[go: up one dir, main page]

EP1117765A2 - Cellules souches portant a leur surface un recepteur fgf - Google Patents

Cellules souches portant a leur surface un recepteur fgf

Info

Publication number
EP1117765A2
EP1117765A2 EP99943982A EP99943982A EP1117765A2 EP 1117765 A2 EP1117765 A2 EP 1117765A2 EP 99943982 A EP99943982 A EP 99943982A EP 99943982 A EP99943982 A EP 99943982A EP 1117765 A2 EP1117765 A2 EP 1117765A2
Authority
EP
European Patent Office
Prior art keywords
cells
fgfr
stem cells
composition
lin
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99943982A
Other languages
German (de)
English (en)
Inventor
E. Lynette Wilson
Patricia E. 9 Spanish Steps Ferguson Road BURGER
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
New York University NYU
Original Assignee
New York University NYU
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by New York University NYU filed Critical New York University NYU
Publication of EP1117765A2 publication Critical patent/EP1117765A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P41/00—Drugs used in surgical methods, e.g. surgery adjuvants for preventing adhesion or for vitreum substitution
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood cells

Definitions

  • the present invention is directed to a new phenotype of stem cells which contain a fibroblast growth factor receptor (FGFR) on the cell surface thereof and further have a phenotype indicative of a primitive state.
  • FGFR fibroblast growth factor receptor
  • the present invention is further directed to subpopulations thereof having a phenotype indicative of endothelial or stromal cells.
  • stem cells also known as progenitor cells
  • progenitor cells stem cells
  • stem cells have been found in the epidermis, the intestinal epithelium, and the hematopoietic system.
  • mesenchymal tissues There is mostly indirect evidence of stem cells in mesenchymal tissues.
  • In vivo and in vi tro studies have provided evidence of osteogenic precursor cells in bone marrow and other stromal cell preparations.
  • the identity of cells in these tissues and their relationship to cells with classical stem cell characteristics have yet to be established.
  • Endothelial cells are part of the normal bone marrow stro a.
  • Long-term cultures of human bone marrow contain a complex mix of stromal cells including adipocytes, fibroblasts, endothelial cells, macrophages, and smooth muscle cells.
  • Endothelial cells and hematopoietic cells are thought to be derived from the common progenitor cells, hemangioblasts .
  • Cell surface molecules on various types of cells, and particularly hematopoietic cells are given a cluster of differentiation (CD) designation in which each CD molecule designation describes a surface molecule (marker) identifiable by a cluster of monoclonal antibodies that display the same cellular reactivity.
  • CD cluster of differentiation
  • CD designations are assigned at regularly held international workshops on human leukocyte differentiation antigens.
  • the CD19 marker is specific to B cells
  • the CD33 marker is specific to myeloid cells.
  • One marker which has been indicated as being present on stem cells is CD34. However, this marker is also found on a significant number of lineage-committed progenitors.
  • markers which are known or thought to be present primarily on stem cells include AC133 (Yin et al, 1997; Buhring et al, 1999), Thy-1 (Murray et al, 1995) and c-kit (D'Arena et al, 1998) .
  • CD34 + hematopoietic stem cells are present in peripheral blood.
  • the major source of CD34 + hematopoietic stem cells in the adult is the bone marrow
  • the purpose of this small, circulating CD34 + cell population is unknown.
  • the bone marrow is "leaky", and the stem cells escape, circulate and return to the marrow.
  • the function of these circulating stem cells is to seed sites, such as the liver and the spleen, which can function as additional sites of hematopoiesis in a crisis.
  • the human CD34 + hematopoietic population isolated from bone marrow, cord blood, and peripheral blood is a heterogeneous population that contains hematopoietic stem cells.
  • circulating CD34 + cells also contain endothelial stem cells, which may also circulate (Asahara et al, 1997; Nieda et al, 1997; Shi et al, 1998; Lin et al, 1998) .
  • Asahara et al (1997) have shown that CD34 + cells isolated from the peripheral blood can be incorporated into the endothelium of ischaemic blood vessels of recipient animals.
  • Purified umbilical cord blood CD34 + cells also give rise to von Willebrand factor-expressing endothelial cells in vi tro, providing additional evidence for a circulating progenitor endothelial cell (Nieda et al, 1997) .
  • bone marrow derived CD34 + cells also contain a transplantable stromal stem cell (Prockop, 1997; Pereira et al, 1998) .
  • Recently, convincing evidence has been presented (Goan et al, 1997) that human CD34 + progenitor cells from peripheral blood or cord blood that were transplanted into NOD/SCID immunodeficient mice gave rise to human stromal cells.
  • the human stromal cells expressed the endothelial cell-specific vascular endothelial growth factor (VEGF) receptor-2 (KDR) and von Willebrand factor, indicating that they were of endothelial origin.
  • VEGF vascular endothelial growth factor
  • KDR vascular endothelial growth factor receptor-2
  • von Willebrand factor indicating that they were of endothelial origin.
  • infusion of whole bone marrow cells into recipient mice results in fibroblasts of donor origin in a number of non-hematopoietic tissues.
  • CD34 has been shown to be expressed by bone marrow stromal precursor cells (Simmons et al, 1991), it is possible that these stromal progenitors reside in the bone marrow within the CD34 + progenitor population.
  • FGFs Fibroblast growth factors
  • ES embryonic stem
  • FGF-2 Fibroblast growth factors
  • FGF-2 acts to antagonize cytokines that induce differentiation
  • low amounts of FGF-2 on the order of 10-100 pg/ml, induce a more primitive phenotype in human K562 leukemic cells.
  • stem cells which are progenitors of endothelial and/or stromal cells.
  • endothelial stem cells and stromal stem cells, or a stem cell which is a progenitor of both, would find many utilities in repairing damaged vasculature and in treating other conditions where endothelial or stromal cells need to be replenished.
