WO2008043181A1 - Augmentation de populations de cellules souches par la modulation de la protéine tyrosine phosphatase des lymphocytes t (tc-ptp) - Google Patents

Augmentation de populations de cellules souches par la modulation de la protéine tyrosine phosphatase des lymphocytes t (tc-ptp) Download PDF

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WO2008043181A1
WO2008043181A1 PCT/CA2007/001819 CA2007001819W WO2008043181A1 WO 2008043181 A1 WO2008043181 A1 WO 2008043181A1 CA 2007001819 W CA2007001819 W CA 2007001819W WO 2008043181 A1 WO2008043181 A1 WO 2008043181A1
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cells
ptp
cell
population
markers
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Michel L. Tremblay
Annie Bourdeau
Sebastien Trop
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Mcgill University
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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
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61P9/10—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
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/599—Cell markers; Cell surface determinants with CD designations not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)

Definitions

  • the present invention relates to methods of propagating stem cells and expanding stem cell populations including endothelial progenitor cells.
  • the invention further provides methods for using stem cells and endothelial progenitor cells for various therapeutic applications including revascularization of ischemic tissues.
  • T-cell protein tyrosine phosphatase is an intracellular enzyme encoded by Ptpn2. It is ubiquitously expressed in embryonic and adult cells, with highest levels in hematopoietic tissues (reviewed in Bourdeau et al., 2005).
  • Human TC-PTP was identified by the screening of a human peripheral T-cell cDNA library with labeled oligonucleotides derived from the catalytic domain of the PTP1 B protein tyrosine phosphatase.
  • the cDNA sequence of human TC-PTP contains a full open reading frame of 1305 base pairs (bp), and a 978 bp 3'-untranslated region (Cool et al. Proc Natl Acad Sci U S A. 1989 Jul;86(14)).
  • Work describing the mapping of human TC-PTP to chromosome 18p11.2-p11.3 also identified two TC-PTP pseudogenes that were localized on chromosomes 1 and 13.
  • mice homologue of TC-PTP termed PTP-2 or MPTP was described (Miyasaka and Li MoI Cell Biochem. 1992 Dec 2;118(1):91-8; Mosinger et al. Proc Natl Acad Sci U S A. 1992 Jan 15;89(2):499-503).
  • the mouse gene is 88.8% identical to human TC-PTP at the nucleotide level, and maps to chromosome 18, a region of synteny with the human TC-PTP locus.
  • the murine cDNA sequence comprises 1570 bp and contains a 5'-untranslated region of 61 bp, a single full open reading frame of 1146 bp and a 3'-untranslated region that includes a polyadenylation signal located 90 bp upstream of the polyadenylation site (Mosinger et al. Proc Natl Acad Sci U S A. 1992 Jan 15;89(2):499-503).
  • TC-PTPa TC-PTP gene at the position where the sequence diverges from that of its murine counterpart.
  • This splicing event generated two distinct mRNAs encoding proteins termed TC-PTPa and TC-PTPb of predicted size of 45 kDa and 48.5 kDa respectively (Champion-Arnaud et al. Oncogene 1991
  • TC-PTPa mRNA contains a unique exon originating from the 3' end of the TC-PTP gene, which encodes a segment of 12 hydrophilic amino acid residues (Mosinger et al. Proc Natl Acad Sci U S A. 1992 Jan 15;89(2):499-503; Lorenzen et al. J. Cell Biol 1995 Nov;131(3):631-43). This transcript is the major gene product found in most human and mouse tissues (Kamatkar et al. J. Biol Chem 1996 Oct 25;271 (43):26755-61). TC-PTPb mRNA is less abundant in human cells and is absent in the mouse.
  • the predicted protein contains an extra hydrophobic segment of 36 amino acids (residues 382-418) at its carboxyl terminus (Mosinger et al. Proc Natl Acad Sci U S A. 1992 Jan 15;89(2):499-503; Lorenzen et al. J Cell Biol. 1995 Nov;131(3):631-43).
  • TC-PTP " ' ' mice appear physically normal until 10-14 days of age, at which time; they progressively develop tissue mononuclear cell infiltrates (You-Ten, 1997). Elevated levels of IFN- ⁇ can be measured at 19 d of age (Heinonen, 2004), and the animals die between 21 and 35 d of age. TC-PTP "7" mice display defective hematopoiesis and immune function, characterized by anemia and splenomegaly secondary to sequestration of erythrocytes and accumulation of myeloid cells (You-Ten, 1997).
  • TC-PTP was also shown to interact with TRAF2 downstream of the TNF proinflammatory cytokine. This interaction inactivated Src and suppressed MAPK signaling (van Vliet, 2005).
  • TC-PTP has been identified as a critical regulator of colony-stimulating factor 1 (CSF-1) signaling and mononuclear phagocyte development.
  • CSF-1 colony-stimulating factor 1
  • TC-PTP Upon CSF-1 stimulation, a deficiency in TC-PTP leads to enhanced tyrosine phosphorylation of the Grb2/Gab2/Shp2 complex by the CSF-1 receptor, and increased activation of
  • the JAK/STAT signaling pathway is important for the development of
  • TC-PTP has been shown to control cytokine signaling events by its negative action on the Janus kinase (Jak) and signal transducer and activator of transcription (Stat) pathways (reviewed in (Bourdeau, 2005).
  • JAK/STAT pathway is widely used by the cytokine receptor superfamily to link receptor stimulation to gene transcription (Aaronson, 2002). This pathway is crucial for hematopoietic and endothelial development, as well as cellular response to growth factors (Aaronson, 2002).
  • TC- PTP substrate-trapping mutant D/A was shown to interact with Jak1 and Jak3 (Simoncic, 2002).
  • Stati , Stat3 and Stat5a/5b were also identified as substrates for TC-PTP (ten Hoeve, 2002; Yamamoto, 2002; Aoki, 2002).
  • HSC Hematopoietic stem cells
  • EPC represent 0.05% of total bone marrow cellularity and are, at the present time, indistinguishable from each other.
  • Long-term HSC (LT-HSC) give rise to short-term HSC (ST-HSC) that have limited self-renewal activity.
  • ST-HSC become committed and can differentiate into common myeloid progenitors (CMP) or common lymphoid progenitors (CLP). CLP further differentiate into all lymphoid lineages including mature T, B and NK cells.
  • CMP common myeloid progenitors
  • CLP common lymphoid progenitors
  • CMP can give rise to granulocyte-monocyte progenitors (GMP), yielding mature monocytic and granulocytic populations, or to megakaryocyte- erythrocyte progenitors (MEP), which produce platelets and erythrocytes.
  • EPC can differentiate into circulating EPC (CEPC) and tissue EPC (TEPC), ultimately giving rise to mature endothelial cells (EC) (see Figure 1).
  • monocytes may also differentiate into TEPC.
  • Cell surface markers that define each marrow stem cell subpopulation are indicated. Figure 1 adapted from references (Akashi, 2000; Hristov, 2004; Iwami, 2004; Kondo, 1997; Urbich, 2004; Urbich, 2004).
  • Vasculogenesis refers to de novo formation of blood vessels from undifferentiated precursor cells. This process contributes to postnatal neovascularization, and may be mediated by bone marrow-derived EPC or HSC (reviewed in 11 12 ).
  • the bone marrow is the major reservoir for these progenitors.
  • these cells can differentiate into endothelial cells, forming capillary-like structures in vitro and acquiring mature endothelial cell markers such as CD31 13"15 .
  • a small percentage of EPC/HSC are released into the circulation (CEPC) and incorporate the vessel structure as a normal process of vascular regeneration after local injury. This vessel regeneration can be enhanced by augmenting the local availability of EPC.
  • the relative number of CEPC in peripheral blood is below 0.1% in normal conditions.
  • EPC are mobilized from the bone marrow and are preferentially recruited to sites of ischemia, where they are incorporated into new blood vessels.
  • This increased number of EPC found at the site of injury correlates with increased cytokine concentrations 21'24 and the activation of STAT3 and STAT5 in vascular smooth muscle cells and in hypoxic endothelial cells, respectively 25"27 .
  • cytokines such as G-CSF increase the number of circulating CD34 + stem cells by enhancing mobilization and recruitment of EPC from the bone marrow 28 .
  • G-CSF improved cardiac function after Ml 29 .
  • GM-CSF Another cytokine, GM-CSF is also known to increase the number of circulating hematopoietic progenitors in humans, including EPC, as shown by improvement of hind limb neovascularization using the hind limb ischemia model 23 ' 30 .
  • Other factors such as vascular endothelial growth factor, angiopoietins, fibroblast growth factors, stromal-derived growth factors and HMG CoA reductase inhibitors also promote the mobilization and incorporation of EPC into new blood vessels 2324 31 - 33 .
  • Agents for, and methods of augmenting local EPC numbers would be useful.
  • the present invention provides a method for expanding stem cells comprising administering an effective amount of an agent that inhibits TC-PTP to a starting population of cells comprising stem cells whereby inhibition of TC-PTP increases stem cell expansion in a final population of cells.
  • the present invention provides a method for isolating expanded stem cells comprising: a) administering an effective amount of an agent that inhibits TC-PTP to a starting population of cells comprising stem cells whereby inhibition of TC-PTP increases stem cell expansion in a final population of cells; and b) isolating a population of expanded stem cells.
  • the present invention also provides a use of a substance that binds CD105 to isolate EPC.
  • the invention further provides a method for isolating a population of endothelial progenitor cells comprising: a) obtaining a starting population of cells comprising stem cells; and b) isolating a final population of CD105+ cells wherein said final population of CD105+ cells comprise endothelial progenitor cells.
  • the invention provides an isolated stem cell that has been expanded and/or isolated using a method of the invention.
  • a culture medium for propagating stem cells including hematopoietic stem cells and endothelial progenitor cells, wherein said culture medium comprises an effective amount of an agent that inhibits TC-PTP.
  • the present invention includes a method of identifying substances which can inhibit TC-PTP comprising the steps of:
  • the invention provides for the therapeutic use of stem cells, hematopoietic stem cells or endothelial progenitor cells that have been isolated or cultured using a method of the invention.
  • Other embodiments provide therapeutic use of agents that inhibit TC-PTP for increasing stem cells in an animal in need thereof. Methods of treatment using an agent that inhibits TC-PTP are also provided.
  • Figure 1 schematically shows hematopoietic and endothelial differentiation in murine adult bone marrow.
  • FIG. 2 shows increased numbers of EPC in TC-PTP ' ' ' bone marrow.
  • TC-PTP +/+ and TC-PTP '7" bone marrow cells were stained with antibodies to lineage (Lin) markers, and to CD105, CD117 and Sca-1 , and analyzed by flow cytometry as described in Materials and Methods.
  • the absolute cell count for the Lin ' CD117 + population per 1 X 10 6 bone marrow cells is indicated (left panels).
  • the Lin " CD117 + Sca-1 + population was further fractionated into 4 subsets (I-IV) based on the level of expression of Sca-1 (low or high), and on surface expression of CD105; the percentage of cells in each subset is indicated (right panels).
