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WO1998000523A1 - Procede de production de compositions enrichies en cellules souches hematopoietiques et anticorps a utiliser dans ledit procede - Google Patents

Procede de production de compositions enrichies en cellules souches hematopoietiques et anticorps a utiliser dans ledit procede Download PDF

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Publication number
WO1998000523A1
WO1998000523A1 PCT/EP1997/003422 EP9703422W WO9800523A1 WO 1998000523 A1 WO1998000523 A1 WO 1998000523A1 EP 9703422 W EP9703422 W EP 9703422W WO 9800523 A1 WO9800523 A1 WO 9800523A1
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WIPO (PCT)
Prior art keywords
cells
antibody
marker
cell
stem cells
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PCT/EP1997/003422
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English (en)
Inventor
Beth L. Hill
Elen Rozler
Benjamin P. Chen
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Novartis Ag
Systemix, Inc.
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Publication date
Application filed by Novartis Ag, Systemix, Inc. filed Critical Novartis Ag
Priority to AU34407/97A priority Critical patent/AU3440797A/en
Publication of WO1998000523A1 publication Critical patent/WO1998000523A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells

Definitions

  • the field of this invention is the isolation of a population of cells enriched for human hematopoietic stem cells.
  • Mammalian hematopoietic cells provide a diverse range of physiological activities. These cells arc divided into lymphoid, myeloid and crythroid lineages.
  • the lymphoid lineage comprising B cells and T cells, provides for the production of antibodies, regulation of the cellular immune system, detection of foreign agents in the blood, detection of cells foreign to the host, and the like.
  • the myeloid lineage which includes monocytes, granulocytes, megakaryocytes as well as other cells, monitors for the presence of foreign bodies, provides protection against neoplastic cells, scavenges foreign materials, produces platelets, and the like.
  • the erythroid lineage provides the red blood cells, which act as oxygen carriers.
  • stem cells a single precursor ceil population
  • stem cells are capable of self- regeneration and can become lineage committed progenitors which are dedicated to differentiation and expansion into a specific lineage.
  • stem cells refers to hematopoietic cells and not stem cells of other cell types.
  • a pluripotent stem cell can be defined by possessing the following characteristics: (1) gives rise to progeny in all defined hematolymphoid lineages; and (2) a limiting numbers of cells are capable of fully reconstituting a seriously immunocompromised host in all blood cell types and their progenitors, including the pluripotent hematopoietic stem cell, by self-renewal.
  • a highly purified population of stem cells is necessary for a variety of in vitro experiments and in vivo indications.
  • a purified population of stem cells will allow for identification of growth factors associated with their self-regeneration.
  • growth factors associated with their self-regeneration there can be as yet undiscovered growth factors associated with: (1) the , ⁇ M , PCT EP97/03422 /00523
  • Stem cells find use in: (1) regenerating the hematopoietic system of a host deficient in any class of hematopoietic cells; (2) a host that is diseased and can be treated by removal of bone ma_ ⁇ ow, isolation of stem cells and treatment with drugs or irradiation prior to re-engraftment of stem cells; (3) producing various hematopoietic cells; (4) detecting and evaluating growth factors relevant to stem cell self- regeneration; and (5) the development of hematopoietic cell lineages and assaying for factors associated with hematopoietic development Stem cells also are important targets for gene therapy, where the inserted genes promote the health of the individual into whom the stem ceils arc transplanted.
  • stem cells can serve in the treatment of lymphoraas and leukemias, as well as other neoplastic conditions where the stem cells are purified from tumor cells in the bone marrow or peripheral blood, and reinfused into a patient after myelosuppressive or myeloablative chemotherapy.
  • myelosuppressive or myeloablative chemotherapy there have been world- ide efforts toward isolating stem cells in substantially pure or pure form.
  • Stem cells constitute only a small percentage of the total number of hematopoietic cells. Hematopoietic cells are identifiable by the presence of a variety of cell surface "markers.” Such markers can be either specific to a particular lineage or progenitor cell or can be present on more than one cell type. Currently, it is not known how many of the markers associated with differentiated cells are also present on stem cells. One marker, which was previously indicated as present solely on stem cells, CD34, is also found on a significant number of lineage committed progenitors. U.S. Pat. No.4,714,680 describes a population of cells expressing the CD34 marker. In view of the small proportion of the total number of cells in the bone marrow
  • rho 10 Decreased rhodami ⁇ e 123 (rhol23) staining of hematopoietic cells appears to correlate to stem cell potential. This so-called “rho 10 " marker is determined not by the initial dye accumulation but by an efflux process sensitive to P-glycoprotein (P-gp) inhibitors. Retention of several P-gp-transported fluorescent dyes, including rho 123, in human bone marrow cells was inversely correlated with the expression of P-gp.
  • Bone marrow cells expressing physical and antige ⁇ ic characteristics of pluripotent stem cells show high levels of P-gp expression and fluorescent dye efflux. Fractions of human bone marrow cells isolated on the basis of either increased rhol23 efflux or P- gp expression contain practically all the primitive progenitor cells of human bone marrow, including long-term culture-initiating cells (LTC-IC). Chaudhary and Roninson (1991) Cell 66:85-94.
  • the mouse stem cell has been obtained in at least highly concentrated, if not purified form, where fewer than about 30 cells obtained from bone marrow were able to reconstitute all of the lineages of the hematopoietic system of a lethally irradiated mouse.
  • Each assayed cell is multipotent for all hematopoietic lineages, while self-renewal is variable amongst these cells.
  • compositions substantially enriched for hematopoietic stem cells employ a separation regimen utilizing antibodies (hereinafter identified as ⁇ lEMl 6) specific for a unique cell smface marker (hereinafter identified as EMI 6) that is expressed on stem and progenitor cells, while being less accessible or absent on more mature cells.
  • ⁇ lEMl 6 antibodies
  • EMI 6 unique cell smface marker
  • Positive selection of stem cells with antibodies that recognize EM16 can be used in combination with selection techniques that isolate cells expressing other stem cell markers and/or techniques that isolate cells or a cell population lacking lineage- specific (LIN”) markers. Enriched populations of cells derived from these methods are also provided. /00523
  • FIG. 1 panels A through D, shows FACSTM analysis of EM 16 and rhod__rn_ne 123 staining (panel D), with isotype IgM control (panel C), of CD34 " Thy-r, viable, low side scatter gated bone marrow cells (panels A and B).