  • a small population of cells having a "primitive phenotype", such as CD34 + or CD34 " lin " has been isolated which express cell surface receptors for fibroblast growth factor (FGF) .
  • FGF fibroblast growth factor
  • the population of cells bearing FGF receptors (FGFR) are designated as FGFR + .
  • the FGFR + primitive phenotype cell population has several unique properties:
  • the CD34 + FGFR + cells are predominantly present in the region of the fluorescence-activated cell sorter profile having low forward scatter (FSC) and low side scatter (SSC) .
  • FSC forward scatter
  • SSC low side scatter
  • the majority of the cells of the population are very small and of low granularity.
  • These small cells are located in the FSC/SSC region of the fluorescence-activated cell sorter profile that is normally not analyzed, as this area contains many of the dead and apoptotic cells.
  • this region has recently been shown to be the site of a mesenchymal stem cell population (Zohar et al,
  • the CD34 ⁇ lin " FGFR + cells have FSC/SSC properties that are similar to those of the CD34 + FGFR + cells.
  • This CD34 " lin " FGFR + population also includes significant numbers of cells with higher FSC properties.
  • the CD34 + FGFR + cells are deeply dormant, which is characteristic of a stem cell population. They do not proliferate in culture until 30-60 days after isolation.
  • the FGFR + primitive phenotype cell population is a unique stem cell population that is a precursor cell for endothelium and/or he atopoiesis and/or stroma.
  • the FGFR + primitive phenotype cell population obtained either from general circulation, the bone marrow, cord blood or embryonic cells, is capable of forming endothelial, blood and stromal cells, depending on the need at the time.
  • Figure 1 is a bar graph summarizing the fourteen experiments of Example 1 and showing the percent of CD34 + FGFR + cells which express the indicated third antigens.
  • Figures 2A-2F are flow cytometry plots for Experiment 7 of Example 1.
  • Figure 2A is a dot plot of FSC versus SSC of all events with region RI drawn to eliminate the area containing most of the cell debris and doublets.
  • Figure 2B is a histogram showing the intensity of staining with the dye 7-aminoactinomycin D (7-AAD) .
  • the region R2 is drawn to delineate live cells.
  • Figure 2C is a dot plot of SSC versus CD34 gated on RI AND R2.
  • the region R3 is drawn to delineate CD34 + cells.
  • Figure 2D is a dot plot of FSC versus SSC gated on RI AND R2 AND R3, thus showing the FSC/SSC characteristics of live CD34 + cells in RI .
  • Figure 2E is a dot plot of CD34 versus FGFR gated on RI AND R2. The region R4 is drawn to delineate CD34 + FGFR + .
  • Figure 2F is a dot plot of FSC versus SSC gated on RI AND R2 AND R4 , thereby showing the characteristics of live CD34 + FGFR + cells in RI .
  • Figure 3 is a bar graph showing the percent of live FGFR + cells co-expressing the indicated antigens.
  • Figure 4 is a bar graph showing the growth of various cell populations in the presence or absence of FGF-2 or a combination of FGF-2 plus VEGF.
  • Stem cells when transplanted, can restore the production of hematopoietic, endothelial and stromal cells to a patient who has lost such production due to, for example, radiation therapy.
  • FGFR + primitive phenotype cells preferably CD34 + FGFR + or CD34 " lin " FGFR +
  • stem cells By isolating FGFR + primitive phenotype cells, preferably CD34 + FGFR + or CD34 " lin " FGFR + , from other cells in the body, it is possible to obtain relatively pure stem cells, preferably separate from contaminating cells and other substances, so that the stem cells can be safely transplanted into a patient in need thereof.
  • the unique isolated cells of the present invention are separated from other cells by virtue of their CD34 + or CD34 ⁇ lin " state and possession of fibroblast growth factor receptors.
  • the cells can be isolated by conventional techniques for separating cells, such as those described in Civin, U.S. Patents 4,714,680, 4,965,204, 5,035,994, and 5,130,144, Tsukamoto et al 5,750,397, and Loken et al, U.S. Patent 5,137,809, all of which are hereby incorporated by reference in their entirety.
  • a CD34- specific monoclonal antibody or an FGFR-specific antibody can be immobilized, such as on a column or on magnetic beads.
  • the entire cell population may then be passed through the column or added to the magnetic beads. Those which remain attached to the column or are attached to the magnetic beads, which may then be separated magnetically, are those cells which contain a marker which is recognized by the antibody used. Thus, if the anti-CD34 antibody is used, then the resulting population will be greatly enriched in CD34 + cells. If the antibody used is FGFR, then the resulting population will be greatly enriched in FGFR + cells. That population may then be enriched in the other marker by repeating the steps using a solid phase having attached thereto an antibody to the other marker.
  • CD34 + FGFR + cells are by means of flow cytometry, most preferably by means of a fluorescence- activated cell sorter (FACS) , such as those manufactured by Becton-Dickinson under the names FACScan or FACSCalibur.
  • FACS fluorescence- activated cell sorter
  • the cells having a CD34 marker thereon are tagged with a particular fluorescent dye by means of an anti-CD34 antibody which has been conjugated to such a dye.
  • the FGFR marker of the cells are tagged with a different fluorescent dye by means of an anti-FGFR antibody which is conjugated to the other dye.
  • This emitted light is detected by a photo- multiplier tube (PMT) specific for the emission wavelength of the fluorochome by virtue of a set of optical filters.
  • the signal detected by the PMT is amplified in its own channel and displayed by a computer in a variety of different forms—e.g., a histogram, dot display, or contour display.
  • fluorescent cells which emit at one wavelength express a molecule that is reactive with the specific fluorochrome- labeled reagent
  • non-fluorescent cells or fluorescent cells which emit at a different wavelength do not express this molecule but may express the molecule which is reactive with the fluorochrome-labeled reagent which fluoresces at the other wavelength.
  • the flow cytometer is also semi-quantitative in that it displays the amount of fluorescence (fluorescence intensity) expressed by the cell. This correlates, in a relative sense, to the number of the molecules expressed by the cell.