  • CFU-EC Endothelial cell colonies obtained from TC-PTP +/+ and TC-PTP ' ' ' whole bone marrow were harvested at day 5 and analyzed by flow cytometry using endothelial cell surface markers.
  • Figure 3 shows increased bone marrow CEPC and peripheral blood stem cells in TC-PTP " ' " bone marrow.
  • CD14 was assessed in the Lin " CD117 + Sca-1 +/hi CD105 + subpopulation by flow cytometry.
  • RCN relative cell number.
  • B TC-PTP +/+ and TC-PTP " ' " white blood cells were stained with antibodies to lineage (Lin) markers, and to
  • CD117 and Sca-1 were analyzed by flow cytometry as described in Materials and Methods.
  • EPC are identified as Lin " CD117 + Sca-1 +/hl and the absolute cell counts per 1 X 10 6 white blood cells is indicated (square).
  • the Lin " CD117 + Sca-1 +/hl population was further fractionated based on the surface expression of CD105 and CD14, identifying CEPC.
  • the absolute cell counts per 1 X 10 6 white blood cells is indicated (square).
  • the representative data shown has been gated on Lin " cells.
  • FIG. 4 Increased phosphorylation of the CD117 receptor in TC-PTP " ' " bone marrow stem cell population.
  • A, C Whole bone marrow was harvested from TC-PTP +/+ and TC-PTP "7" mice. Ex vivo cells were stained for surface expression of Lin " CD117 + Sca-1 +/hl (identifying EPC; panel A) or Lin " CD117 + Sca-1 +/hi CD105 + CD14 + (identifying CEPC; panel C), and intracellular expression of phosphorylated CD117 (p-CD117; phospho c-kit receptor; thick line) or intracellular nonspecific IgG (thin line), for flow cytometry analysis.
  • Contour plot analysis (left panels) were gated on Lin " cells (EPC; panel A) and on Lin " CD117 + Sca-1 +/hi (CEPC; panel C). Histograms (middle panels) are showing mean fluorescent intensity (MFI) of p-CD117 (thick line) compared to IgG (thin line) on EPC or CEPC. Histograms (right panels) are showing MFI of total CD117 protein either in p-CD117 reaction (thick line) or in IgG reaction (thin line) on EPC or CEPC.
  • MFI mean fluorescent intensity
  • Figure 5 shows increased stem cell population after treatment of whole bone marrow with sodium orthovanadate.
  • Figure 6 shows treatment of murine bone marrow cells with TC-PTP blocking agents augments EPC population.
  • a 1 D Balb/c, TC-PTP + ' " or PTP1 B " ' " bone marrow cells were electroporated with a control scramble sequence (SCR) or TC-PTP specific RNAi sequence (panel A) or cultured in the presence of indicated concentrations of small molecule inhibitor (panel D). Both culture types were analyzed by flow cytometry 48 h post-treatment. Cells were stained with antibodies to lineage (Lin) markers, and to CD117 and Sca-1 , as described in Materials and Methods.
  • Contour plot analysis was gated on Lin " cells and the absolute EPC count (per 1 X 10 6 marrow cells) for the CD117 + Sca-1 + fraction is indicated.
  • B, E The average number of stem cells (per 1 X 10 6 marrow cells) are shown for both the RNAi experiments (panel B) and the small molecule inhibitor (panel E).
  • C, F Balb/c bone marrow was harvested, treated with either RNAi sequence (panel C) or with small molecule inhibitor (panel F) and incubated in EndoCult culture medium to allow the development of endothelial cell colonies (CFU-EC), as described in Materials and Methods. * p ⁇ 0.001.
  • PTP blocking agents augments EPC population.
  • A, C Human bone marrow cells were electroporated with a control scramble sequence (SCR) or TC-PTP specific RNAi sequence (panel A) or cultured in the presence of indicated concentrations of small molecule inhibitor (panel C). Both culture types were analyzed by flow cytometry 48 h post-treatment. Cells were stained with Lin " CD34 + CD133 + CD117 + to identify EPC and Lin " CD34 + CD133 + CD117 + CD105 + CD14 + for CEPC as described in Materials and Methods.
  • T cell protein tyrosine phosphatase (TC-PTP) activity increases stem cell populations including hematopoietic stem cell (HSC) and endothelial progenitor cell (EPC) populations, including circulating EPC (CEPC).
  • HSC hematopoietic stem cell
  • EPC endothelial progenitor cell
  • CEPC circulating EPC
  • agents that inhibit TC-PTP including gene ablation, RNAi silencing, non-selective phosphatase inhibitors (including both inorganic molecules such as sodium orthovanadate and conventional small organic molecules) that inhibit TC-PTP are useful for augmenting stem cell populations including HSC, EPC and CEPC populations.
  • the inventors have demonstrated these results in a variety of cells including murine and human cells.
  • EPC and CEPC are increased in the bone marrow and in the periphery where they can promote vasculogenesis.
  • CD105 cell surface marker can be used to isolate and/or enrich endothelial progenitor cells.
  • mouse stem cells expanded by a method of the invention can be isolated using a method that comprises fractionation according to the level of Sca-1 expression and CD117 expression.
  • Murine CEPC can be isolated using these and CD14.
  • expanded human stem cells, including EPC can be isolated using cell surface markers such as CD133, CD34, and CD117 and human CEPC can be isolated using these and CD14.
  • the present invention provides methods of expanding stem cell populations including EPCs and HSCs using an agent that inhibits TC-PTP.
  • the invention also provides methods for isolating stem cell populations including EPCs.
  • the invention provides methods for using stem cells and endothelial progenitor cells for various therapeutic applications including revascularization of ischemic tissues.
  • the present invention provides a method for expanding stem cells, said method comprising: a) administering an effective amount of an agent that inhibits TC-PTP to a starting population of cells comprising stem cells, whereby inhibition of TC-PTP increases stem cell expansion in a final population of cells.
  • stem cells as used herein means immature cells that are capable of giving rise to different cell types and/or capable of differentiation and includes but is not limited to totipotent cells, hemagioblasts, pluripotent cells and multipotent progenitor cells, including endothelial progenitors cells, circulating endothelial progenitor cells, short-term hematopoietic cells and long-term hematopoietic cells.
  • Endothelial progenitor cell or EPC is used interchangeably with endothelial stem cell and is a progenitor cell or stem cell that gives rise to endothelial cells.
  • EPC includes circulating EPC (CEPC) and tissue EPC (TEPC).
  • CEPC circulating EPC
  • TEPC tissue EPC
  • EPC can be characterized by EPC cell marker expression.
  • a murine EPC is optionally characterized by LJn-CDI 17+Sca- 1+/hiCD105+ expression
  • a human EPC is optionally characterized by LJn- CD34+CD133+CD117+CD105+.
  • CEPC are characterized by surface expression of CD14 in addition to EPC markers.
  • Hematopoietic stem cell or HSC is a stem cell that gives rise to hematopoietic cells and includes but is not limited to, long-term HSC (LT- HSC) and short-term HSC (ST-HSC).
  • LT- HSC long-term HSC
  • ST-HSC short-term HSC
  • expanding stem cells means that the number of stem cells is higher in the final cell population after administering the agent that inhibits TC-PTP as compared to the starting population of cells. In one embodiment the total number of stem cells is increased 1.5 fold, preferably 2 fold, more preferably 2-3 fold, or 5 fold over the numbers in the starting cell population.
  • a starting population of cells as used herein can be comprised in an animal or any sample that contains stem cells including, but not limited to, blood, bone marrow, umbilical cord, lymphoid tissue, epithelia, thymus, liver, spleen, cancerous tissues, lymph node tissue, infected tissue, fetal tissue and fractions or enriched portions thereof.
  • the starting population or sample is preferably bone marrow or blood including peripheral blood or umbilical cord blood or fractions thereof, including buffy coat cells, mononuclear cells and low density mononuclear cells (LDMNC).
  • the starting population is optionally patient blood or bone marrow, or any tissue sample that may be obtained for purposes of culture, or cell fractionation for purification of resident stem cells followed by culture.
  • animal includes all members of the animal kingdom, especially mammals, including human.
  • the animal is preferably human such as a patient.
  • the sample comprising the starting population can be obtained using techniques known in the art.
  • final population of cells refers to the post treatment population of cells that comprises augmented or increased stem cells, such as EPC and HSC, as compared to the starting population of cells.
  • Post treatment refers to post administration of an agent that inhibits TC-PTP.
  • the final population in certain embodiments is bone marrow. In other embodiments, the final population is peripheral blood.
  • the final population of cells can also be an ex vivo treated tissue or cell culture.
  • the starting sample or fraction thereof may be enriched for certain cell types and/or depleted for other cell types.
  • the starting sample or fraction thereof may be enriched for endothelial progenitor cells and/or depleted of mature cells and/or other multipotent progenitor cells.
  • the sample may be enriched or depleted of certain cell types using techniques known in the art.
  • the cells of a particular phenotype may be depleted by incubating the starting sample or fraction thereof with a substance that binds a cell marker or substances that bind cell markers such as an antibody cocktail containing antibodies specific for markers on the cells to be depleted.
  • the antibody cocktail can comprise one or more antibodies to specific cell markers present on cells to be depleted.
  • Cell markers that may be used to deplete a starting sample or fraction thereof include but are not limited to "terminal differentiation" cell markers and multipotent progenitor cell markers.
  • Terminal differentiation cell markers are expressed on mature or differentiated cell lineages and multipotent progenitor cell markers are expressed on, but are not limited to, ST-HSC, CLP, CMP, GMP, and MEP (see figure 1).
  • Terminal differentiation cell markers suitable for use in the present invention comprise but are not limited to CD3, CD4, CD5, CD8, CD11b, CD19, CD31 , CD49b, Ter119; and mutipotent progenitor cell markers suitable for use in the present invention comprise but are not limited to CD127 and CD135. Other names for these markers are provided in Table 1.
  • a person skilled in the art will recognize that other markers that are expressed on differentiated cell lineages and multipotent progenitors can also be used with the methods of the invention.
  • TeM 19 is a murine marker of erythroid cells. As indicated in Table 1 , CD235a can be used to replace this marker in humans.
  • the term "Lin markers” is used interchangeably with terminal differentiation cell markers.
  • the Lin marker antibody cocktail is also enriched with antibodies to multipotent progenitor cell markers. A person of ordinary skill in the art would understand which cell markers can be used to deplete a starting sample. Further, such a person would recognize that cell marker homologues or orthologues or functionally equivalent cell markers may be used with cells of the corresponding species.
  • the cells of a particular phenotype may be enriched by incubating a starting sample or fraction thereof with an antibody cocktail containing antibodies specific for markers on the cells to be enriched.
  • the cells to be enriched are a population of stem cells. In a preferred embodiment, the cells to be enriched are EPC. In another embodiment, the cells to be enriched are HSC.
  • Cell markers that may be used to enrich a starting sample of the present invention include but are not limited to CD133, CD34, CD117, Sca-1 and CD105.