  • FIG. 2 panel A through E, shows FACS TM analysis of CD34 * bone marrow cells (panel A) staining with I-CD38 and an a ⁇ ti-Thy-1 (panel C), with IgGl isotypc control (panel B), and staining with I-CD38 and I-EM16 (panel E), with IgM isotype control (panel D).
  • the y-axis shows CD38 staining; the x-axis shows staining of the antibody being tested (Thy-1, EM16, IgM isotype or IgG isotype).
  • FIG. 3 panels A through C, shows FACS analysis of CD34* bone marrow cells (control, panel A), stained with ⁇ -EMl6 (y-axis) and Thy-1 (x-axis) (panel C), with IgM isotype control (panel B).
  • FIG 4 panels A through D, compares Thy-1 versus CD34 expression (panel B), with EM16 versus CD34 expression (panel D). Panels A & C show isotype controls.
  • the x-axis is CD34 expression; the y-axis is the antibody being tested (Thy-1 , EM16, IgM isotypc or IgG isotypc).
  • Figure 5 is a graph plotting CAFC frequency after 3 to 5 weeks in culture.
  • a hybridoma cell line producing an antibody that specifically recognizes the EM 16 marker was deposited with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, RockvjJlc, Maryland, 20852 U.S.A., on May 1, 1996, under the provisions of the Budapest Treaty for the International Recognition for the Deposit of Microorganisms for the Purposes of Patent Procedure, and has been assigned accession number HB-12100.
  • the present invention provides methods for isolating a population of hematopoietic cells highly enriched for hematopoietic stem cells expressing, or being characterized as having the EM 16 marker.
  • EM 16 the cell surface marker recognized by antibodies specific for EMI 6
  • ⁇ EM1 the cell surface marker recognized by antibodies specific for EMI 6
  • Antibodies to the EMI 6 marker bind about 50% of CD34 + cells obtained from freshly ficolled ( ⁇ ⁇ 1.077) cadaveric bone marrow and 3-4% of total mononuclear cells on average.
  • ⁇ EM16 has the following characteristics.
  • binding to CD34 cells was 24% (See
  • the SCTD-hu thymus assay indicates that EM 16+ cells have the potential to develop into T cells, indicative of T-lymphoid potential (see Table I).
  • the SCID-hu bone assay indicates that EM16 *" cells have SCTD-hu bone repopulating activity, generating B-cells and myeloid cells, indicative of primitiveness and pluripotentiality (see Table 1). Thus.
  • ⁇ EM16 can be used in place of antibodies specific to other stem cell markers e.g., CD34, to purify the majority of progenitor cells and pluripotent stem cells from heterogeneous cell populations containing hematopoietic stem cells.
  • Antibodies to EM 16 bind to a larger subset of CD34 + cells than do antibodies to Thy- 1.
  • EM 16 marker defines a population of cells which is enriched in CFU-GM, in addition to the CAFC activity, which provides a desirable bone marrow graft composition.
  • the composition obtained by selection based on the EM 16 marker is more rapidly regenerated and differentiated into mature lineages than a composition based on selection utilizing Thy-1.
  • the EM16 + CD34 + cell composition includes a majority of T cell progenitors and therefore, lymphoid progenitor activity.
  • the present invention provides methods of obtaining a composition substantially enriched in hematopoietic stem cells.
  • substantially enriched is intended to mean a population of cells, wherein greater than 65%, preferably 70%, and more preferably greater than 75%, of the cell population comprises hematopoietic stem cells characterized in being EM16 + .
  • the methods involve combining a cell population containing hematopoietic cells with an antibody that specifically recognizes and binds to EM 16 under conditions which allow the antibody to specifically bind to EM 16 and separating the cells recognized by the antibody.
  • the antibody is labeled with a detectable marker and after combining the cell population with the antibody, the antibody-bound cells are selected based on the presence of bound, labeled antibody on the cell or cells.
  • Cell populations useful in this method include, and are not limited to, cell populations obtained from bone marrow, both adult and fetal, mobilized peripheral blood (MPB) and umbilical cord blood.
  • EM 16 * hematopoietic stem cells can be isolated from any known source of hematopoietic stem cells, including, but not limited to, bone marrow, both adult and fetal, mobilized peripheral blood (MPB) and umbilical cord blood.
  • MPB mobilized peripheral blood
  • umbilical cord blood is discussed, for instance, in Issaragrishi et al. (1 95) N. End. J. Med. 332:367-369.
  • bone marrow cells can be obtained from a source of bone marrow, including but not limited to, ilium (e.g., from the hip bone via the iliac crest), tibia, femora, vertebrate, or other bone cavities.
  • Other sources of stem cells include, but are not limiied to, embryonic yolk sac, fetal liver, and fetal spleen.
  • the methods can include further enrichment or purification procedures or steps for stem cell isolation by positive selection for other stem cell specific markers. Suitable posi tive stem cell markers include, but are not limited to, CD34 + and Thy- 1 + .
  • an appropriate solution can be used to flush the bone, including, but not limited to, salt solution, conveniently supplemented with fetal calf serum (FCS) or other naturally occu ⁇ ng factors, in conjunction with an acceptable buffer at low concentration, generally from about 5-25 ⁇ r ⁇ M.
  • Convenient buffers include, but are not limited to, HEPESTM, phosphate buffers and lactate buffers. Otherwise bone marrow can be aspirated from the bone in accordance with conventional techniques. 8/00523
  • the invention also provides the antibodies useful to carry out the above method and the hybridoma cell lines that produce monoclonal antibodies with the requisite specificity.
  • the invention encompasses antibodies which specifically recognize the cell surface marker having the epitope recognized by ⁇ EM16.
  • c_EM16 encompasses any antibody or fragment thereof, either native or recombinant, chimeric synthetic or naturally-derived, which retains sufficient specificity to bind specifically to the cell surface marker having the epitope recognized by ocEM16.
  • the terms “antibody” or “antibodies” include the entire antibody and antibody fragments containing functional portions thereof.
  • antibody includes any monospecific, bispecific or chimeric compound comprised of a sufficient portion of the light chain variable region and or the heavy chain variable region of o_EM16 to effect binding to the epitope to which the whole C-EM1 antibody has binding specificity.
  • the fragments can include the variable region of at least one heavy or light chain immunoglobulin polypeptide, and include, but are not limited to, Fab fragments, F(ab') 2 fragments, and Fv fragments.
  • the monospecific domains can be attached by any method known in the art to another suitable molecule.
  • the attachment can be, for instance, chemical or by genetic engineering.