  • Flow cytometers are also equipped to measure non- fluorescent parameters, such as cell volume or light scattered by the cell as it passes through the laser beam.
  • Cell volume is usually a direct measurement.
  • the light scatter PMTs detect light scattered by the cell either in a forward angle (forward scatter; FSC) or at a right angle (side scatter; SSC) .
  • FSC is usually an index of size
  • SSC is an index of cellular complexity, although both parameters can be influenced by other factors.
  • FACS machines may have the additional capability to physically separate the various fractions by deflecting the cells of different properties into different containers.
  • Any other method for isolating the CD34 + FGFR + population of a starting material, such as bone marrow, peripheral blood or cord blood, may also be used in accordance with the present invention.
  • the various subpopulations of the present invention may be isolated in similar manners.
  • the isolated cell population of this invention can be used in therapeutic methods, such as stem cell transplantation, as well as other therapeutic methods as described below, as well as others that are readily apparent to those skilled in the art.
  • the isolated cell populations can be administered directly by intravenous route to a mammalian patient requiring a bone marrow transplant in an amount sufficient to reconstitute the patient's hematopoietic and immune system.
  • effective quantities can be readily determined by those skilled in the art and will depend, of course, upon the exact condition being treated by the therapy. In many applications, however, an amount containing approximately the same number of stem cells found in one-half to one liter of aspirated marrow should be adequate.
  • a suspension of human cells from marrow or blood comprising cells which are positive both for CD34 and for fibroblast growth factor receptors, preferably substantially free of cells that are not positive for both CD34 and fibroblast growth factor receptors, can restore the production of hematopoietic cells to a human lacking production of these cells.
  • a suspension of these isolated cells is administered to a patient in need thereof in an effective amount to restore production of hematopoietic/ endothelial/stromal cells.
  • the patients in need of this product are those with a specific requirement for hematopoietic, endothelial or stromal cells.
  • patients with vascular injury persons with genetic defects in their hematopoietic, stromal or endothelial cells, such as collagen deficiency, adenosine deaminase deficiency, or clotting factor deficiency. It is expected that the circulating stem cells will selectively home to sites of hematopoietic, endothelial or stromal cell damage/deficiency.
  • CD34 + phenotype indicators besides CD34 + are also known. These include AC133 + (Buhring et al, 1994; Yin et al, 1997), ⁇ hy-1 (Murray et al, 1995) and c-kit + (Buhring et al, 1994; D'Arena et al, 1998) .
  • Another population of primitive cells is CD34 ⁇ lin " .
  • the present inventors have discovered that the CD34 + FGFR + population of the preferred embodiment of the present invention contains significant amounts of the AC133 marker (approximately 64%) , the Thy-1 marker (approximately 52%) or the c-kit marker (approximately 57%) . Many of these cells have more than one primitive marker.
  • markers are known to be endothelial markers. These include VE-Cadherin (also known as CD144) (Vittet et al, 1996), TIE-1 (also known as TIE) (Suda et al, 1997), TEK (also known as TIE-2) (Suda et al, 1997; Hamaguchi et al, 1999) and CD31 (also known as PECAM) (Watt et al, 1993) . Significant quantities of each of these markers were also found on the CD34 + FGFR + population.
  • CD34 + FGFR + population includes a primitive population of cells which are precursors of endothelial cells.
  • the subpopulation with these endothelial markers can be isolated and are also part of the present invention.
  • Certain additional markers are known to be stromal cell markers, such as STRO-1 (Gronthos et al, 1994) .
  • a subpopulation of the CD34 + FGFR + cells of the present invention which is also STRO-l + is a primitive population of cells which are precursors of stromal cells. The subpopulation with these stromal markers can be isolated and are also part of the present invention.
  • results as to co-expression of additional markers were obtained from fluorescence-activated cell sorter (FACS) analysis using specific antibodies to cell surface antigens.
  • the antibodies were labeled with three or four different fluorochromes .
  • the results show that 100% of live FGFR + Thy-1 + cells co-express VE-Cadherin, 97% of live FGFR + AC133 + cells co-express VE-Cadherin, 91% of live
  • FGFR + AC133 + cells co-express Thy-1
  • 67% of live FGFR + TEK + cells co-express Thy-1.
  • Cell sorter systems using additional fluorochromes will be able to allow the separation of those cells in the CD34 + FGFR + population which also express two or more of the various other primitive or endothelial markers discussed above.
  • other markers such as the vascular endothelial growth factor-receptor (VEGF-R) (also known as KDR) , which is a marker for endothelial cells, can also be included in such analyses.
  • VEGF-R vascular endothelial growth factor-receptor
  • CD34 + FGFR + Thy-1 + VEGF-R + cells which subpopulation can be identified and isolated by such systems by one of ordinary skill in the art without undue experimentation.
  • This subpopulation represents a progenitor population capable of developing into either or both of the hematopoietic and endothelial lineages.
  • the isolated CD34 + FGFR + cells grow exceedingly slowly in culture with a long lag of 4-6 weeks.
  • the cells grow in an FGF-dependent manner, as shown in Example 3 and Table 5.
  • a long dormant period is associated with a stem cell phenotype, indicating that these cells have growth characteristics compatible with stem cells.
  • CD34 + FGFR + cells can be isolated in substantial purity, i.e., in a substantially homogeneous population, by the methods discussed above, such as, for example, by means of the FACS apparatus, it is not always necessary that the CD34 + FGFR + stem cell population of the present invention be present in substantial purity. For example, for most purposes, it is sufficient if the population of cells contains greater than 90% of human stem cells characterized as CD34 + and FGFR + or FGFR + with another indication of primitive phenotype. Other aspects of the present invention include subpopulations of the FGFR + primitive phenotype population which are substantially homogeneous for other markers.