  • Sca-1 is a murine stem cell marker.
  • CD133 and CD34 can be used in humans.
  • CD117 and Sca-1 can be used to enrich for murine HSC and EPC.
  • CD117, CD133 and CD34 can be used to enrich for human HSC and EPC.
  • cells expressing CD117 and Sca-1 cell markers are enriched by incubating a starting sample or fraction thereof with an antibody cocktail containing antibodies specific for CD117 and Sca-1.
  • cells expressing CD117, CD133 and/or CD34 cell markers are enriched by incubating a starting sample or fraction thereof with an antibody cocktail containing antibodies specific for CD117, CD133 and/or CD34.
  • the cell marker CD105 can be used to further enrich for EPC.
  • EPCs are enriched by incubating a starting sample or fraction thereof with an antibody cocktail containing antibodies specific for CD105.
  • the antibody cocktail comprises antibodies specific for CD105, CD117 and Sca-1.
  • cell markers can also be used to enrich a starting population.
  • Other cell markers that can be used comprise CD34 and
  • the antibody cocktail comprises antibodies specific for CD105, CD117, CD133 and CD34.
  • the cell marker CD14 can be used to further enrich for circulating EPC (CEPC).
  • CEPCs are enriched by incubating a starting sample or fraction thereof with an antibody cocktail containing antibodies specific for CD14.
  • the antibody cocktail comprises antibodies specific for CD14, CD105, CD117 and Sca-1.
  • the antibody cocktail comprises antibodies specific for CD14, CD105, CD117, CD133 and CD34.
  • Antibodies specific for a particular cell marker are preferably used to deplete and/or enrich a starting sample of particular cell types.
  • Other molecules that bind cell markers may optionally be used, such as aptamers.
  • Molecules that bind cell markers are typically tagged with a detection unit.
  • the detection unit can comprise a fluorochrome tag and permits cells that have bound the molecule to be separated or fractionated by flow cytometry. Flow cytometry techniques are well known to persons skilled in the art.
  • One skilled in the art would recognize that other methods know in the art could also be used to deplete and/or enrich for specific cell types.
  • magnetic beads can be used to enrich or deplete a staring population of cells. Methods employing magnetic beads include but are not limited to use of commercially available products such as EasySep or CellSep.
  • Another method employs ficoll or other similar separation gradients to isolate bone marrow mononuclear cells. Further, it will be readily recognized by one skilled in the art that such methods can be further combined resulting in progressive sounds of enrichment.
  • a starting population is enriched using magnetic beads.
  • a starting population is depleted using magnetic beads.
  • methods employing magnetic beads are combined with other enrichment and/or depletion methods.
  • the other enrichment and/or depletion method comprises flow cytometry methods.
  • depletion and/or enrichment of a starting population comprises separation of cells using ficoll gradients.
  • methods employing ficoll are combined with other enrichment and/or depletion methods.
  • flow cytometry is the other enrichment or depletion method.
  • stem cell markers such as HSC and/or EPC stem cell markers and methods known in the art such as flow cytometry.
  • TC-PTP refers to T-cell protein tyrosine phosphatase and includes but is not limited to variants and homologs of TC-PTP from all species and sources.
  • TC-PTP includes but is not limited to TC-PTPa and TC-PTPb.
  • TC-PTP includes but is not limited to a TC-PTP gene (ptpn2), protein, mRNA transcript or cDNA.
  • Example of TC- PTP include but are not limited to mouse TC-PTP sequences NW_000134, NW 001030635, NT 039674 and human TC-PTP sequences NT 010859, NW 926940.
  • an agent that inhibits TC-PTP is used herein interchangeably with "TC-PTP inhibitor” and/or "TC-PTP blocking agent” and means an agent that inhibits TC-PTP including all variants and homologs of TC-PTP from all species and sources.
  • Agents that inhibit TC-PTP comprise agents that inhibit TC-PTP enzyme activity, as reflected for example in increased phosphorylation of downstream molecules such as CD117, agents that reduce TC-PTP protein expression, agents that reduce TC-PTP mRNA expression and agents that prevent normal intracellular localization or promote the intracellular mislocalization of TC-PTP affecting its normal function.
  • Agents that inhibit TC-PTP comprise pharmacological and biological agents including antisense nucleic acids and RNAi nucleic acid molecules.
  • "An agent that inhibits TC-PTP” may comprise one or more TC-PTP inhibitors.
  • TC-PTP inhibitor comprises phosphatase inhibitors.
  • the phosphatase inhibitor can be a nonspecific phosphatase inhibitor.
  • the phosphatase inhibitor is optionally an organic molecule or inorganic molecule.
  • the agent that inhibits TC-PTP comprises a tyrosine phosphatase inhibitor.
  • the tyrosine phosphatase inhibitor is a vanadium containing tyrosine phosphatase inhibitor.
  • Vanadium containing tyrosine phosphatase inhibitors include but are not limited to vanadate, orthovanadate, pervanadate; vanadate dimer, vanadate tetramer, vanadate pentamer, vanadate hexamer, vanadate heptamer, vanadate octamer, vanadate nonamer, vanadate decamer, vanadate polymer, vanadyl sulfate, bis (6, ethylpicolinato) (H(2)0) oxovanadium(IV) complex, bis (1-oxy-2- pyridinethiolato) oxovanadium (IV), bis (maltolato) oxovanadium (IV), bis (biguanidato) oxovanadium (IV), bis (N'N'-dimethylbiguanidato) oxovanadium (IV), bis (beta-phenethyl-biguanidato)
  • the vanadium tyrosine phosphatase inhibitor is orthovanadate or a salt thereof.
  • the concentration of orthovanadate is preferably 10-100 ⁇ M. More preferably the concentration is 10-50 ⁇ M.
  • An agent that inhibits TC-PTP is in one embodiment a small molecule inhibitor.
  • Thioxothiazolidinone Compounds [0058] One class of small molecule inhibitor that inhibits TC-PTP comprises thioxothiazolidinone compounds. The inventors have previously identified thioxothiazolidinone compounds that inhibit PTP1 B 40 . The inventors now show that these compounds inhibit TC-PTP activity and are useful for augmenting stem cell populations. Thioxothiazolidinone compounds useful for augmenting stem cells comprise compounds of Formula I:
  • X and Y are independently an oxygen or sulfur;
  • Z is sulfur, oxygen, nitrogen or a methylene group;
  • Ri is H, an alkyl, a cycloalkyl, a substituted or unsubstituted cycloalkenyl, an aryl, a m-halophenyl, a p-halophenyl, a m-alkylphenyl, a m- alkoxyphenyl, a p-alkylphenyl, a p-alkoxyphenyl, a hydroxyphenyl, a dichlorophenyl, a pyrrole, a furan, a pyridine, a piperazine, or a morpholino ring group; and R 2 is a substituted aryl, a substituted or unsubstituted cycloalkyl, or a substituted or unsubstituted cycloalkenyl.
  • the agent that inhibits TC-PTP is a thioxothiazolidinone compound.
  • the alkyl of Ri is a
  • the cycloalkyl of Ri is a C 3 - 8 cycloalkyl.
  • the cycloalkenyl of Ri is a C-i- ⁇ .
  • the aryl of R 2 has 3-nitro, 4-hydroxy, and 5-methoxybenzene substituents; 3-nitro, 4,5-dihydroxy benzene substituents; 3-nitro, 4-hydroxy,
  • 5-alkoxy substituents or 3-nitro, 4-hydroxy, 5-cycloalkyl substituents.
  • the 5-alkoxy is a C 2- s alkoxy.
  • the 5-cycloalkyl is a C 3 - 8 cycloalkyl.
  • the chiral centers of carbon atoms of compounds of Formula I can independently of one another have R or S configurations.
  • enantiomers, isomers or tautomers, as well as any derivatives or analogs of compounds of Formula I that retain the same biological activity of inhibiting TC-PTP will be useful as agents for methods disclosed herein.
  • a halogen group of a compound provided herein can be substituted with another halogen group such as a fluoro, chloro, bromo or iodo group and retain biological activity.
  • a pharmaceutically acceptable salt of a compound of Formula I can be obtained using methods well-known to those skilled in the art.
  • a salt can be obtained by combining a compound of Formula I with an inorganic or organic acid or base in a solvent or diluent, or from other salts by cation exchange or anion exchange.
  • Salt-forming groups in a compound of Formula I are groups or radicals having basic or acidic properties.
  • Compounds having at least one basic group or at least one basic radical can form acid addition salts with, for example, inorganic acids such as hydrochloric acid, sulfuric acid, a phosphoric acid, or with suitable organic carboxylic or sulfonic acids.
  • Suitable organic carboxylic or sulfonic acids may include aliphatic mono- or di-carboxylic acids (e.g., trifluoroacetic acid, acetic acid, propionic acid, glycolic acid, succinic acid, maleic acid, fumaric acid, hydroxymaleic acid, malic acid, tartaric acid, citric acid, oxalic acid); amino acids (e.g., arginine, lysine); aromatic carboxylic acids (e.g., benzoic acid, 2- phenoxy-benzoic acid, 2-acetoxy-benzoic acid, salicylic acid, 4-aminosalicylic acid); aromatic aliphatic carboxylic acids (e.g., mandelic acid, cinnamic acid); heteroaromatic carboxylic acids (e.g., nicotinic acid, isonicotinic acid); aliphatic sulfonic acids (e.g., methane-, ethane- or 2-hydroxyethane
  • metal or ammonium salts such as alkali metal or alkaline earth metal salts (e.g., sodium, potassium, magnesium or calcium salts) or ammonium salts with ammonia or suitable organic amines such as tertiary monoamines (e.g., triethylamine or tri-(2- hydroxyethyl)-amine), or heterocyclic bases (e.g., N-ethyl-piperidine or N 1 N 1 - dimethylpiperazine).
  • alkali metal or alkaline earth metal salts e.g., sodium, potassium, magnesium or calcium salts
  • suitable organic amines such as tertiary monoamines (e.g., triethylamine or tri-(2- hydroxyethyl)-amine), or heterocyclic bases (e.g., N-ethyl-piperidine or N 1 N 1 - dimethylpiperazine).
  • pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, i.e., the material can be administered to a subject without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
  • prodrugs of compounds of Formula I are also embraced by the present invention.
  • a prodrug of Formula I or a pharmaceutically acceptable salt thereof is intended to include any covalently bonded carrier which releases the active parent drug according to Formula I in vivo when such prodrug is administered to a mammalian subject.
  • Prodrugs of a compound of Formula I are prepared by modifying functional groups present in the compound in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compound.
  • Prodrugs include compounds of Formula I wherein a hydroxy, amino, or sulfhydryl group is bonded to any group that, when the prodrug of a compound of Formula I is administered to a mammalian subject, cleaves to form, for example a free hydroxy I.
  • the agent that inhibits TC-PTP is compound 1.
  • Compound 1 inhibits TC-PTP with an IC50 of 5.3+0.1 at pH 7.0 40 .