  • the ⁇ EM16 can be produced by any recombinant means known in the art.
  • recombinant antibodies include, but are not limited to, fragments produced in bacteria and non-human antibodies in which the majority of the constant regions have been replaced by human antibody constant regions.
  • humanized antibodies can be obtained by host vertebrates genetically engineered to express the recombinant antibody.
  • ⁇ EMl ⁇ includes the monoclonal antibody designated ATCC
  • HB-12100 or any monoclonal antibody orpolyclo ⁇ al antibody, that binds specifically to EM 16 in such a manner as to recognize, preferentially, hematopoietic progenitor and hematopoietic stem cells.
  • This also includes any o_EM16 having the same antigenic specificity as ATCC HB-12100. If a monoclonal or polyclonal antibody being tested specifically binds with EMI 6, then the antibody being tested and the antibodies provided by the hybridomas of this invention are equivalent. It also is possible to determine without undue experimentation, whether an antibody has the same specificity as the monoclonal antibody of this invention by determining whether the antibody being tested prevents a monoclonal antibody of this invention from binding EMI 6.
  • the antibody being tested competes with a monoclonal antibody of the invention as shown by a decrease in binding by the monoclonal antibody of this invention, then it is likely that the two antibodies bind to the same or a closely related epitope.
  • the antibody being tested binds all EM16 + cells and shows the same staining patterns as described in the Examples herein, the antibody being tested likely binds a different epitope of the EM 16 antigen and, therefore is also useful in selecting the EMI 6 + cells of the present inventio ⁇ .
  • the EMI 6 antibodies or functional portions thereof are obtained by methods known in the art for production of antibodies. The actual methods used are described in the Examples presented herein although any method known in the art of antibody production can be used.
  • Such methods include, but arc not limited to, separating B cells with cell-surface antibodies of the desired specificity, cloning the DNA expressing the variable regions of the light and heavy chains and expressing the recombinant genes in a suitable host cell.
  • Standard monoclonal antibody generation techniques can be used wherein the antibodies are obtained from immortalized antibody-producing hybridoma cells.
  • These hybridomas can be produced by immunizing animals with stem cells, and fusing B lymphocytes from the immunized animals, preferably isolated from the immunized host spleen, with compatible immortalized cells, preferably a B cell myeloma. The resulting hybridomas are screened for their binding specificity to EMI 6 as described above.
  • the hybridoma or continuous cell lines producing these antibodies are further provided by this in ention.
  • the antibodies can be conjugated to other compounds including, but not limited to, enzymes, magnetic beads, colloidal magnetic beads, haptens, fluorochromes, metal compounds, radioactive compounds or drugs.
  • the enzymes that can be conjugated to the antibodies include, but are not limited to, alkaline phosphatase, 8/00523
  • the fluorochromes that can be conjugated to the antibodies include, but are not limited to, fluorescein isothiocyanate, tetr___ie_hylrho_ _mine isothiocyanate, phycoerythrin, allophycocyanins and Texas Red.
  • fluorescein isothiocyanate tetr___ie_hylrho_ _mine isothiocyanate
  • phycoerythrin allophycocyanins and Texas Red.
  • the metal compounds that can be conjugated to the antibodies include, but are not limited to, ferritin, colloidal gold, and particularly, colloidal superparamagnetic beads.
  • the haptens that can be conjugated to the antibodies include, but are not limited to, biotin, digoxigenin, oxazalone, and nitrophenol.
  • the radioactive compounds that can be conjugated or incorporated into the antibodies are known to the art, and include but are not limited to technetium 99m ("Tc), 125 1 and amino acids comprising any radionuclides, including, but not limited to, ,4 C, 3 H and 3 S.
  • the cell population is initially subject to negative selection techniques to remove those cells that express lineage specific markers and retain those cells which are lineage negative (“LIN " "”).
  • LIN " cells generally refer to cells which lack markers such as those associated with T cells (such as CD2, 3, 4 and 8), B cells (such as CDIO, 19 and 20), myeloid cells (such as CD14, 15, 16 and 33), natural killer (“NK”) cells (such as CD2, 16 and 56), RBC (such as glycophorin A), megakaryocytes (CD41), mast cells, eosinoph ⁇ s or basophils.
  • T cells such as CD2, 3, 4 and 8
  • B cells such as CDIO, 19 and 20
  • myeloid cells such as CD14, 15, 16 and 33
  • natural killer (“NK”) cells such as CD2, 16 and 56
  • RBC such as glycophorin A
  • megakaryocytes CD41
  • mast cells eosinoph ⁇ s or basophils.
  • the lineage specific markers include, but are not limited to, at least one of CD2, CD14, CDI5, CD16, CD19, CD20, CD38, HLA-DR and CD71; more preferably, at least CD14 and CD 15.
  • LIN refers to a cell population selected based on the lack of expression of at least one lineage specific marker.
  • a highly enriched composition can be obtained by selective isolation of cells that are CD34 EM16UN " .
  • Monoclonal antibodies are particularly useful for identifying markers associated with particular cell lineages and/or stages of differentiation.
  • the antibodies can be attached to a solid support to allow for crude separation.
  • separation techniques employed should ma imize the retention of viability of the fraction to be collected.
  • Various techniques of different efficacy can be employed to obtain "relatively crude” separations. Such separations are up to 10%, usually not more than about 5%, preferably not more than about 1%, of the total cells present not having the marker can remain with the cell population to be retained.
  • the particular technique employed will depend upon efficiency of separation, associated cytotoxicity, ease and speed of performance, and necessity for sophisticated equipment and/or technical skill.
  • Procedures for separation can include, but are not limited to, physical separation, magnetic separation, using antibody-coated magnetic beads, affinity chromatography, cytotoxic agents joined to a monoclonal antibody or used in conjunction with a monoclonal antibody, including, but not limited to, complement and cytotoxins, and "panning" with antibody attached to a solid matrix, e.g., plate, elutriation or any other convenient technique.
  • the use of physical separation techniques include, but are not limited to, those based on difrerenccs in physical (density gradient centrifugation and counter-flow centrifugal elutriation), cell surface (lectin and antibody affinity), and vital staining properties (mitochondria-binding dye rho 123 and DNA-binding dye Hoechst 33342). These procedures are well known to those of skill in this art. Techniques providing accurate separation include, but are not limited to, flow cytometry, which can have varying degrees of sophistication, e.g., a plurality of color channels, low angle and obtuse light scattering detecting channels, impedance channels, etc.