  • the subpopulation may be a substantially homogeneous population or a composition in which greater than 90% of the cells therein are CD34 + FGFR + (or CD34 " lin ⁇ FGFR + ) and TIE-1 + .
  • the subpopulation may also be a substantially homogeneous population or a composition in which greater than 90% of the cells therein are CD34 + FGFR + (or CD34 " lin " FGFR + ) and CD31 + and/or TEK + and/or VEGFR + and/or VE- Cadherin "1" and/or positive for any other endothelial marker.
  • the subpopulation may be a substantially homogeneous population or a composition in which greater than 90% of the cells therein are CD34 + FGFR + (or CD34 " lin " FGFR + ) and positive for one or more of the other primitive markers, such as AC133, Thy-1 and c-kit.
  • the subpopulation may also be a substantially homogeneous population or a composition in which greater than 90% of the cells therein are CD34 + FGFR + (or CD34 " lin ⁇ FGFR + ) and also positive for one or more of the other primitive markers and further positive for one or more of the endothelial markers.
  • the subpopulation may also be a substantially homogeneous population or a composition in which greater than 90% of the cells therein are CD34 + FGFR + (or CD34 " lin ⁇ FGFR + ) and also positive for one or more of stromal markers, such as CD34 + FGFR + (or CD34 " lin " FGFR + ) and STRO-l + .
  • Such subpopulations are also contemplated by the present invention.
  • the stem cell population may be a substantially smaller percent of the total cell count being administered.
  • the remaining cells may be filler cells, which may be cells incapable of replicating.
  • the remaining cells may be any of the other types of cells from which the cells of the present invention are originally separated.
  • CD34 + FGFR + cells having other indications of primitive phenotype are also contemplated in accordance with the present invention.
  • CD34 " cells which are also negative for lineage markers (lin " ) may be even more primitive than CD34 + cells (see Zanjani et al, 1999) .
  • the CD34 " lin " phenotype is also considered to be an indication of primitiveness in accordance with the present invention.
  • embryonic stem cells are used as the source of cells from which the population of the present invention is to be separated, all such cells, by definition, have a primitive phenotype.
  • FGFR + cells separated from an embryonic stem cell source will inherently be FGFR + cells with a primitive phenotype.
  • other primitive markers such as AC133, Thy-1 and c-kit, may also be used as markers for primitiveness.
  • the present invention relates to phenotypes which are FGFR + , as well as positive for any phenotype indicating primitive state cells, including, but not limited to, CD34 + , CD34 " lin " , being embryonic stem cells, AC133 + , Thy-1 + and c-kit + .
  • the present invention further relates to subpopulations thereof as described above.
  • the pluripotent FGFR + primitive phenotype stem cells, or pluripotent stem cells of any of the other phenotypes of the present invention have considerable commercial use, including one or more of the following:
  • Endothelial stem cells of the present invention can be used to target the delivery of angiostatic agents and anti-tumor agents to the rapidly proliferating vascular bed associated with tumors. Endothelial cells are long-lived, and the stem cells can be used as vectors to deliver angiostatic/antitumor agents to the rapidly expanding vascular bed associated with tumors without affecting the stable endothelium of established blood vessels .
  • the endothelial stem cells of the present invention can be used to coat valves and implant devices, eliminating many of the clotting problems currently associated with these devices.
  • the endothelial stem cells can be cultured from specific individuals so that valves, implant devices, etc., may be coated with autologous endothelial cells. Panels of HLA-matched endothelial stem cells can also be produced for these purposes.
  • Genetically engineered stem cells "homing" to the endothelium, bone marrow, or connective tissue stroma are long-lived and can secrete proteins, such as adenosine deaminase or clotting factors, as well as other proteins, such as t-PA, which promote thrombolysis .
  • a wild-type gene can be incorporated into the endothelial stem cells, either by homologous or random recombination. With allogeneic endothelial stem cells, normal cells lacking the genetic defect can be used therapeutically.
  • Other indications for gene therapy are introduction of drug resistance genes to enable normal stem cells to have an advantage and be subject to selective pressure, e.g., the multiple drug resistance gene.
  • Endothelial diseases other than those associated with endothelial cells may also be treated, where the disease is related to the lack of a particular excreted product, such as a hormone, enzyme, interferon, factor, or the like.
  • a particular excreted product such as a hormone, enzyme, interferon, factor, or the like.
  • a ribozyme, antisense or other message can be inserted to inhibit particular gene products or susceptibility to disease.
  • Engineered endothelial stem cells secreting such factors as tissue plasminogen activator, designed to help prevent restenosis after balloon angioplasty, can be infused. Asahara et al (1997) have demonstrated that endothelial stem cells selectively home to sites of vascular damage.
  • the stem cells of the present invention can be expanded in number by long-term in vi tro culture with minimal differentiation until needed. However, the stem cells can produce blood cells when treated with the appropriate hematopoietic growth and differentiation factors, or form tubular network structures characteristic of endothelial cells upon treatment with the appropriate agents, or form fibroblast-like stromal cells upon treatment with the appropriate agents. Additionally, the cultured stem cells of the present invention can mature into functionally competent blood cells in vi vo, capable of mediating antigen-specific immune responses, repopulating lympho-hematopoietic organs, and prolonging survival of animals with a destroyed hematopoietic system.