  • compounds of Formula I or pharmaceutically acceptable salts thereof, or prodrugs thereof are generally combined with pharmaceutically acceptable carriers before use.
  • pharmaceutically acceptable carriers examples of such carriers and methods of formulation of pharmaceutically acceptable compositions containing inhibitors and carriers can be found in Remington:
  • compositions suitable for effective administration, such compositions will contain an effective amount of the inhibitor.
  • an inhibitor of PTP-Ib may also inhibit TC-PTP.
  • a non-selective inhibitor of PTP-Ib may also be useful for inhibiting TC-PTP.
  • Agents that inhibit TC-PTP also comprise nucleic acid molecules that reduce TC-PTP mRNA levels, TC-PTP protein expression and/or that directly inhibit TC-PTP activity.
  • nucleic acid molecule refers to an oligomer or polymer of nucleotide or nucleoside monomers consisting of naturally occurring bases, sugars, and intersugar (backbone) linkages.
  • the term also includes modified or substituted oligomers comprising non-naturally occurring monomers or portions thereof, which function similarly. Such modified or substituted nucleic acid molecules may be preferred over naturally occurring forms because of properties such as enhanced cellular uptake, or increased stability in the presence of nucleases.
  • the term also includes chimeric nucleic acid molecules that contain two or more chemically distinct regions. For example, chimeric nucleic acid molecules may contain at least one region of modified nucleotides that confer beneficial properties (e.g. increased nuclease resistance, increased uptake into cells), or two or more nucleic acid molecules of the invention may be joined to form a chimeric nucleic acid molecule.
  • the nucleic acid molecules of the present invention may be ribonucleic or deoxyribonucleic acids and may contain naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil.
  • the nucleic acid molecules may also contain modified bases such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and other alkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza uracil, 6-aza cytosine and 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine, 8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyl adenine and other 8-substituted adenines, 8- halo guanines, 8-amino guanine, 8-thiol guanine, 8-thiolalkyl guanines, 8- hydroxyl guanine and other 8-substituted guanines, other aza and deaza uracils, thymidines, cytosines, adenines, or
  • nucleic acid molecules of the invention may contain modified phosphorous, oxygen heteroatoms in the phosphate backbone, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages.
  • the nucleic acid molecules of the invention may also comprise nucleotide analogs that may be better suited as therapeutic or experimental reagents.
  • An example of an nucleic acid molecule analogue is a peptide nucleic acid (PNA) wherein the deoxyribose (or ribose) phosphate backbone in the DNA (or RNA), is replaced with a polyamide backbone which is similar to that found in peptides (P.E. Nielsen, et al Science 1991 , 254, 1497). PNA analogues have been shown to be resistant to degradation by enzymes and to have extended lives in vivo and in vitro.
  • PNA peptide nucleic acid
  • PNAs also bind stronger to a complimentary DNA sequence due to the lack of charge repulsion between the PNA strand and the DNA strand.
  • Other oligonucleotides may contain nucleotides containing polymer backbones, cyclic backbones, or acyclic backbones.
  • the nucleotides may have morpholino backbone structures (U.S. Patent No. 5,034,506).
  • Nucleic acid molecules may also contain groups such as reporter groups, a group for improving the pharmacokinetic properties of a nucleic acid molecule, or a group for improving the pharmacodynamic properties of a nucleic acid molecule.
  • Nucleic acid molecules may also have sugar mimetics.
  • the nucleic acid molecules may be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • the nucleic acid molecules of the invention or a fragment thereof may be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed with mRNA or the native gene (e.g. phosphorothioate derivatives and acridine substituted nucleotides).
  • the nucleic acid molecules may be produced biologically using an expression vector introduced into cells in the form of a recombinant plasmid, phagemid or attenuated virus in which the nucleic acid molecules are produced under the control of a high efficiency regulatory region, the activity of which may be determined by the cell type into which the vector is introduced.
  • the nucleic acid molecules of the invention include but are not limited to antisense nucleic acid molecules and double stranded nucleic acid molecules as well as nucleic acid molecules that inhibit the enzymatic activity of TC-PTP and which are unrelated to the sequence of TC-PTP.
  • antisense nucleic acid molecule as used herein means an oligomer or polymer of nucleotide or nucleoside monomers that is complementary to its target, e.g. the TC-PTP mRNA transcript.
  • an agent that inhibits TC-PTP comprises an antisense nucleic acid molecule.
  • the antisense nucleic acid molecule is minimally 15 nucleotides and can be 15-20, 20-30, 30-40, 40-50 or 50-100 nucleotides in length.
  • the antisense nucleic acid molecule can be greater than 100 nucleotides and is maximally the number of nucleotides present in a TC-PTP transcript.
  • double stranded nucleic acid molecule means a nucleic acid molecule comprising two strands of oligomers or polymers of nucleotide monomers, wherein the oligomers or polymers are electrostatically bonded.
  • the double stranded nucleic acid molecule comprises a nucleic acid molecule suitable for reducing TC-PTP expression by RNA interference (RNAi).
  • RNAi RNA interference
  • the nucleic acid molecule suitable for RNAi methods may be double stranded RNA and may be an oligomer that is composed of 20-30 nucleotides, or greater than 30 nucleotides.
  • RNAi technology are well known in the art.
  • RNAi (e.g RNAi sequences) inhibition of TC-PTP increase stem cell numbers.
  • TC-PTP directed RNAi sequence having sense sequence GCCCAUAUGAUCACAGUCG (SEQ ID NO: 1) which targets exon 2 in human and mouse TC-PTP, increases EPC and CEPC numbers in human and mouse final populations.
  • TC-PTP directed RNAi sequence having sense sequence GGCACAAAGAAGUUACAUC (SEQ ID NO: 2) which targets exon 3 in mouse TC-PTP, increases HSC, EPC and CEPC in mouse final poulations. Scrambled RNAi sequences (SCR) did not increase EPC cell numbers.
  • RNAi sequences at least 15 nucleotides are optionally used to inhibit TC-PTP.
  • Two thymidine nucleotides are optionally added to the sequence to create a two nucleotide overhang.
  • the inventors have used TC1 having sequnce GCCCAUAUGAUCACAGUCGtt (SEQ ID NO: 3) in mouse and human cells and TC2, having sequence GGCACAAAGAAGUUACAUCtt (SEQ ID NO: 4) in mouse cells to inhibit TC-PTP.
  • the agent that inhibits TC-PTP is an RNAi sequence.
  • the RNAi sequence is GCCCAUAUGAUCACAGUCG (SEQ ID NO: 1).
  • the RNAi sequence is GGCACAAAGAAGUUACAUC (SEQ ID NO:2).
  • the RNAi sequence is GCCCAUAUGAUCACAGUCGtt (SEQ ID NO:3).
  • the RNAi sequence is GGCACAAAGAAGUUACAUCtt (SEQ ID NO:4).
  • a person skilled in the art will recognize that other RNAi sequences that target TC-PTP can be identified using methods known in the art and used in the methods described herein.
  • multiple RNAi sequences can be used simultaneously. RNAi sequences can be used in series to reduce TC- PTP levels in a starting population.
  • TC-PTP comprise aptamers.
  • Aptamers are short strands of nucleic acids that can adopt highly specific 3-dimensional conformations. Aptamers can exhibit high binding affinity and specificity to a target molecule. These properties allow such molecules to specifically inhibit the functional activity of enzymes and are included as agents that inhibit TC-PTP.
  • the agent that can inhibit TC-PTP is a
  • Antibodies to TC-PTP may be prepared using techniques known in the art such as those described by Kohler and Milstein, Nature 256, 495 (1975) and in U.S. Patent Nos. RE 32,011 ; 4,902,614; 4,543,439; and 4,411 ,993, which are incorporated herein by reference. (See also Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.), 1980, and Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988, which are also incorporated herein by reference). Within the context of the present invention, antibodies are understood to include but are not limited to monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, and F(ab')2) and recombinantly produced binding partners.
  • monoclonal antibodies e.g., Fab, and F(
  • the present invention also includes a method of identifying substances which can inhibit TC-PTP comprising the steps of:
  • an "effective amount of an agent that inhibits TC-PTP” means as used herein, an amount effective to inhibit TC-PTP activity or expression and can be determined using routine methods known to one of ordinary skill in the art. Further, two or more agents that inhibit TC-PTP may be used in combination. In addition, subsequent additions of an agent that inhibits TC-PTP
  • PTP may be added at various time points during the incubation of the starting population of cells.
  • an agent that inhibits TC-PTP is added one or more times after the initial incubation with an agent that inhibits TC- PTP.
  • the agent may be administered one or more times after the initial administration.
  • the agent may be administered contemporaneously with one or more other agents that increase stem cell numbers and/or a therapeutic agent.
  • administering or “administration” as used herein, means that the starting population is exposed to an agent that inhibits TC- PTP under suitable conditions to expand the stem cells. The “administering" can be done in vitro or in vivo.
  • the term "administering” as used herein, is synonymous with “incubating” especially for in vitro expansion of stem cells.
  • the “administering” can further comprise local or systemic administration of an agent that inhibits TC-PTP.
  • Local administration is, in one embodiment, accomplished by direct injection.
  • vanadate, a small molecule inhibitor such as compound 1 , or a RNAi sequence specific for TC-PTP is injected directly to a desired site.
  • the injection is optionally an intramuscular injection.
  • the desired site is optionally an ischemic site such as a myocardial infarct site.
  • Other methods of local administration are known in the art and discussed elsewhere herein.
  • suitable conditions to expand the stem cells comprise using a suitable culture medium, in a suitable cell chamber, under suitable ambient conditions, for a suitable period of time to expand the stem cells.
  • a "suitable culture medium” is preferably any culture medium that supports stem cell growth.
  • a suitable culture medium may comprise animal serum, including fetal bovine serum (FBS), ⁇ -mercaptoethanol and/or antibiotics.
  • FBS fetal bovine serum
  • ⁇ -mercaptoethanol ⁇ -mercaptoethanol
  • antibiotics antibiotics.
  • the culture medium is Iscove's modified Dulbecco's medium (IMDM) supplemented with 10% FBS and 0.02% v/v ⁇ -mercaptoethanol.
  • IMDM Iscove's modified Dulbecco's medium
  • the culture medium is a long-term bone marrow culture medium.
  • the long term bone marrow culture medium is DMEM high glucose, 1% antibiotics 15% horse serum, 5% fetal calf serum, 10E-6 M hydrocortisone, 10E-4M ⁇ -mercaptoethanol, transferrin 400 ⁇ g/ml and WEHI supernatant (17%, final concentration).
  • EndoCultTM (Stem Cell Technologies) growth medium is also used.
  • One skilled in the art would be aware of the different culture media available and their suitability for different stem cell cultures. For example a skilled person would know that some animal sera may not be useful for propagating human stem cells which are to be introduced into a human. An alternative culture medium would be required.
  • a suitable cell chamber comprises a culture plate or flask that is permissive for maintaining stem cells. Different culture plates or flasks may be used to incubate different starting samples or populations of cells and during different segments of the incubation. The types of cell chambers suitable for different starting samples or populations of cells are well known to someone skilled in the art. Similarly someone skilled in the art would know what cell chambers are suitable for different segments of the incubation.