  • Cells also can be selected by flow cytometry based on light scatter characteristics, where stem cells are selected based on low side scatter and low to medium forward scatter profiles. Cytospin preparations show the enriched stem cells to have a size between mature lymphoid cells and mature gra ⁇ ulocytes.
  • ⁇ EM16 in a first separation, typically starting with about 1 x 10 8"9 , preferably at about 5 x 10 s"9 cells, ⁇ EM16 can be labeled with a first fluorochrome. while the antibodies for the various dedicated lineages, can be conjugated to a fluorochrome with different and distinguishable spectral characteristics from the first fluorochrome. While each of the lineages can be separated in more than one "separation" step, desirably (he lineages are separated at the same time as one is positively selecting forEMl ⁇ and/or other stem cell markers, e.g. (CD34 + , Thy-l*).
  • stem cell markers e.g. (CD34 + , Thy-l*).
  • the cells can be selected and isolated from dead cells, by employing dyes associated with dead cells (including but not limited to, propidium iodide (PI)).
  • the cells are collected in a medium comprising 2% FCS. hile it is believed that the particular order of separation is not critical to this invention, the following order is preferred.
  • the cells are initially separated by a coarse separation, e.g., density gradient ce ⁇ trifugation, elutriation, or chemical depletion, followed by a fine separation, e.g., magnetically stabilized fluidized bed (U.S. Patent No. 5,409,813) or flow cytometry with positive selection with ⁇ EM16.
  • the cells obtained as described above can be used immediately or frozen at liquid nitrogen temperatures and stored for long periods of time, being thawed and capable of being reused.
  • the cells usually will be stored in 10% DMSO. 50% fetal calf serum (FCS), 40% RPMI 1640 medium. Once thawed, the cells can be expanded by use of growth factors and/or stromal cells associated with stem cell proliferation and differentiation.
  • a composition highly enriched or containing a substantially purified population of stem cells is provided.
  • the results presented herein indicate that antibodies to EM 16 recognize and bind with high specificity to a cell surface antigen found on human hematopoietic cells, and exposed to a high degree on hematopoietic stem cells. This specificity can be used to isolate and purify these stem cells.
  • Such a composition also has utility in reconstituting hematopoietic systems and in studying various parameters of hematopoietic cells.
  • the cells generated fromEM16 + cells and obtained from these cultures give rise to B cells.
  • T cells, erythroid cells and myelomonocytic cells in the in vivo assays described below.
  • In vitro analyses for hematopoietic progenitor cells have also been reported by W itlock and Witte (1982) Proc. Natl. Acad. Sci. USA 79:3608-3612; and Whitlock et al. (1987) £eU 48:1009-1021.
  • the epitope bound by EM 16 is novel as determined by comparison with a large database (MLDVA V, (1993)) describing staining patterns of hundreds of antibodies to hematopoietic cells.
  • MLDVA V large database
  • Several cell lines as well as peripheral blood lymphocytes (PBL) and HUVEC have been tested for the expression of the epitope bound by EMI 6 (See Table 3).
  • the patterns of epitope expression observed indicate that EM16 binds to an epitope that is distinct from those bound by monoclonal antibodies to CD34,or Thy-1 , and from any other antigens that have been described on hematopoietic stem cells. No binding to peripheral blood mononuclear cells is observed. The level of epitope expression is modest on both bone marrow and other positively stained cells tested. The treatment of bone marrow cells with neuraminadase or glycoprotease does not diminish the binding of these monoclonal antibodies indicating that carbohydrate components are not a major aspects of the epitope defined by the monoclonal antibodies.
  • EM16 + population contains all cells that are CD34 + /Thy-r, including both rhol23 l0 and rho 123 ⁇ subsets.
  • This population of cells has been shown to include the most primitive pluripotential hematopoietic stem cells.
  • EMI 6 is expressed on the subset of CD34 + cells that contains virtually all primitive hematopoietic stem cells and D ⁇ M16 enables the purification and isolation of this subset of hematopoietic stem cells.
  • compositions having greater than 90%, usually greater than about 95% of EM 16 * cells can be achieved in this manner.
  • the desired stem cells can be further enriched by selection for UN " and or CD34* and or Thy-I + and/or rho 10 , or combinations of the markers described herein.
  • the long- term culture activity is enriched in the EMI 6 * fraction compared to EM16 .
  • the long- term culture initiating cell (LTCIC) assay or the CAFC (cobblestone area forming cell assay) which shows a direct correlation is currently the best in vitro assay for stem cell activity.
  • the EM 16 + cells will generate both B and myeloid cells in long-term cultures.
  • the methods can be used as a diagnostic assay for the number of stem cells in a sample of hematopoietic cells.
  • periodic leukapheresis samples may be tested forEMl ⁇ expression, where increased EM16-positive cells in the sample indicate increased stem cell content and, therefore, optimal time to harvest blood samples having maximal stem cell content.
  • the cells and antibody are combined under conditions sufficient to allow specific binding of the antibody to EM 16 and the EM16 + cells are then quanti ⁇ ated.
  • the EM 16* cells can be isolated or further purified.
  • the grafts are analyzed for the presence of donor HLA + lymphoid and myeloid progeny.
  • EM 16 * ccDs are found to engraft in the SCTD-hu bone model, and to give rise to myeloid and lymphoid progeny.
  • fetal thymus is isolated and cultured from 4-7 days at about 25°C, so as to deplete substantially the lymphoid population.
  • the cells to be tested for T cell activity are then microi ⁇ jccted into the thymus tissue, where the HLA of the population which is injected is mismatched with the HLA of the thymus cells.
  • the thymus tissue can then be transplanted into a scid/scid mouse as described in US Patent No. 5,147,784, particularly transplanting under the kidney capsule.
  • a sorted population of EM 16* cells can be microinjccted into HLA mismatched thymus fragments.
  • assays of the thymus fragments injected with EM16* cells show that EM16 cells can generate donor- derived T cells.
  • composition comprising a substantially enriched population of human hematopoietic stem cells, wherein the cells express the Em 16 marker.
  • compositions obtained by the methods also can be used to fully reconstitute an immunocompromised host such as an irradiated host and/or a host subject to chemotherapy; or as a source of cells for specific lineages, by providing for their maturation, proliferation and differentiation into one or more selected lineages by employing a variety of factors, including, but not limited to, erythrop ⁇ ietin, colony stimulating factors, e.g., GM-CSF, G-CSF, or M-CSF, interleukins, e.g., IL-1, -2, -3, - 4, -5, -6, -7, -8, etc., or the like, or stromal cells associated with the stem cells becoming committed to a particular lineage, or with their proliferation, maturation and differentiation.