  • Fresh or frozen cells from bone marrow, cytokine mobilized peripheral blood or cord blood were obtained from donors after informed consent.
  • Bone marrow or cord blood samples were diluted with 4 volumes RPMI 1640 medium containing 10% fetal calf serum (FCS) .
  • Mononuclear cells were separated on a Histopaque®-1077 density gradient (Sigma Diagnostics, St. Louis, MO) and washed x 2 with PBS-citrate (PBS containing 13.6 mmol/L sodium citrate, 1 mmol/L adenosine and 2 mmol/L theophylline) .
  • Histopaque®-1077 density gradient centrifugation but were washed X 2 with PBS-citrate before proceeding to the filtration step.
  • PBS-citrate washed cells (from all sources) were filtered through a 40 ⁇ m nylon cell strainer (Falcon 2340, Becton-Dickinson, New Jersey) . Cells were resuspended in 2 ml PBS-citrate and overlaid on a 3 ml PBS-citrate/10% bovine serum albumin (BSA) cushion and then centrifuged for 10 minutes at 200 g at room temperature, to remove platelets (Thoma et al, 1994). This step was repeated once or twice.
  • BSA bovine serum albumin
  • DNase was used to digest DNA from cell debris.
  • Samples were enriched for CD34 + cells by one of the following methods:
  • CD34-FITC CD34-PE
  • CD34-RPE-CY5 c-kit-PE
  • CD38-FITC Mouse IgG-FITC
  • Mouse IgG-PE Mouse IgG- RPE-Cy5
  • Mouse IgG2a streptavidin-PE and goat-anti-mouse-RPE
  • Thy- 1-FITC and Thy-1-PE were obtained from Immunotech (Immunotech, France) .
  • CD31-FITC, Mouse IgG Biotin , CD34-APC and Mouse IgG APC were obtained from Caltag (Caltag Laboratories,
  • HLA-DR-FITC Mouse IgG and Goat IgG were purchased from Sigma (Sigma®, St. Louis MO) and CD34-APC, CD31-PE, Mouse IgG-APC from Becton-Dickinson (Becton- Dickinson, San Jose, CA) .
  • AC133-PE was obtained from Miltenyi (Miltenyi Biotec, Auburn, CA) .
  • VE-Cadherin-FITC was a kind gift from Dr. W. A. Muller, Cornell University, New York.
  • TIE 1-FITC, TIE 1-PE, biotinylated TIE 1 and biotinylated TEK were generous gifts from Dr. T.
  • CD34 + selected cells were analyzed using CellQuest software (Becton-Dickinson, San Jose, CA) .
  • the dye, 7- aminoactinomycin D (7-AAD) was used in some experiments to identify dead cells. This was done to ensure that the CD34 + FGFR + population was a viable population.
  • CD34 + FGFR + Co-Expression of Other Antigens CD34-enriched cells were purified from cytokine mobilized peripheral blood (PB) , bone marrow (BM) , or cord blood (CB) , using magnetic separation techniques (Dynal or MiniMacs) . Fluorescent-labeled antibodies were used to assay for CD34 + FGFR + cells and a percentage of CD34 + FGFR + was determined for each experiment. The results of the fourteen experiments which were run are shown in Table 1. For the fourteen experiments, a mean of 4.4% ( ⁇ 2.3%) of CD34 + cells expressed FGFR.
  • the presence of a third antigen on the CD34 + FGFR + cells was also ascertained using fluorescent antibodies.
  • the mean percentage of CD34 + FGFR + cells expressing a particular third antigen is shown in Table 1 and is shown graphically in Figure 1.
  • the error bars indicate the standard deviation (SD) and the numbers show the number of experiments assaying a particular antigen.
  • Figure 2B is a histogram showing the intensity of staining with the dye 7-AAD (Philpott et al, 1996) .
  • 7-AAD is a large molecule which is not readily taken up by cells with intact cell membranes and which stains dead cells intensely.
  • the histogram is gated on RI, and the region R2 is drawn to delineate live cells.
  • Figure 2C is a dot plot of SSC versus CD34 gated on RI AND R2.
  • the gates are set using Boolean logic in which the convention used for RI AND R2 is R1*R2, and in this instance the gate delineates live cells in RI .
  • the region R3 is drawn to delineate CD34 + cells in this plot.
  • FITC was used to label the CD34 + cells.
  • Figure 2D is a dot plot of FSC versus SSC gated on
  • RI AND R2 AND R3 (R1*R2*R3), i.e., this plot uses "backgating" to show the FSC/SSC characteristics of live CD34 + cells in RI .
  • Figure 2E is a dot plot of CD34 versus FGFR gated on RI AND R2, i.e., gated on live cells in RI .
  • the region R4 is drawn to delineate CD34 + FGFR + cells.
  • Figure 2F is a dot plot of FSC versus SSC gated on RI AND R2 AND R4 (R1*R2*R4), i.e., this plot uses "backgating" to show the characteristics of live CD34 + FGFR + cells in RI . It is noted that the majority of live CD34 + FGFR + cells have low FSC and low SSC, i.e., they are small cells with low granularity. The position of these cells on the scatter dot plot is somewhat unusual, as it appears in the region which is conventionally ignored.
  • Example 2 CD34 ⁇ lin ⁇ FGFR * Co-Expression of Other Antigens The following experiment was conducted using the techniques described for Example 1. Lineage depletion to obtain lin " cells was conducted as described in Bhatia et al, 1997, and Bhatia et al, 1998, using Dynal beads and magnetic separation. The results obtained are summarized in Table 4.
  • CD34 lin FGFR + cells co-express CD31
  • 40% of CD34 ⁇ lin ⁇ FGFR + cells co-express c-kit 47% of CD34 " lin " FGFR + cells co-express VE-Cadherin
  • 18% of CD34 " lin " FGFR + cells co-express TIE-1.
  • Cytokine mobilized peripheral blood cells were enriched for CD34 + cells using magnetic separation.
  • the cells were stained with fluorescent-labeled antibodies to CD34 and FGFR and sorted on a fluorescence-activated cell sorter (FACS) into FGFR + and FGFR " populations.
  • FACS fluorescence-activated cell sorter
  • Cells were seeded at 1000 cells per well into collagen/gelatin coated wells containing long-term culture medium (LTCM) with either (a) no additions, (b) 10 ng/ml FGF-2 or (c) 10 ng/ml FGF-2 plus 10 ng/ml VEGF, and incubated at 37 °C in a humidified incubator. After 4-6 weeks, the number of cells per well were counted.
  • Table 5 and shown graphically in Figure 4. It can be seen that the FGFR + population grows in an FGF dependent manner and that the addition of VEGF together with FGF-2 further increases the growth of the cells.
  • Vittet et al "Embryonic stem cells differentiate in vitro to endothelial cells through successive maturation steps", Blood 88:3424-3431 (1996).
  • Wood et al, "CD34 expression patterns during early mouse development are related to modes of blood vessel formation and reveal additional sites of hematopoiesis", Blood 90:2300-2311 (1997) .