  • the culture plate or flask may be coated to promote cell adherence of particular cell types.
  • the suitable cell chamber is a plate or flask that is coated with fibronectin.
  • Suitable ambient conditions for expanding stem cells comprise maintaining cells with sufficient carbon dioxide, and at a suitable temperature. Suitable ambient conditions may be maintained using an incubation unit that regulates carbon dioxide level and temperature. The suitable ambient conditions for expanding stem cells are known to a person skilled in maintaining in vitro cell cultures.
  • a suitable period of time is any period of time where inhibition of TC-PTP increases the number of stem cells in the final cell population.
  • a suitable period of time is minimally the minimum period of TC-PTP inhibition required to increase stem cell numbers.
  • the suitable period of time will vary with the inhibitor.
  • the suitable period is one cell cycle of a stem cell in the starting cell population.
  • the minimum period of time is at least 12 hours or 24 hours.
  • stem cells are expanded in the presence of a TC-PTP inhibitor for at least 24 hours.
  • stem cells are expanded in the presence of orthovanadate for at least 24 hours.
  • stem cells are incubated with or expanded in the presence of a small molecule inhibitor for at least 24 hours, 24-48 hours, or at least 48 hours.
  • the small molecule inhibitor is a thioxothiazolidinone compound.
  • stem cells are incubated with or expanded in the presence of an RNAi sequence directed at TC-PTP.
  • the RNAi sequence is TC1 (sense: GCCCAUAUGAUCACAGUCG (SEQ ID NO: 1), exon 2, human and mouse).
  • the RNAi sequence is TC2 (sense: GGCACAAAGAAGUUACAUC (SEQ ID NO: 2), exon 3, mouse).
  • stem cells are incubated with or expanded in the presence of TC1 and TC2. Subsequently, stem cells may be cultured in the absence of a TC-PTP inhibitor.
  • the present invention provides a method for isolating expanded stem cells comprising: a) administering an effective amount of an agent that inhibits TC-PTP to a starting population of cells comprising stem cells, whereby inhibition of TC-PTP increases stem cell expansion in a final population of cells; and b) isolating a population of expanded stem cells from the final population of cells.
  • Isolating is used interchangeably with “fractionating” and as used herein refers to the separation of stem cells from the final population of cells.
  • a population of expanded stem cells may be isolated using cell markers.
  • Cell markers may be used to isolate or fractionate cells, from the final population of cells. Stem cells that are positive or express a particular cell marker or set of cell markers can be fractionated from the final population of cells using flow cytometry techniques, which are well established in the art. [0095] Several cell markers are known to be associated with stem cells.
  • CD117 and Sca-1 are cell markers that have previously been shown to be present on hematopoietic stem cells. Accordingly, cell markers that may be used to isolate a population of expanded stem cells of the present invention comprise CD117 and Sca-1. In one embodiment CD117 and Sca-1 cell are markers are used to isolate expanded hematopoietic stem cells. In another embodiment, CD117 and Sca-1 are used to isolate expanded endothelial progenitor cells.
  • the inventors have made the novel finding that the cell marker CD105 cells can be used to isolate and/or enrich for endothelial progenitor cells.
  • CD105 + cells are used to isolate endothelial progenitor cells.
  • the invention provides a method for isolating a population of expanded stem cells enriched for endothelial precursor cells, said method comprising fractionating a final population of cells with cell markers comprising CD105.
  • the method comprises fractionating a final population of cells with stem cell markers comprising CD117, Sca-1 and CD105.
  • stem cell markers include but are not limited to CD34 and CD133.
  • the antibody cocktail can comprise various combinations of antibodies. Other antibodies to cell markers not herein mentioned may be used and are considered within the scope of the invention.
  • cell markers can be used to separate stem cell populations that express different levels of a cell marker.
  • a stem cell population may express a high level or a low level of a particular cell marker.
  • Flow cytometry techniques which are well known in the art, can be used to separate cells expressing a high level of a cell marker from cells expressing a low level of a cell marker.
  • expanded stem cells are isolated using an antibody cocktail containing antibodies specific for the expression of Sca-1 and separating cells expressing a high level of Sca-1.
  • cell markers can be used to deplete the final population of cells of certain cell types.
  • Terminal differentiation cell markers and multipotent progenitor cell markers previously described, can be used to isolate or fractionate stem cells that do not express or express low amounts of particular terminal differentiation cell markers and/or multipotent progenitor cell markers.
  • Cell markers that are expressed on mature cell lineages and multipotent progenitors can also be used with the methods of the invention. A person of ordinary skill in the art would understand which cell markers can be used to deplete the final population of cells.
  • Isolating a population of expanded stem cells can comprise both a depletion step using terminal differentiation cell markers and/or multipotent progenitor cell markers and a positive selection step using cell markers expressed on stem cells.
  • the step of isolating a population of expanded stem cells comprises: i. fractionating a final population of cells to obtain a sub-population of cells negative for one or more terminal differentiation cell markers and/or multipotent progenitor cell markers; and ii. fractionating said sub-population of cells to obtain a stem cell enriched fraction using cell markers selected from the group CD117, Sca-1 and CD105 (in the mouse); or CD117, CD34, CD133 and CD105 (in the human).
  • the final population is fractionated using terminal differentiation markers and multipotent progenitor cell markers, CD3, CD4, CD5, CD8, CD11b, CD19, CD49b, Ter119 or CD235a, CD31 , CD127 and CD135 and the sub-population is fractionated using CD117 and Sca-1 , or CD117, CD133 and CD34, obtaining a stem cell enriched fraction comprising HSC.
  • the final population is fractionated using terminal differentiation and multipotent progenitor markers, CD3, CD4, CD5, CD8, CD11b, CD19, CD49b, Ter119 or CD235a, CD31 , CD127 and CD135 and the sub-population is fractionated using CD117, Sca-1 and CD105, or CD117, CD133, CD34 and CD105, obtaining a stem cell enriched fraction comprising EPC.
  • the presence and relative expansion of EPCs obtained using a method of the invention can be determined by performing various endothelial progenitor cell colony-forming assays.
  • the presence and relative expansion of hematopoietic stem cells expanded using a method of the invention can be determined by performing various differentiation assays. These methods are routine and well known in the art to a person of ordinary skill.
  • the present invention also provides a method for isolating endothelial progenitor cells and circulating endothelial progenitor cells, said method comprising: a) obtaining a starting population of cells comprising stem cells; and b) isolating a final population of CD105+ cells wherein said final population of CD105+ cells comprise endothelial progenitor cells.
  • CD105 is a cell marker that the inventors have shown can be used to isolate and/or enrich for endothelial progenitor cells.
  • Other cell markers including CD117 and Sca-1 , CD133 and CD34, can also be used in combination with CD105 to further isolate a CD105+ enriched final population comprising endothelial precursor cells.
  • the method comprises isolating CD105+, CD117+ and Sca-1+ cells.
  • Sca-1 expression is high.
  • Additionally terminal differentiation markers and multipotent progenitor cell markers can be used to deplete the starting population of particular cell types.
  • the method comprises fractionating the starting population of cells comprising stem cells with terminal differentiation cell markers and/or multipotent progenitor cell markers. In another embodiment, the method further comprises fractionating cells expressing CD117 and Sca-1. In a more specific embodiment, the method comprises fractionating cells expressing a high level of Sca-1.
  • the invention also provides for isolated stem cells that have been expanded and/or isolated using a method of the invention.
  • the isolated stem cell is a CD105+, CD117+ Sca-1 + EPC.
  • the isolated stem cell is a CD105+, CD117+, Sca-1+,
  • the isolated stem cell is a CD105+
  • the isolated stem cell is a CD105+, CD117+, CD133+, CD34+, CD14+ CEPC.
  • the invention provides in another embodiment, a method of establishing a stem cell line.
  • the stem cell line is a hematopoietic stem cell line.
  • the stem cell line is an endothelial stem cell line.
  • One aspect of the invention provides a culture medium for propagating hematopoietic stem cells and/or endothelial progenitor cells, wherein said culture medium contains an agent that inhibits TC-PTP.
  • the culture medium is a conditioned medium which is made from cells secreting virus comprising nucleic acid molecules that inhibit TC-PTP.
  • the nucleic acid molecule comprises an antisense molecule.
  • the nucleic acid molecule comprises a molecule that inhibits TC-PTP through RNA interference.
  • the culture medium and inhibiting agent are provided separately, to be combined either prior to use, or during cell culture. V. Therapeutic Uses
  • one aspect of the invention provides for the therapeutic use of an agent that inhibits TC-PTP and/or stem cells that have been isolated or cultured using a method of the invention.
  • one embodiment provides a method of increasing stem cells comprising administering an agent that inhibits TC-PTP to an animal in need thereof. Another embodiment provides use of an agent that inhibits TC-PTP for increasing stem cells in an animal in need thereof.
  • Another embodiment provides use of an agent that inhibits TC-PTP for the manufacture of a medicament for increasing stem cells in an animal in need thereof.
  • an animal in need thereof is any animal that would benefit from receiving stem cells. In a particular embodiment the animal is human.
  • stems cells can be used to treat diseases and disorders that comprise vessel damage.
  • augmenting stem cells is useful in a patient with a hematopoietic disorder.
  • augmenting stems cells is useful in a bone marrow transplant recipient.
  • one embodiment provides a method or use of an agent that inhibits TC-PTP for treating a vessel disease.
  • Another embodiment provides a method or use of an agent that inhibits TC-PTP for treating a hematopoietic cell disorder.
  • Another embodiment provides a method or use in an animal wherein the animal receives a bone marrow transplant.
  • the agent that inhibits TC-PTP is a small molecule inhibitor.
  • the small molecule inhibitor is a thioxothiazolidinone compound.
  • the agent that inhibits TC-PTP is a RNAi sequence.
  • agent that is a vanadate compound is administered locally. In other embodiments the agent is administered systemically.
  • an inhibitor of TC-PTP is administered to an animal.
  • an inhibitor of TC-PTP is used on a patient's bone marrow-derived stem cells post-purification, prior to reinfusion, to augment the stem cell pool.
  • an inhibitor of TC-PTP is used on a patient's bone marrow directly to expand stem cells and the resulting population of cells is injected into the patient.
  • Stem cells expanded or isolated using a method of the invention can be reinfused using methods known in the art including systemic reinfusion, percutaneous intra-coronary infusion, left ventricular catheter- based intramyocardial injection, surgical intramyocardial injection and intradermal reinfusion. Stem cells can also be injected into peripheral sites of ischemia such as in injured peripheral blood vessels.
  • Hematopoietic stem cell transplantation can be used to treat hematopoietic dyscrasias and malignancies (reviewed in Copelan E ⁇ A. 2006. Hematopoietic Stem-Cell Transplantation. New Engl. J. Med.