  • an immunocompromised host such as an irradiated host and/or a host subject to chemotherapy
  • M-CSF e.g., interleukins, e.g., IL-1, -2, -3
  • the EM 16 * cells also can be used in the isolation and evaluation of factors associated with the differentiation and maturation of hematopoietic cells.
  • the invention encompasses the use of EMI 6 * cells in assays to determine the activity of media, such as conditioned media, or to evaluate fluids for cell growth activity, involvement with dedication of particular lineages, or the like.
  • the invention also encompasses treatment of diseases or amelioration of symptoms associated with disease, amenable to gene transfer into EM 16 + cell populations obtained by the methods disclosed herein.
  • gene therapy or “gene transfer” is defined as the insertion of genes into cells for the purpose of medicinal therapy.
  • gene therapy or “gene transfer” is defined as the insertion of genes into cells for the purpose of medicinal therapy.
  • stem cell genetic insertion There are many applications of gene therapy, particularly via stem cell genetic insertion, and thus are well known and have been extensively reviewed.
  • Gene therapy using HSCs is useful to treat a genetic abnormality in lymphoid and myeloid cells that results generally in the production of a defective protein or abnormal levels of expression of the gene.
  • these diseases the introduction of a normal copy or functional homolog of the defective gene and the production of even small amounts of the missing gene product would have a beneficial effect.
  • overexpression of the gene product would not be expected to have deleterious effects.
  • diseases for which gene transfer into HSCs is potentially useful These diseases generally include bone marrow disorders, erythroid cell defects, metabolic disorders and the like.
  • Hematopoietic stem cell gene therapy is beneficial for the treatment of genetic disorders of blood cells such as a and ⁇ -thalassemia, sickle cell anemia and hemophilia A and B in which the globin gene or clotting factor gene is defective.
  • SCUDS severe combined immunodeficiency disease
  • ADA adenosine deaminase
  • Such patients are ideal candidates to receive gene therapy by introducing the ADA gene into their HSCs instead of the patient's lymphocytes as done in the past.
  • Other diseases include chronic granulomatosis where the neutrophils express a defective cytochrome b and Gaucher disease resulting from an abnormal glucocerebrosidase gene product in macrophages.
  • the retroviral vector can carry a gene that encodes, for eg., a toxin or an apoptosis inducer effective to specifically kill the cancerous cells.
  • Specific killing of tumor cells can also be accomplished by introducing a suicide gene to cancerous hematopoietic cells under conditions that only the tumor cells express the suicide gene.
  • the suicide gene product confers lethal sensitivity to the cells by converting a normally nontoxic drug to a toxic derivative.
  • the enzyme cytosine deaminase converts the nontoxic substance 5'-fluorocytosine to a toxic derivative, 5-fluorouracil (Mullen et al.(1992) Proc. natl. Acad. Sci.
  • Tumor-specific lymphocytes can be geneticaDy modified for example, to locally deliver gene products with anti-tumor activity to sites of the tumor to circumvent the toxicity associated with the systemic delivery of these gene products.
  • a gene therapy approach can also be applied to render bone marrow cells resistant to the toxic effects of chemotherapy.
  • Gene therapy can also be used to prevent or combat viral infections such as
  • HSCs can be genetically modified to render them resistant to infection by HTV.
  • One approach is to inhibit viral gene expression specifically by using antisense RNA or by subverting existing viral regulatory pathways.
  • Antisense RNAs complementary to retroviral RNAs have been shown to inhibit the rephcation of a number of retroviruses (To et al. (1986) Mol. Cell. Biol. 6:4758-4762; Hopper and Coffin (1988) ImGluzman and Hughes (eds.), Current Communications in Biology. Viral Vectors. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. pp 139-145) including HTV * (Rhodes and James (1991) AIDS 5:145-151) and HTLV-1 (von Reuden et al. (1991) J. Virol. 63:677-682).
  • the therapeutic gene can encode, eg., aB or T cell signalling molecule capable of reconstituting the normal apoptotic signal that results in the death and elimination of autoreactive cells.
  • Diseases other than those associated with hematopoietic cells can also be treated by genetic modification, where the disease is related to the lack of a particular secreted product including, but not limited to, hormones, enzymes, interferons, growth factors, or the like.
  • a particular secreted product including, but not limited to, hormones, enzymes, interferons, growth factors, or the like.
  • inducible production of the deficient protein can be achieved, so that production of the protein will parallel natural production, even though production will be in a different cell type from the cell type that normally produces such protein.
  • the therapeutic gene is transduced into the cell by any number of methods, e.g., using adenoviral vectors, adeno-associated viral vectors, and hposomes.
  • Adenoviral, vaccinia, canaiyp ⁇ x viral, canonic hposomes and plasmids are useful to achieve transient expression.
  • retroviral vectors such as, Moloney Murine Leukemia Virus (MoMLV), Myeloproliferative Sarcoma Virus (MPSV), Murine
  • Embryonic Stem Cell Virus and adenovirus, are useful to achieve stable and sustained expression of the transferred gene or gene product.
  • Direct physical methods also arc available. These methods include the use of the "gene gun” or calcium phosphate transfection method. As noted above, any method of gene transfer is encompassed by this invention.
  • viral vector is defined as a recombinantly produced virus or viral particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro.
  • a vector construct refers to the polynucleotide comprising the retroviral genome or part thereof, and a therapeutic gene.
  • retroviral mediated gene transfer or “retroviral transduction” carries the same meaning and refers to the process by which a gene or nucleic acid sequences are stably transferred into the host cell by virtue of the virus entering the cell and integrating its genome into the host cell genome.
  • retroviral vector refers to a viral particle capable of introducing exogenous nucleic acid into a cell through a viral or viral-like entry mechanism. Retroviruses carry their genetic information in the form of RNA; however, once the virus infects a cell, the RNA is reverse-transcribed into the DNA form which integrates into the genomic DNA of the infected cell. The integrated DNA form is called a provirus.
  • Retroviral vectors useful in the methods of this invention are produced recombinantly by procedures already taught in the art.
  • WO 94/29438 describes the construction of retroviral packaging plasmids and packaging cell lines.
  • the retroviral vectors useful in the methods of this invention are capable of infecting HSCs.