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Hematology (AREA)
  • General Engineering & Computer Science (AREA)
  • Public Health (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Veterinary Medicine (AREA)
  • Biochemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Rheumatology (AREA)
  • Immunology (AREA)
  • Surgery (AREA)
  • Vascular Medicine (AREA)
  • Diabetes (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Composition de cellules souches pluripotentes sensiblement purifiées présentant un récepteur du facteur de croissance des fibroblastes (FGFR) et un phénotype indiquant un état primitif telles que des cellules souches CD34?+, CD34-lin-, Thy-1+, AC133+¿ ou c-kit+. L'état de cellule souche embryonnaire est également un phénotype qui indique un état primitif. Cette population peut également être définie par des sous-populations de cette dernière portant un autre marqueur qui indique des cellules endothéliales, telles que des cellules TIE-1?+, TEK+, CD31+¿, VE-Cadhérine+ ou VEGFR+ ou qui indique des cellules de stroma, telles que des cellules STRO-1+.
EP99943982A 1998-08-31 1999-08-31 Cellules souches portant a leur surface un recepteur fgf Withdrawn EP1117765A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US9848098P 1998-08-31 1998-08-31
US98480P 1998-08-31
PCT/US1999/019730 WO2000012683A2 (fr) 1998-08-31 1999-08-31 Cellules souches portant a leur surface un recepteur fgf