  • Stem cells and agents that expand stem cells can be used for treating numerous genetic and degenerative disorders. Among them, age-related functional defects, hematopoietic and immune system disorders, heart failures, chronic liver injuries, diabetes, Parkinson's and Alzheimer's diseases, arthritis, and muscular, skin, lung, eye, and digestive disorders as well as aggressive and recurrent cancers could be successfully treated by stem cell-based therapies (Mimeault M et al, Clin Pharmacol Ther. 2007 Sep;82(3):252-64).
  • Bone marrow-derived stem cells which also include marrow stromal cells (also termed mesenchymal stem cells), can be used for the repair of joint tissues such as articular cartilage, subchondral bone, menisci and tendons, thereby enhancing reparative signals in traumatic, degenerative and inflammatory joint disorders (De Bari C et al. Clin Sci (Lond). 113:339-48 2007).
  • tissue-resident stem cells have been shown to contribute in muscle regeneration and repair, implying a role for these cells in aging and neuromuscular diseases (Musaro A et al. Eur J Histochem. 2007;51 Suppl 1 :35-43). Enhanced revascularization of ischemic limbs, both in animal models and in clinical trials in humans has been documented.
  • Recent applications also include enhancing revascularization after myocardial ischemia, as well as neuroprotection after cerebral ischemia, among others (Nat Clin Pract Cardiovasc Med. 2006 Mar;3 Suppl 1 :S23-8. See also S65, S69, S73 and S101).
  • EPC can be also used to enhance endothelial cell repair after focal endothelial damage in atherosclerosis or diabetes as well as for other endothelial cell disfunctions (J Cell. MoI. Med. VoI 10, 2006, pp318-332) (Arterioscler. Thromb. Vase. Biol. 2006;26;758- 764).
  • EPC can be used in cases of retinopathy and heart failure.
  • EPC can also be used for valve tissue engineering (Clrculation2006;114;132-137).
  • the therapeutic methods of the invention can be used to treat any condition wherein it is desirable to use stem cells.
  • one embodiment provides a method of enhancing revascularization comprising administering an agent that inhibits TC-PTP to an animal in need thereof. Another embodiment provides use of an agent that inhibits TC-PTP for enhancing revascularization. A further embodiment provides use of an agent that inhibits TC-PTP for the manufacture of a medicament for enhancing revascularization. In certain embodiments, the ischemia is acute.
  • Revascularization is useful for treating ischemia such as results from myocardial infarction.
  • a method of treating ischemia comprising administering an agent that inhibits TC-PTP to an animal in need thereof.
  • Another embodiment provides use of an agent that inhibits TC-PTP for treating ischemia.
  • a further embodiment provides use of an agent that inhibits TC-PTP for the manufacture of a medicament for treating ischemia.
  • Certain embodiments provide a method of treating myocardial infarction comprising administering an agent that inhibits TC-PTP to an animal in need thereof.
  • Another embodiment provides use of an agent that inhibits TC-PTP for treating myocardial infarction.
  • a further embodiment provides use of an agent that inhibits TC-PTP for the manufacture of a medicament for treating myocardial infarction.
  • beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission
  • Treating can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • the present invention also includes pharmaceutical compositions containing an agent that inhibits TC-PTP for use in the methods of the invention. Accordingly, the present invention provides a pharmaceutical composition for expanding stem cells comprising an effective amount of an agent that inhibits TC-PTP in admixture with a suitable diluent or carrier.
  • compositions containing compounds of Formula I can contain pure enantiomers or pure diastereomers or mixtures of enantiomers, for example in the form of racemates, or mixtures of diastereomers. Mixtures of two or more stereoisomers of compounds are further contemplated with varying ratios of stereoisomers in the mixtures.
  • Compositions of compounds of Formula I can also contain trans- or c/s-isomers including pure c/s-isomers, pure frans-isomers or c/s/ ⁇ rans-isomer mixtures with varying ratios of each isomer.
  • diastereomers e.g., c/s/fra/?s-isomers
  • diastereomers can be separated into the individual isomers (e.g, by chromatography) or racemates (e.g., separated using standard methods such as chromatography on chiral phases or resolution by crystallization of diastereomeric salts obtained with optically active acids or bases).
  • Stereochemically uniform compositions of compounds identified herein can also be obtained by employing stereochemically uniform reactants or by using stereoselective reactions.
  • compositions are administered to a subject in amounts sufficient augment stem cell numbers.
  • the compositions are administered to augment EPC.
  • the effective amount can vary according to a variety of factors such as the subject's condition, weight, sex and age. Other factors include the mode of administration. The appropriate amount can be determined by a skilled physician. In general, an effective amount is one which alleviates one or more signs or symptoms of the disease or condition being controlled or treated.
  • compositions can be used alone at appropriate dosages. Alternatively, co-administration or sequential administration of other agents may be desirable.
  • compositions can be administered in a wide variety of therapeutic dosage forms in conventional vehicles for administration.
  • the compositions can be administered in such oral dosage forms as tablets, capsules (each including timed release and sustained release formulations), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups and emulsions, or by injection.
  • they can also be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form, all using forms well-known to those of ordinary skill in the pharmaceutical arts.
  • compositions can be administered in a single daily dose, or the total daily dosage can be administered in divided doses of two, three or four times daily for example.
  • compositions can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well- known to those of ordinary skill in that art.
  • the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.
  • the dosage regimen utilizing the compositions is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the subject; the severity of the condition to be treated; the route of administration; the renal, hepatic and cardiovascular function of the subject; and the particular composition thereof employed.
  • a physician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the composition required to prevent, counter or arrest the progress of the condition.
  • Optimal precision in achieving concentrations of composition within the range that yields efficacy without toxicity requires a regimen based on the kinetics of the composition's availability to target sites. This involves a consideration of the distribution, equilibrium, and elimination of a composition.
  • Such pharmaceutical compositions can be for intralesional, intravenous, topical, rectal, parenteral, local, inhalant or subcutaneous, intradermal, intramuscular, intrathecal, transperitoneal, oral, and intracerebral use.
  • the composition can be in liquid, solid or semisolid form, for example pills, tablets, creams, gelatin capsules, capsules, suppositories, soft gelatin capsules, gels, membranes, tubelets, solutions or suspensions.
  • the present invention also provides pharmaceutical compositions comprising stem cells.
  • the stem cells may be endothelial progenitor cells or hematopoietic stem cells.
  • compositions of the invention can be intended for administration to humans or animals. Dosages to be administered depend on individual needs, on the desired effect and on the chosen route of administration.
  • the pharmaceutical compositions can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions which can be administered to patients, and such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle. Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985). [00135] On this basis, the pharmaceutical compositions include, albeit not exclusively, the active compound or substance in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids. The pharmaceutical compositions may additionally contain other agents such as other agents that can prevent the inhibition of apoptosis or that are used in treating inflammatory conditions or sepsis.
  • mice [00137] Generation of TC-PTP and PTP1 B mutant mice was described previously 2 . Experiments were performed with mice on a mixed Balb/c- 129SJ background and with mice backcrossed for 8 generation on Balb/c. All procedures were approved by the McGiII University Research and Ethics Animal Committee, and experiments were carried out according to the Canadian Council on Animal Care ethical regulations. Flow cytometry (murine cells)
  • Bone marrow cell suspensions were prepared from tibia, femur, humerus and ulna of TC-PTP +/+ and TC-PTP ' ' " mice (age 14-19 d) or only from femurs of adult Balb/c mice in PBS containing 2% FBS, and filtered through a 70 ⁇ m cell strainer. Mononuclear cells were also obtained from peripheral blood and were isolated using "Lympholyte" (Cedarlane, Hornby, Ontario, Canada) according to the manufacturer's instructions and resuspended in PBS containing 2% FBS.
  • Lineage (Lin) markers comprised the following combination of antibody-PE conjugates: CD3 ⁇ (145-2C11 ; BD Biosciences), CD4 (RM4-5; BD Biosciences), CD5 (53-7.3; Biolegend), CD8 (53-6.7; BD Biosciences), CD11 b (M1-70; BD Biosciences), CD19 (1 D3; BD Biosciences), CD31 (MEC 13.3; BD Biosciences), CD49b (9C10; Biolegend), CD127 (SB/199; BD Biosciences), CD135 (A2F10; eBioscience), Ter-119 (BD Biosciences).
  • Bone marrow cells from healthy donors were obtained from Stem Cell Technologies. Cell suspensions were prepared in PBS containing 2% FBS, and filtered through a 70 ⁇ m cell strainer. Cell suspensions were then stained with the indicated antibodies. Reactions were incubated at 4 0 C for 30 min, in 100 ⁇ l PBS containing 2% FBS, followed by washing in the same solution. When appropriate, cells were then stained with streptavidin- Pacific Blue at 4°C for 20 min, in 100 ⁇ l PBS containing 2% FBS, followed by washing in the same solution. Data acquisition was performed using a FACSAria or FACS LSRII flow cytometer (BD Biosciences), and analysis was done with FIoJo software (BD Biosciences).
  • Antibody-fluorochrome conjugates used were as follows: CD14-PECy7 (M5E2; Biolegend), CD34- FITC (AC136; Miltenyi Biotec; Auburn, CA), CD105-biotin (166707; Cedarlane Laboratories; Horby, ON, Canada), CD117-PECy5 (A3C6E2; Biolegend), CD133-APC (AC133; Miltenyi Biotec) streptavidin-Pacific Blue (Molecular Probes, Burlington, ON, Canada).
  • Lineage (Lin) markers comprised the following combination of antibody-PE conjugates: CD3 (UCHT1 ; Biolegend), CD4 (RPA-T4; Biolegend), CD5 (UCHT2; Biolegend), CD8 (RPA-T8; Biolegend), CD11b (ICRF44; Biolegend), CD19 (HIB19; Biolegend), CD31 (WM59; Biolegend), CD127 (HCD127; Biolegend), CD135 (BV10A4H2; Biolegend), CD235ab (HIR2; Biolegend).).
  • Intracellular staining was performed post-surface staining of the cells.
  • Cells were fixed and permeabilized with CytoPerm CytoFix according to the to the manufacturer's instructions (BD Biosciences).
  • the phospho-CD117 antibody was obtained from Cell Signaling Technology (Danvers, MA), the non-specific IgG from BD Biosciences, and the anti-rabbit Alexa 488 conjugate from Invitrogen-Molecular Probes (Burlington, ON, Canada). Endothelial progenitor cell colony-forming assay
  • Sodium orthovanadate treatment of bone marrow cells [00142] Whole bone marrow was obtained from TC-PTP +/+ and TC-PTP " ' ⁇ mice aged 14 to 19 d, and from adult Balb/c mice. Cells, 4 X 10 5 , were plated in 6-well plates in 2 ml Iscove's modified Dulbecco's medium (Stem Cell Technologies) supplemented with 10% FBS and 0.02% v/v ⁇ -mercaptoethanol for 24 h or 48 h. Where indicated, sodium orthovanadate (Sigma-Aldrich, Oakville, ON, Canada) was added at the start of culture to a final concentration of 2 ⁇ M, 10 ⁇ M or 50 ⁇ M. After incubation, cells were harvested and analyzed by flow cytometry.