  • the techniques used to construct vectors, a ⁇ cl transfect and infect cells are widely practiced in the art.
  • retroviral vectors are those derived from murine, avian or primate retroviruses.
  • Retroviral vectors based on the Moloney (Mo) murine leukemia virus (MuLV) are the most commonly used because of the availability of retroviral variants that efficiently infect human cells.
  • Suitable vectors include those based on the Gibbon Ape Leukemia Virus (GALV) or HTV.
  • GLV Gibbon Ape Leukemia Virus
  • HTV High Speed Downlink Transcription Tube
  • the viral gag, pol and env sequences are removed from the virus, creating room for insertion of foreign DNA sequences. Genes encoded by the foreign DNA are usually expressed under the control of the strong viral promoter in the LTR. Such a construct can be packed into viral particles efficiently if the gag, pol and env functions arc provided in trans by a packaging cell line.
  • the gag-pol and env proteins produced by the cell assemble with the vector RNA to produce infectious virions that are secreted into the culture medium.
  • the virus thus produced can infect and integrate into the DNA of the target cell, but does not produce infectious viral particles since it is lacking essential packaging sequences.
  • Most of the packaging cell lines currently in use have been transfected with separate plasmids, each containing one of the necessary coding sequences, so that multiple recombination events arc necessary before a replication competent virus can be produced.
  • RNA produced from the recombinant virus is packaged.
  • the virus stock released from the packaging cells thus contains only recombinant virus.
  • the range of host cells that may be infected by a retrovirus or retroviral vector is determined by the viral envelope protein.
  • the recombinant virus can be used to infect virtually any other cell type recognized by the env protein provided by the packaging cell, resulting in the integration of the viral genome in the transduced cell and the stable production of the foreign gene product.
  • murine ecotropic env of MoMLV allows infection of rodent cells
  • amphotropic env allows infection of rodent, avian and some primate cells, including human cells.
  • Amphotropic packaging cell lines for use with MoMLV systems are known in the art and commercially available and include, but are not limited to, PA12 and PA317. Miller et al. (1985) Mol. Cell. Biol. 5:431-437; Miller et al. (1986) Mol. CeU. Biol. 6:2895-
  • Xenotropic vector systems exist which also allow infection of human cells.
  • VSV-G vesicular stomatitis virus
  • the vectors will contain at least two heterologous genes or gene sequences: (i) the therapeutic gene to be transferred; and (ii) a marker gene that enables tracking of infected cells.
  • therapeutic gene can be an entire gene or only the functionally active fragment of the gene capable of compensating for the deficiency in the patient that arises from the defective endogenous gene.
  • Therapeutic gene also encompasses antisense oligo ⁇ ucleotides or genes useful for antisense suppression and ribozymes for ribozyme-mediated therapy.
  • Therapeutic genes that encode dominant inhibitory oligonucleotides and peptides as weD as genes that encode regulatory proteins and oligonucleotides also are encompassed by this invention.
  • gene therapy will involve the transfer of a single therapeutic gene although more than one gene may be necessary for the treatment of particular diseases.
  • the therapeutic gene is a normal, i.e. wild-type, copy of the defective gene or a functional homolog.
  • the therapeutic gene is a dominant inhibiting mutant of the wild-type.
  • the therapeutic gene can include the regulatory and untranslated sequences.
  • the therapeutic gene will generally be of human origin although genes from other closely related species that exhibit high homology and biologically identical or equivalent function in humans may be used, if the gene product does not induce an adverse immune reaction in the recipient.
  • a primate insulin gene whose gene product is capable of converting glucose to glycogen in humans would be considered a functional equivalent of the human gene.
  • the therapeutic gene suitable for use in treatment will vary with the disease. For example, a suitable therapeutic gene for treating sickle ceil anemia is a normal copy of the ⁇ -globin gene. A suitable therapeutic gene for treating SCTD is the normal ADA gene.
  • Nucleotide sequences for the therapeutic gene will generally be known in the art or can be obtained from various sequence databases such as GenBank.
  • the therapeutic gene itself will generally be available or can be isolated and cloned using the polymerase chain reaction PCR (Perkin-Eimer) and other standard recombinant techniques.
  • the skilled artisan will readily recognize that any therapeutic gene can be excised as a compatible restriction fragment and placed in a vector in such a manner as to allow proper expression of the therapeutic gene in hemaotpoietic cells.
  • a marker gene can be included in the vector for the purpose of monitoring successful transduction and for selection of cells into which the DNA has been integrated, as against cells which have not integrated the DNA construct.
  • Various marker genes include, but are not limited to, antibiotic resistance markers, such as resistance to G4] 8 or hygromycin. Less conveniently, negative selection may be used, including, but not limited to, where the marker is the HSV-tk gene, which will make the cells sensitive to agents such as acyclovir and gancyclovir.
  • selections could be accomplished by employment of a stable cell surface marker to select for transgene expressing stem cells by FACSTM sorting.
  • the NcoR (neomycin /G418 resistance) gene is commonly used but any convenient marker gene whose sequences arc not already present in the recipient cell, can be used.
  • the viral vector can be modified to incorporate chimeric envelope proteins or nonviral membrane proteins into retroviral particles to improve particle stability and expand the host range or to permit cell type-specific targeting during infection.
  • the production of retroviral vectors that have altered host range is taught, for example, in WO 92/14829 and WO 93/14188.
  • Retroviral vectors that can target specific cell types in vivo are also taught, for example, in Kasahar et al. (1994) Science 266:1373-1376. Kasahara et al.
  • MoMLV Moloney leukemia virus
  • EPO human erythropoieti ⁇
  • This hybrid virus shows tissue tropism for human red blood progenitor cells that bear the receptor for EPO, and is therefore useful in gene therapy of sickle cell anemia and thalassemia.
  • Retroviral vectors capable of specifically targeting infection of HSCs are preferred for in vivo gene therapy.
  • the viral constructs can be prepared in a variety of conventional ways. Numerous vectors are now available which provide the desired features, such as long terminal repeats, marker genes, and restriction sites, which may be further modified by techniques known in the art.
  • the constructs may encode a signal peptidc sequence to ensure that genes encoding cell surface or secreted proteins are properly processed post-tra ⁇ slationally and expressed on the cell surface if appropriate.
  • the foreign gene(s) is under the control of a cell specific promoter.
  • the introduced gene may be put under the control of a promoter that will cause the gene to be expressed constitutively, only under specific physiologic conditions, or in particular cell types.