Publications (1)

Publication Number Publication Date
EP1117765A2 true EP1117765A2 (fr) 2001-07-25

Family

ID=22269466

Family Applications (1)

Application Number Title Priority Date Filing Date
EP99943982A Withdrawn EP1117765A2 (fr) 1998-08-31 1999-08-31 Cellules souches portant a leur surface un recepteur fgf

Country Status (7)

Country Link
EP (1) EP1117765A2 (fr)
JP (1) JP2002523085A (fr)
AU (1) AU774581B2 (fr)
CA (1) CA2341754A1 (fr)
IL (1) IL141683A0 (fr)
WO (1) WO2000012683A2 (fr)
ZA (1) ZA200101622B (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013023801A1 (fr) 2011-08-16 2013-02-21 Seco Tools Ab Plaquette de coupe amovible à double face et outil de coupe comportant une telle plaquette

Families Citing this family (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030125615A1 (en) * 1998-02-05 2003-07-03 Yitzhack Schwartz Homing of autologous cells to a target zone in tissue using active therapeutics or substances
US6468794B1 (en) 1999-02-12 2002-10-22 Stemcells, Inc. Enriched central nervous system stem cell and progenitor cell populations, and methods for identifying, isolating and enriching for such populations
US7217565B2 (en) 1999-02-12 2007-05-15 Stemcells California, Inc. Enriched central nervous system stem cell and progenitor cell populations, and methods for identifying, isolating and enriching for such populations
AU780256B2 (en) * 1999-02-12 2005-03-10 Boco Silicon Valley, Inc. Enriched central nervous system stem cell and progenitor cell populations, and methods for identifying, isolating and enriching for such populations
US7105150B2 (en) 1999-02-12 2006-09-12 Stemcells California, Inc. In vivo screening methods using enriched neural stem cells
US7037719B1 (en) 1999-02-12 2006-05-02 Stemcells California, Inc. Enriched central nervous system stem cell and progenitor cell populations, and methods for identifying, isolating and enriching for such populations
FR2808803B1 (fr) * 2000-05-11 2004-12-10 Agronomique Inst Nat Rech Cellules es modifiees et gene specifique de cellules es
AU2001274624A1 (en) 2000-06-26 2002-01-08 Renomedix Institute Inc. Cell fraction containing cells capable of differentiating into nervous system cells
IL154159A0 (en) 2000-08-01 2003-07-31 Yissum Res Dev Co Directed differentiation of ebryonic cells
US7247477B2 (en) * 2002-04-16 2007-07-24 Technion Research & Development Foundation Ltd. Methods for the in-vitro identification, isolation and differentiation of vasculogenic progenitor cells
US7470538B2 (en) 2002-12-05 2008-12-30 Case Western Reserve University Cell-based therapies for ischemia
ITNA20040043A1 (it) * 2004-07-28 2004-10-28 Francesco Carinci Tecnica di ingegneria tissutale ottenibile mediante isolamento di una nuova sottopopolazione di cellule staminali mbp-shed ed mbp-dpsc, isolate da polpa di denti decidui e permanenti in grado di produrre in vitro tessuto osseo umano.
US9034316B2 (en) 2006-10-24 2015-05-19 Amorcyte, Llc Infarct area perfusion-improving compositions and methods of vascular injury repair
ES2485387T3 (es) 2005-11-07 2014-08-13 Amorcyte, Inc. Composiciones y métodos de reparación de lesiones vasculares
US8637005B2 (en) 2005-11-07 2014-01-28 Amorcyte, Inc. Compositions and methods of vascular injury repair
US20110076255A1 (en) 2005-11-07 2011-03-31 Pecora Andrew L Compositions and methods for treating progressive myocardial injury due to a vascular insufficiency
BR112015018923B1 (pt) 2013-02-06 2022-03-03 Nc Medical Research Inc Métodos in vitro de produção de subpopulação heterogênea de células de medula óssea e para otimizar protocolo experimental para isolar população de células que trata neurodegeneração causada por acidente vascular cerebral isquêmico