  • the inventors have previously demonstrated multiple hematopoietic defects in the bone marrow of TC-PTP '7" mice, suggesting a primary anomaly of HSC.
  • bone marrow was obtained from TC-PTP +/+ and TC-PTP " ' " mice. Cells were then analyzed by flow cytometry after staining for surface expression of CD117 and
  • Typical Lin markers were used to exclude mature progenitor cells.
  • CD31 , CD127 and CD135 were used to exclude mature endothelial cells (EC), common lymphoid progenitors
  • CD105 is generally considered a marker of endothelial cells, but is also expressed on a subset of circulating CD34 + human hematopoietic progenitors; its expression on bone marrow HSC has not been characterized.
  • Pluripotent stem cells were defined as Lin " CD117 + . This subpopulation was further subdivided based on surface expression of Sca " 1 and CD105 ( Figure 2A). In wild type marrow, two distinct populations could be identified: Sca-1 + CD105 " (I) and Sca-1 + CD105 + (II). However, in TC-PTP " ' " bone marrow, two additional populations were observed: Sca-1 h 'CD105 " (III) and Sca-1 hl CD105 + (IV). In addition, there was a distinct increase in the percentage of Sca-1 +/hl CD105 + cells in TC-PTP "7" bone marrow compared to control.
  • FIG. 2 shows increased numbers of EPC in TC-PTP " ' " bone marrow.
  • TC-PTP +/+ and TC-PTP "7" bone marrow cells were stained with antibodies to lineage (Lin) markers, and to CD105, CD117 and Sca-1 , and analyzed by flow cytometry as described in Materials and Methods.
  • the absolute cell count for the Lin " CD117 + population per 1 X 10 6 bone marrow cells is indicated (left panels).
  • the Lin " CD117 + Sca-1 + population was further fractionated into 4 subsets (I-IV) based on the level of expression of Sca-1 (low or high), and on surface expression of CD105; the percentage of cells in each subset is indicated (right panels).
  • B Absolute cell counts (per 1 X 10 6 bone marrow cells) for the total Lin " CD117 + population and its CD105 " and CD105 + subsets were obtained by flow cytometry. P ⁇ 0.005.
  • CD117 in TC " PTP +/+ EPC is not phosphorylated, as demonstrated by equivalent mean fluorescent intensity (MFI) of phosphorylated CD117 and IgG control (MFI 30 v. 29).
  • MFI mean fluorescent intensity
  • TC- PTP "7" EPC showed constitutive phosphorylation of CD117, as demonstrated by a 2-fold increase in MFI compared to IgG control (MFI 48 v. 24), and a 1.6- fold increase compared to TC-PTP +7+ EPC (MFI 48 v. 30).
  • This differential phosphorylation pattern is significant, as equivalent amounts of total CD117 protein were detected in TC-PTP +7+ and TC-PTP "7” EPC ( Figure 4A).
  • TC-PTP and potentially other protein tyrosine phosphatases, are implicated in the regulation of hematopoietic and endothelial stem cell proliferation, and support the notion that pharmacological inhibitors of these enzymes may be employed to augment the stem cell population, with obvious clinical implications.
  • Murine or human bone marrow cells (5 X 10 6 ), were electroporated (320 V, 960 ⁇ F) with 1 ⁇ M of TC-PTP specific RNAi sequence
  • TC1 sense: GCCCAUAUGAUCACAGUCG (SEQ ID NO: 1), exon 2, human and mouse; Ambion Austin, TX)
  • TC2 sense: GGCACAAAGAAGUUACAUC
  • RNAi sequences are molecules known to degrade in a specific fashion their target RNA, and they have been reported to successfully achieve TC-PTP inhibition 38 .
  • Two TC-PTP-specific RNAi sequences were developed and demonstrated increased efficiency by using them in combination.
  • Whole bone marrow was obtained from adult Balb/c mice and from control TC-PTP +7" and PTP1 B " ' " animals.
  • TC-PTP-specific RNAi sequences as well as a control scramble sequence (SCR) were delivered to bone marrow progenitors by electroporation 39 . On average, at least 80% of stem cells take up the RNA with this method.
  • Electroporation of TC-PTP-specific RNAi sequences in Balb/c bone marrow cells produced a 3.1-fold increase in the number of Lin " CD117 + Sca-1 +/hl EPC compared to PBS and SCR controls (p ⁇ 0.01).
  • CFU-EC endothelial cell colony assay
  • Murine or human bone marrow cells (5 X 10 6 ), were plated in 6- well plates coated with fibronectin in EndoCult media (Stem Cell Technologies) for 48 h. Where indicated, a small molecule inhibitor previously described 40 , was added at the start of culture to a final concentration of 10 ⁇ M or 50 ⁇ M. After incubation, cells were harvested and analyzed by flow cytometry.
  • Small chemical inhibitors of protein tyrosine phosphatases have potential applications in the treatment of several human diseases and possess the advantage of direct uptake by target cells.
  • the inventors have identified a unique uncharged thioxothiazolidinone derivative, which is capable of inhibiting PTP1 B and TC-PTP. This small molecule can penetrate cells, inhibit the catalytic pocket of these two enzymes, and cause hyperphosphorylation of a known substrate of PTP1 B and TC-PTP 40 . Since the absence of PTP1 B does not affect bone marrow progenitor cell numbers, the inventors reasoned that this compound could have great potential in this system.
  • CFU-EC endothelial cell colony assay
  • Ter-119 is used to exclude erythroid cells; in humans, CD235a can be used to replace this marker. Accordingly, human EPC were defined as Lin " CD34 + CD133 + CD117 + . This subpopulation was further subdivided based on surface expression of CD105 and CD14 to identify CEPC.
  • Table 1 Selected CD nomenclature and expression pattern. The existence of a human homolog corresponding to each surface marker is indicated.
  • TC1 GCCCAUAUGAUCACAGUCG SEQ ID NO: 1
  • SCR control scramble sequence
  • EPCs Endothelial progenitor cells
  • TC-PTP deficient mice have an increased EPC and CEPC in the bone marrow as well as in the peripheral blood.
  • Progenitor cells from TC-PTP " ' " mice or normal stem cells treated with TC-PTP blocking agents are used to promote repair of blood vessels or ischemic heart after injury in a mouse model.
  • Animals are first subjected to femoral artery ligation or left anterior descending (LAD) coronary artery ligation and then treated with stem cells.
  • LAD left anterior descending
  • Hindlimb ischemia (femoral artery ligation)
  • Revascularization in a hindlimb ischemia model (ligation of the femoral artery) and recovery is accomplished using bone marrow stem cell transplant or administration of an agent that inhibits TC-PTP.
  • the right femoral artery is exposed using a longitudinal skin incision of ⁇ 1 cm, and ligated with 6-0 silk, just distal to the profunda femoris branch, and proximal to the genus descendens artery.
  • the muscles and skin are closed layer by layer with 6-0 absorbable and nylon sutures, respectively.
  • the contralateral leg is used as the unoperated control. Mice are placed under a heat lamp to recover.
  • Myocardial infarction (left anterior descending [LAD] coronary artery ligation) is accomplished using bone marrow stem cell transplant or administration of TC-PTP blocking agent.
  • This operation is performed under general anesthesia and the mouse is ventilated artificially with a respirator.
  • An oblique 8 mm incision is made 2 mm away from the left sternal border, towards the left armpit.
  • the muscles are separated.
  • the rib cage and moving left lung are then visualized.
  • the 4 th intercostal space is then opened taking caution not to damage the lung.
  • the chest retractor is inserted and opened gently to spread the wound 8-10 mm in width.
  • the pericardium is gently picked up with curved and straight forceps, pulled apart and placed behind the arms of the retractor.
  • the LAD artery is ligated 1-2 mm below the tip of the left auricle in its normal position, which induces roughly 40-50% ischemia of the left ventricle.
  • a 7-0 silk ligature on a tapered needle is passed underneath the LAD coronary artery.
  • the ligature is then tied with three knots.
  • a syringe fitted with a 30 ga needle is then used to inject 0.1 ml cell suspension in the border zone of the infarct.
  • the retractor is removed and the lungs are reinflated by shutting off the ventilator outflow for 1-2 seconds.
  • the chest cavity is closed by bringing together the 4 th and the 5 th ribs with one or two 6-0 nylon sutures (with pressure applied to the chest wall to reduce the volume of free air).
  • the muscles and skin are closed layer by layer with 6-0 absorbable and nylon sutures, respectively.
  • Test animals are administered either: 1) genetically modified stem cells (using TC-PTP "7" bone marrow), 2) ex-vivo culture enhanced EPC and CEPC with TC-PTP blocking agent, 3) local administration of TC-P TP blocking agent or 4) systemic administration of TC-PTP blocking agent.
  • Control animals are injected with a suitable carrier and/or TC-PTP +/+ stem cells.
  • Revascularization and repair or ischemic heart is assessed by comparing the degree of recovery from the induced ischemic damage in operated animals treated with TC-PTP deficient cells versus control.
  • Echocardiography may be used to assess improvement in ventricular function.
  • Baseline echocardiography may be performed 72 h following coronary artery ligation; subsequent testing may be performed 4 weeks later to evaluate ventricular function.
  • Necropsy examination upon sacrifice of the test animal allows for histological evaluation of capillary density, as well as extent of infarction and scar formation. Revascularization and repair of ischemic damage in limbs is assessed, for example, by comparing capillary density and blood flood in animals treated with TC-PTP deficient cells versus control. Animals administered TC-PTP deficient cells have increased capillary density and better blood flow than control animals injected with PBS and TC- PTP +/+ bone marrow cells.
  • angiography consisting of intravenous injection of radiopaque contrast agent followed by X-ray imaging, may be used to visualize neovascularization. Necropsy examination supplements the above imaging modalities upon sacrifice of the test animal.
  • Ex- vivo cultured EPC and CEPC [00173] Operated mice are injected with cultured wild type Balb/c bone marrow, treated 48h with TC-PTP-specific RNAi sequence to increase the number of bone marrow EPC and CEPC, one day post-surgery. Cells are injected i.v. (tail vein; 1 x 10E6 cells in PBS). The control mice are injected with PBS or Scrambled (SCR) RNAi sequence treated bone marrow cells.
  • SCR Scrambled
  • mice are injected with cultured wild type Balb/c bone marrow, treated 48h with small molecule inhibitor of TC-PTP (10 ⁇ M and 50 ⁇ M concentration) to increase the number of bone marrow EPC and CEPC, one day post-surgery.
  • Cells are injected i.v. (tail vein; 1 x 10E6 cells in PBS).
  • the control mice are injected with DMSO.
  • Revascularization and repair or ischemic heart is assessed by comparing the degree of recovery from the induced ischemic damage in animals treated with cells inhibited for TC-PTP versus control. Echocardiography may be used to assess improvement in ventricular function.
