20060601], DOI: DOI:10.1016/J.EXGER.2006.03.019 *
VAN DE RIJN, M.; HEIMFELD, S.; SPANGRUDE, G. J.; WEISSMAN, 1. L.: "Mouse hematopoietic stem-cell antigen Sca-1 is a member of the Ly-6 antigen family", PROC NATL ACAD SCI U S A, vol. 86, 1989, pages 4634 - 8
WEISMAN ET AL., ANNU. REV. CELL. DEV. BIOL., vol. 17, pages 387 403
XU ET AL., J MOL CELL CARDIOL, vol. 32, 2000, pages 2339 - 2347
YODER, M. C. ET AL.: "Redefining endothelial progenitor cells via clonal analysis and hematopoietic stem/progenitor cell principals", BLOOD, vol. 109, 2007, pages 1801 - 9
ZAMMARETTI P ET AL: "Adult 'endothelial progenitor cells' - Renewing vasculature", INTERNATIONAL JOURNAL OF BIOCHEMISTRY AND CELL BIOLOGY, EXETER, GB, vol. 37, no. 3, 1 March 2005 (2005-03-01), pages 493 - 503, XP004691890, ISSN: 1357-2725, DOI: DOI:10.1016/J.BIOCEL.2004.06.018 *
ZUK ET AL., TISSUE ENGINEERING, vol. 7, 2001, pages 211 228

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015119642A1 (fr) * 2014-02-10 2015-08-13 The Johns Hopkins University Amélioration du devenir endothélial de cellules souches pluripotentes humaines par une faible pression d'oxygène
US11352606B2 (en) 2014-02-10 2022-06-07 The Johns Hopkins University Low oxygen tension enhances endothelial fate of human pluripotent stem cells
CN108060116A (zh) * 2018-02-12 2018-05-22 重庆医科大学附属儿童医院 一种胎鼠内皮祖细胞的提取分离培养方法
CN108060116B (zh) * 2018-02-12 2020-03-10 重庆医科大学附属儿童医院 一种胎鼠内皮祖细胞的提取分离培养方法
CN113552363A (zh) * 2020-04-03 2021-10-26 中国科学院广州生物医药与健康研究院 Cd44作为造血干/祖细胞的标志物及其应用
CN113552363B (zh) * 2020-04-03 2024-03-08 中国科学院广州生物医药与健康研究院 Cd44作为造血干/祖细胞的标志物及其应用

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