  • the retroviral LTR (long terminal repeat) is active in most hematopoietic cells in vivo and will generally be relied upon for transcription of the inserted sequences and their constitutive expression (Ohashi et al. (1992) Proc. Natl. Acad. Sci. 89: 11332; Correll et al. (1992) Blood 80:331).
  • Other suitable promoters include the human cytomegalovirus (CMV) immediate early promoter and the U3 region promoter of the Moloney Murine Sarcoma Vims (MMSV), Rous Sarcoma Virus (RS V) or Spleen
  • SFFV Focus Forming Virus
  • promoters examples include Granzyme A for expression in T-cells and NK cells, the CD34 promoter for expression in stem and progenitor cells, the CD8 promoter for expression in cytotoxic T-cells, and the C 1 lb promoter for expression in myeloid cells.
  • I ⁇ ducible promoters may be used for gene expression under certain physiologic conditions.
  • an elcctrophile response element may be used to induce expression of a chemoresistance gene in response to electrophilic molecules.
  • the therapeutic benefit may be further increased by targeting the gene product to the appropriate cellular location, for example the nucleus, by attaching the appropriate localizing sequences.
  • the vector construct is introduced into a packaging cell line which will generate infectious virions.
  • Packaging cell lines capable of generating high titers of replication-defective recombinant viruses are known in the art, see for example, WO
  • Viral particles are harvested from the cell supernatant and purified for in vivo infection using methods known in the art such as by filtration of supematants 48 hours post transfection.
  • the viral titer is determined by infection of a constant number of appropriate cells (depending on the retrovirus) with titrations of viral supematants.
  • the transduction efficiency can be assayed 48 hours later by both FACS and Southern blotting.
  • PCR can be performed to detect the marker gene or other virally transduced sequences. Generally, periodic blood samples are taken and PCR conveniently performed using eg. NeoR probes if the
  • NeoR gene is used as marker.
  • the presence of virally transduced sequences in bone marrow cells or mature hemotopoietic cells is evidence of successful reconstitution by the transduced HSCs.
  • PCR techniques and reagents are well known in the art, See, generally, PCR Protocols. A Guide to Methods and Applications. Innis, Gelfand,
  • HSCs are harvested from the bone marrow or peripheral blood of the donor and the ceil population is enriched for EMI 6 + .
  • the cell population is then infected with the retroviral vector carrying a suitable therapeutic gene.
  • mice C57 B1 mice were tolerized to CD34" bone marrow cells by injection with cyclophospha ide (200 mg/kg) 24 and 48 hours after i.p. injection with 10 7 CD34-bone marrow cells. After 4 weeks, this tolerization regimen was repeated. Starting approximately 1 month later, mice were immunized i.p. with CD34 + cells (4 x 10 6 cells/mouse) or a stem cell enriched subset of CD34 * cells (e.g., CD34 ⁇
  • CD38 " 0.5 - 1 x 10 6 cells/mouse
  • Eusj_on to create hybridomas Splenocytes from immuiiized mice were fused to the myeloma partner P3xAg8.653 (ATCC) using standard PEG mediated fusion as described in Galfre et al. (1977) Nature 266:550-552). Hybridomas from a single spleen were plated into 19 x 96-weIl tissue culture plates in RPMI + 10% FCSTM containing HATTM to kill non-hybridoma cells. Fourteen days after fusion, supematants from the hybridoma wells containing secreted antibodies were harvested for testing.
  • hybridomas secreting the EMI 6 mAb was subcloned three to four times by limiting dilution in the presence of Hybridoma Enhancing Supplement (Sigma) to ensure monoclonality.
  • Cadaveric Bone Marrow and Mobilized Peripheral Blood Cadaveric bone marrow cell suspensions derived from multi-organ donor vertebral bodies were obtained from Northwest Tissue Center (Seattle, WA). Patient peripheral blood samples were obtained after informed consent and chemothcrapeutic regimens designed to mobilize primitive hematopoietic cells into the periphery. Multiple myeloma patients were mobilized with a high dose of cyclophosphamide and GM-CSF according to standard techniques.
  • Non-Hodgkins lymphoma (NHL) patients were mobilized with VP16 and G-CSF. Normal, healthy, volunteer donors were mobilized with G-CSF. Cells were separated over IsoPrep (Robbins Scientific, Sunn vale, CA) harvesting the low density mononuclear cells ( ⁇ ⁇ 1.068 g mL for cadaveric bone marrow, ⁇ ⁇ 1.077 g/mL for peripheral blood) and were further stained with antibodies for FACSTM and analysis.
  • IsoPrep Robots Scientific, Sunn vale, CA
  • Dulbecco's modified phosphate buffered saline (Ca** and Mg 4" * free) supplemented with 2% fetal bovine scrum, 10 mM HEPESTM, 10 U/mL heparin, and 1 mg/mL human gamma globulin (GamimuneTM, Miles, Elkhart, IN) was used.
  • Cells were incubated at 1 x 10 7 /mL in EM 16 hybridoma supernatant diluted 1:2 in buffer for 30 minutes on ice. Cells were washed and EM 16 binding was detected by adding PE- labeled rabbit anti-mouse IgM (Zymed, 1/100 dilution) and incubating for 30 minutes on ice.
  • Sort gates were established to collect cells that were sulforhodamine positive (CD34 + ), propidium iodide*" (viable). These cells were further divided into subsets based upon the level of PE staining (EM16 + and EM16 " ).
  • cells were stained at 4°C with EM16 or control IgM (detected with PE-labeled rabbit anti-mouse IgM, followed by PE-labeled goat anti-rabbit IgG, both from Zymed and diluted 1/100, FL2), anti-Thy-1 (GM201, detected with Texas Red-labeled goat anti-mouse Igd, FL5) and anti-CD34 (Tuk 3, Cy5-labeled FL4) and propidium iodide (FL3). Rhodamine staining is detected in the FL1 channel.
  • Figure 1 shows a FACScanTM analysis of bone marrow cells gated on CD34 " Thy- (Panel A), live, low side scatter events (Panel B), stained with an IgM isotype control (Panel C) or EM 16 (Panel D) and rhoda nine 123 (Panel D).
  • the results show that EM16 stains a population containing nearly all CD34 4 Thy-l 'cells, which are known to contain stem cells, including both rhodamine 10 and rhodamine' 1 ' subsets.