  • Baseline echocardiography may be performed 72 h following coronary artery ligation; subsequent testing may be performed 4 weeks later to evaluate ventricular function.
  • Necropsy examination upon sacrifice of the test animal allows for histological evaluation of capillary density, as well as extent of infarction and scar formation. Revascularization and repair of ischemic damage is assessed, for example, by comparing capillary density and blood flood in animals treated with cells inhibited for TC-PTP versus control. Animals administered cells inhibited for TC-PTP have increased capillary density and better blood flow than control animals injected with PBS and TC-PTP +/+ bone marrow cells. This may be evaluated using Doppler ultrasonography, performed immediately after surgery and repeated every 3 to 5 d up to 5 weeks post surgery. Alternatively, angiography, consisting of intravenous injection of radiopaque contrast agent followed by X-ray imaging, may be used to visualize neovascularization. Necropsy examination supplements the above imaging modalities upon sacrifice of the test animal.
  • mice After the ligation (either femoral artery or LAD) and before closing the muscles and skin, are administered TC-PTP "7" bone marrow cells, which are injected directly at the ligation site (1 x 10E6 cells in PBS).
  • the control mice undergo the same procedure with PBS or TC-PTP +/+ bone marrow cells injected at the ligation site instead of cell suspension.
  • mice after the ligation (either femoral artery or LAD) and before closing the muscles and skin, are administered cultured wild type Balb/c bone marrow, treated 48h with TC-PTP-specific RNAi sequence to increase the number of bone marrow EPC and CEPC, directly at the ligation site (1 x 10E6 cells in PBS).
  • the control mice undergo the same procedure with PBS, or Balb/c bone marrow treated with SCR RNAi sequence injected at the ligation site instead of RNAi-treated cell suspension.
  • mice after the ligation (either femoral artery or LAD) and before closing the muscles and skin, are administered cultured wild type Balb/c bone marrow, treated 48h with small molecule inhibitor of TC-PTP (10 ⁇ M and 50 ⁇ M concentration) to increase the number of bone marrow EPC and CEPC are injected directly at the ligation site (1 x 10E6 cells in PBS).
  • the control mice undergo the same procedure with DMSO injected at the ligation site instead of a treated cell suspension.
  • Revascularization and repair or ischemic heart is assessed by comparing the degree of recovery from the induced ischemic damage in animals treated with TC-PTP inhibited cells versus control.
  • Echocardiography may be used to assess improvement in ventricular function.
  • Baseline echocardiography may be performed 72 h following coronary artery ligation; subsequent testing may be performed 4 weeks later to evaluate ventricular function.
  • Necropsy examination upon sacrifice of the test animal allows for histological evaluation of capillary density, as well as extent of infarction and scar formation. Revascularization and repair of ischemic damage is assessed, for example, by comparing capillary density and blood flood in animals treated with TC-PTP inhibited cells versus control. Animals administered TC-PTP inhibited cells have increased capillary density and better blood flow than control animals injected with TC-PTP +/+ bone marrow cells.
  • angiography consisting of intravenous injection of radiopaque contrast agent followed by X-ray imaging, may be used to visualize neovascularization. Necropsy examination supplements the above imaging modalities upon sacrifice of the test animal.
  • TC-PTP blocking agent either RNAi sequence or small molecule inhibitor
  • Control animals are administered an appropriate control agent (e.g. PBS and/or DMSO).
  • Revascularization and repair or ischemic heart is assessed by comparing the degree of recovery from the induced ischemic damage in animals treated with TC-PTP blocking agent or inhibitor versus control.
  • Echocardiography may be used to assess improvement in ventricular function. Baseline echocardiography may be performed 72 h following coronary artery ligation; subsequent testing may be performed 4 weeks later to evaluate ventricular function. Necropsy examination upon sacrifice of the test animal allows for histological evaluation of capillary density, as well as extent of infarction and scar formation.
  • Revascularization and repair of ischemic damage is assessed, for example, by comparing capillary density and blood flood in animals treated with TC-PTP blocking agent versus control.
  • Animals with coronary artery ligation administered TC-PTP blocking agent will have increased capillary density and better blood flow than control animals injected with PBS or DMSO. These animals will also have increased left ventricular function.
  • Animals with femoral artery ligation administered TC-PTP blocking agent will regain use of their leg. This may be evaluated using Doppler ultrasonography, performed immediately after surgery and repeated every 3 to 5 d up to 5 weeks post surgery.
  • angiography consisting of intravenous injection of radiopaque contrast agent followed by X-ray imaging, may be used to visualize neovascularization. Necropsy examination supplements the above imaging modalities upon sacrifice of the test animal.
  • Tissue resident stem cells exist and are thought to play an important role in regeneration.
  • cardiac resident stem cells can be isolated with cell surface markers by flow cytometry and contribute to the recovery post-ischemia.
  • small molecule inhibitor are injected directly at the ligation site to augment the pool of resident cardiac stem cells. The control mice will undergo the same procedure, but DMSO is injected at the ligation site instead of the compound.
  • HSC Hematopoietic stem cells
  • EPC endothelial progenitor cells
  • TC-PTP T cell protein tyrosine phosphatase
  • stem cells are defined by surface expression of CD117 and Sca-1 , and by lack of expression of an arbitrary set of surface markers found on terminally differentiated cells, together termed lineage (Lin) markers and multipotent progenitor cell markers.
  • Lin markers included CD3, CD4, CD5, CD8, CD11b, CD19, CD31 , CD49b, and Ter119, and multipotent progenitor markers are CD127 and CD135.
  • This pluripotent stem cell population was further fractionated based on surface expression of CD105. The total number of stem cells was increased 5-fold, and the number of CD105 + stem cells was increased 9-fold in TC-PTP " ' " bone marrow compared to control. In addition, a new population of stem cells expressing high levels of Sca-1 was observed.
  • EPC endothelial progenitor cells
  • nucleic acid molecules suitable to inhibit TC-PTP are first chosen and tested. Suitable nucleic acid molecules are chosen using rules and/or computer programs known in the art. Electroporation of antisense or RNAI nucleic acids is accomplished using methods known in the art such as those described in Genesis 2003 Aug: 36(4):203-8. Decreased expression of TC-PTP is confirmed using methods known in the art such as northern blot and RT-PCR.
  • T cell protein tyrosine phosphatase is a negative regulator of janus family kinases 1 and 3. Curr Biol. 2002; 12:446-453.
  • a nuclear protein tyrosine phosphatase TC-PTP is a potential negative regulator of the PRL-mediated signaling pathway: dephosphorylation and deactivation of signal transducer and activator of transcription 5a and 5b by TC-PTP in nucleus. MoI Endocrinol. 2002; 16:58-69.
  • VEGF/JAK2/STAT5 axis may partially mediate endothelial cell tolerance to hypoxia.
  • Placental growth factor reconstitutes hematopoiesis by recruiting VEGFR1(+) stem cells from bone-marrow microenvironment.

Abstract

L'invention concerne un procédé d'augmentation de l'expansion de cellules souches et d'isolement de cellules souches. Le procédé d'expansion des cellules souches comprend l'incubation des cellules avec un agent inhibant la protéine tyrosine phosphatase des lymphocytes T (TC-PTP).
PCT/CA2007/001819 2006-10-12 2007-10-12 Augmentation de populations de cellules souches par la modulation de la protéine tyrosine phosphatase des lymphocytes t (tc-ptp) WO2008043181A1 (fr)

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WO2011073521A1 (fr) * 2009-12-15 2011-06-23 Petri Salven Méthodes pour l'enrichissement de cellules progénitrices endothéliales adultes et leurs utilisations
WO2010147395A3 (fr) * 2009-06-16 2011-08-25 Korea Research Institute Of Bioscience And Biotechnology Composition de milieu comprenant un neuropeptide y pour la génération, le maintien, la croissance non différenciée prolongée de cellules souches pluripotentes et procédé de culture de cellules souches pluripotentes l'utilisant
EP2670414A2 (fr) * 2011-01-31 2013-12-11 Royal Medical Group, PLLC Cellules souches pluripotentes et procédé de stimulation et d'extraction de cellules souches pluripotentes non embryonnaires à partir de cellules provenant du sang d'un animal et utilisation des cellules souches pluripotentes reconstituées pour traiter les maladies telles que la pneumopathie obstructive chronique
US8628963B2 (en) 2009-06-16 2014-01-14 Korea Research Institute Of Bioscience And Biotechnology Medium composition comprising neuropeptide Y for the generation, maintenance, prolonged undifferentiated growth of pluripotent stem cells and method of culturing pluripotent stem cell using the same
WO2015188228A1 (fr) * 2014-06-10 2015-12-17 Monash University Procédé de production de leucocytes par utilisation de l'inhibition du ptpn2 pour transfert adoptif de cellules
WO2021108867A1 (fr) * 2019-12-04 2021-06-10 Monash University Procédés d'activation de leucocytes cytotoxiques à l'aide d'inhibiteurs de ptp1b et de ptpn2

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WO2010147395A3 (fr) * 2009-06-16 2011-08-25 Korea Research Institute Of Bioscience And Biotechnology Composition de milieu comprenant un neuropeptide y pour la génération, le maintien, la croissance non différenciée prolongée de cellules souches pluripotentes et procédé de culture de cellules souches pluripotentes l'utilisant
US8628963B2 (en) 2009-06-16 2014-01-14 Korea Research Institute Of Bioscience And Biotechnology Medium composition comprising neuropeptide Y for the generation, maintenance, prolonged undifferentiated growth of pluripotent stem cells and method of culturing pluripotent stem cell using the same
WO2011073521A1 (fr) * 2009-12-15 2011-06-23 Petri Salven Méthodes pour l'enrichissement de cellules progénitrices endothéliales adultes et leurs utilisations
EP2670414A2 (fr) * 2011-01-31 2013-12-11 Royal Medical Group, PLLC Cellules souches pluripotentes et procédé de stimulation et d'extraction de cellules souches pluripotentes non embryonnaires à partir de cellules provenant du sang d'un animal et utilisation des cellules souches pluripotentes reconstituées pour traiter les maladies telles que la pneumopathie obstructive chronique
EP2670414A4 (fr) * 2011-01-31 2014-08-20 Lacy John Cellules souches pluripotentes et procédé de stimulation et d'extraction de cellules souches pluripotentes non embryonnaires à partir de cellules provenant du sang d'un animal et utilisation des cellules souches pluripotentes reconstituées pour traiter les maladies telles que la pneumopathie obstructive chronique
WO2015188228A1 (fr) * 2014-06-10 2015-12-17 Monash University Procédé de production de leucocytes par utilisation de l'inhibition du ptpn2 pour transfert adoptif de cellules
JP2017524348A (ja) * 2014-06-10 2017-08-31 モナッシュ ユニバーシティ 養子細胞移入のためのｐｔｐｎ２阻害を用いた白血球の産生方法
WO2021108867A1 (fr) * 2019-12-04 2021-06-10 Monash University Procédés d'activation de leucocytes cytotoxiques à l'aide d'inhibiteurs de ptp1b et de ptpn2

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