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5690926A (en) * 1992-10-08 1997-11-25 Vanderbilt University Pluripotential embryonic cells and methods of making same
US5505908A (en) * 1994-09-01 1996-04-09 Halozone Technologies, Inc. Recycling and recovery of methyl bromide fumigant
WO1996031598A1 (fr) * 1995-04-06 1996-10-10 Regeneron Pharmaceuticals, Inc. Ligands tie-2, procedes d'obtention de ces ligands et leurs utilisations
ES2260784T3 (es) * 1996-01-23 2006-11-01 Indevus Pharmaceuticals, Inc. Metodos de utilizacion del gen obese y su producto genico para estimular el desarrolo hematopoyetico.
FR2756570B1 (fr) * 1996-12-03 2002-09-27 Commissariat Energie Atomique Promoteur de la ve-cadherine et ses utilisations

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0012683A2 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013023801A1 (fr) 2011-08-16 2013-02-21 Seco Tools Ab Plaquette de coupe amovible à double face et outil de coupe comportant une telle plaquette

Also Published As

Publication number Publication date
CA2341754A1 (fr) 2000-03-09
ZA200101622B (en) 2001-09-17
AU5696799A (en) 2000-03-21
WO2000012683A3 (fr) 2000-06-29
JP2002523085A (ja) 2002-07-30
AU774581B2 (en) 2004-07-01
WO2000012683A9 (fr) 2000-07-27
WO2000012683A2 (fr) 2000-03-09
IL141683A0 (en) 2002-03-10

Similar Documents

Publication Publication Date Title
US6767737B1 (en) Stem cells bearing an FGF receptor on the cell surface
AU774581B2 (en) Stem cells bearing an FGF receptor on the cell surface
US8986744B2 (en) Stem cell populations and methods of use
Broxmeyer et al. Cord blood stem and progenitor cells
US5599703A (en) In vitro amplification/expansion of CD34+ stem and progenitor cells
Burger et al. Fibroblast growth factor receptor-1 is expressed by endothelial progenitor cells
Zimmerlin et al. Stromal vascular progenitors in adult human adipose tissue
Shaw et al. Hematopoietic stem cells and endothelial cell precursors express Tie-2, CD31 and CD45
US6537807B1 (en) Hematopoietic stem cells
US20150072420A1 (en) Therapeutic use of cd31 expressing cells
JPH07313150A (ja) ヒト造血幹細胞
CN102333861A (zh) 用于分离非常小的胚胎样(vsel)干细胞的方法
US7833697B2 (en) Endothelial stem cells, populations, methods of isolation and use thereof
Parmar et al. Sca+ CD34− murine side population cells are highly enriched for primitive stem cells
Chou et al. In utero transplantation of human bone marrow‐derived multipotent mesenchymal stem cells in mice
JP2008531007A (ja) ヒト造血幹細胞集団を取得する方法
Hu et al. Analysis of origin and optimization of expansion and transduction of circulating peripheral blood endothelial progenitor cells in the rhesus macaque model
Haruta et al. Role of Flk-1 in mouse hematopoietic stem cells
Yoshimoto et al. Bone marrow engraftment but limited expansion of hematopoietic cells from multipotent germline stem cells derived from neonatal mouse testis
AU780468B2 (en) Method for enhancing hematopoiesis
Fang et al. Hemangioblastic characteristics of human adipose tissue-derived adult stem cells in vivo
Cohen Regenerative potential of human CD34+ stem cells mobilized and enriched from bone marrow

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20010330

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

17Q First examination report despatched

Effective date: 20030422

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

RIN1 Information on inventor provided before grant (corrected)

Inventor name: BURGER, PATRICIA E.,9 SPANISH STEPS FERGUSON ROAD

Inventor name: WILSON, E., LYNETTE

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20050412