  • Figure 2 shows the staining of bone marrow cells with either Thy-1 or EM 16 (Panels C and E, respectively), with isotype controls (Panels B and D), of CD34* gated cells (Panel A). Staining with either Thy-1 or EM 16 antibody is shown on the x-axis, and CD38 antibody staining is shown on the y-axis.
  • FIG. 1 shows the results of EMI , like Thy-l, stains nearly all CD34 D38 " cells, which is the population that includes stem cells.
  • Figure 3 shows the staining of EM16 and Thy-l on CD34 + gated cells.
  • Panel A is CD34/Tby-1 staining alone.
  • Panel B is with an IgM isotype control.
  • Panel C is CD34/EM16 staining. The results show that EM 16 stains more CD34 + cells than does
  • FIG. 4 compares EMI 6 versus CD34 staining (Panel D) to Thy-l versus CD34 staining (Panel B) of bone marrow, with isotype controls (Panels A and C).
  • EM 16 binds to slightly more than half of the CD34 * population, similar to Thy-l , and EM 16 stains very few CD34 * cells.
  • EMI 6 binds 50% of CD34* cells from bone marrow.
  • SCID-hu thymus assay Sorted cell populations were microinjected into depleted fetal thyraic pieces and implanted under the kidney capsule of SCID-hu mice as described in Galy, et al. 1994 Blood 84: 104-110. Six weeks after implantation, thymic pieces were recovered and analyzed for the presence of T cell progeny. Grafts were scored as positive if ⁇ l % thymocytes were donor-derived. Data shown in Table 1 demonstrate that the EM 16 * population contains the T-cell potential.
  • SCID-hu bone assay Sotted cell populations were injected into freshly irradiated fetal bone pieces that had been implanted subcutaneously into SCID-hu mice as described in Kyoizumi, et al. (1992) filfiod 79:1704-1711. Eight weeks after injection the bone grafts were analyzed for myeloid and lymphoid progeny. Grafts were scored as positive for donor cell engraftment if ⁇ 1% of the cells harvested from the graft were donor-derived. Results are shown in Table 1.
  • Grafts reconstituted with CD34 " ⁇ M 16 + contained from 4-12% CD33 * cells indicative of myeloid development and from 4-36% CD19 + cells indicative of lymphoid development, and 1-8% CD34* cells indicative of primitiveness of the engrafting population.
  • Methylcellulose assay Sorted cell populations were plated into methylcellulose cultures to determine colony forming cell activity as described in Brandt, et al. (1992) Blood 79:634-641. Added growth factors were kit ligand (100 ng/ml), erythropoietin (2 U/ml), GM-CSF (10 ng/ml) and IL-3 (10 ng ml). The data in Table 1 show that the EM16 + population contains nearly all day 14 CFU-GM and CFU-Mix, while both the
  • EM16 * and EM16 " populations contain similar numbers of BFU-E.

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Abstract

L'invention concerne des procédés de production de compositions sensiblement enrichies en cellules souches hématopoiétiques, ainsi que des compositions obtenues par la mise en oeuvre desdits procédés. Lesdits procédés consistent en une technique de séparation au moyen d'anticorps qui reconnaissent spécifiquement un nouveau marqueur des cellules souches hématopoiétiques, désigné sous le nom de EM16, et le fixent. L'invention concerne également des anticorps monoclonaux dirigés contre ledit marqueur, ainsi que les lignées cellulaires produisant ces anticorps monoclonaux.
PCT/EP1997/003422 1996-07-01 1997-07-01 Procede de production de compositions enrichies en cellules souches hematopoietiques et anticorps a utiliser dans ledit procede WO1998000523A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6632620B1 (en) 2000-06-22 2003-10-14 Andrew N. Makarovskiy Compositions for identification and isolation of stem cells
EP1270593A3 (fr) * 2001-06-19 2004-01-21 Kaneka Corporation L'antigène HSCA-3 et anticorps monoclonal qui reconnait cet antigène
US6852533B1 (en) 1998-01-23 2005-02-08 Cornell Research Foundation, Inc. Purified populations of stem cells
CN100459413C (zh) * 2002-01-24 2009-02-04 高通股份有限公司 低if或零if接收机中用于i-q失配补偿的装置和方法

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995003693A1 (fr) * 1993-07-29 1995-02-09 Systemix, Inc. Nouveau marqueur de cellules souche
WO1996015229A1 (fr) * 1994-11-14 1996-05-23 Systemix, Inc. Procedes d'obtention de compositions enrichies en cellules souches hematopoietiques, compositions derivees et leurs procedes d'utilisation
WO1996040874A1 (fr) * 1995-06-07 1996-12-19 Novartis Ag Procedes d'obtention de compositions enrichies en cellules souches hematopoietiques et anticorps utilises a cet effet
WO1996040875A1 (fr) * 1995-06-07 1996-12-19 Novartis Ag Procedes d'obtention de compositions enrichies en cellules souches hematopoietiques et anticorps utilises a cet effet

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995003693A1 (fr) * 1993-07-29 1995-02-09 Systemix, Inc. Nouveau marqueur de cellules souche
WO1996015229A1 (fr) * 1994-11-14 1996-05-23 Systemix, Inc. Procedes d'obtention de compositions enrichies en cellules souches hematopoietiques, compositions derivees et leurs procedes d'utilisation
WO1996040874A1 (fr) * 1995-06-07 1996-12-19 Novartis Ag Procedes d'obtention de compositions enrichies en cellules souches hematopoietiques et anticorps utilises a cet effet
WO1996040875A1 (fr) * 1995-06-07 1996-12-19 Novartis Ag Procedes d'obtention de compositions enrichies en cellules souches hematopoietiques et anticorps utilises a cet effet

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6852533B1 (en) 1998-01-23 2005-02-08 Cornell Research Foundation, Inc. Purified populations of stem cells
US6632620B1 (en) 2000-06-22 2003-10-14 Andrew N. Makarovskiy Compositions for identification and isolation of stem cells
US7223549B2 (en) 2000-06-22 2007-05-29 Andrew N. Marakovskiy Compositions for identification and isolation of stem cells
EP1270593A3 (fr) * 2001-06-19 2004-01-21 Kaneka Corporation L'antigène HSCA-3 et anticorps monoclonal qui reconnait cet antigène
US7067314B2 (en) 2001-06-19 2006-06-27 Kaneka Corporation Monoclonal antibody, its immunoreactive fragment and hybridoma
CN100459413C (zh) * 2002-01-24 2009-02-04 高通股份有限公司 低if或零if接收机中用于i-q失配补偿的装置和方法

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