EP0695346A1 - Population de cellules souches erythroides humaines - Google Patents

Population de cellules souches erythroides humaines

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Publication number
EP0695346A1
EP0695346A1 EP94913419A EP94913419A EP0695346A1 EP 0695346 A1 EP0695346 A1 EP 0695346A1 EP 94913419 A EP94913419 A EP 94913419A EP 94913419 A EP94913419 A EP 94913419A EP 0695346 A1 EP0695346 A1 EP 0695346A1
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EP
European Patent Office
Prior art keywords
cells
population
progenitor cells
cell
erythroid progenitor
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Withdrawn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
EP94913419A
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German (de)
English (en)
Inventor
James G. Bender
Dennis E. Van Epps
Kristen L. Unverzagt
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Baxter International Inc
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Baxter International Inc
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Application filed by Baxter International Inc filed Critical Baxter International Inc
Publication of EP0695346A1 publication Critical patent/EP0695346A1/fr
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Classifications

    • 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
    • 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
    • A61P7/06—Antianaemics
    • 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
    • C12N5/0641—Erythrocytes
    • 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
    • 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
    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/02—Drug screening

Definitions

  • the present invention relates to a method of isolating human blood cells, in particular, blood cells of the erythroid lineage, specifically erythroid lineage progenitor cells.
  • the present invention also relates to compositions of erythroid progenitor cells produced in accordance with the present inventive method.
  • the red cells which circulate through the circulatory system, and their precursors may be considered to be a functional unit.
  • This functional unit has been designated the "erythron.”
  • the erythron is comprised of a restricted group of cellular elements that range from pronormoblasts in bone marrow to mature enucleated red cells that circulate in blood.
  • the red blood cells which owe their color to the presence of hemoglobin, carry oxygen throughout the body by way of complexes that form between an iron atom located in the heme group of the hemoglobin and oxygen.
  • a human When a human has a substantially lower than normal number of circulating red blood cells, the patient is considered to be anemic or to be suffering from a selective red cell aplasia.
  • Selective red cell aplasias are a group of myelopathies that are characterized by the selective depletion of the erythroid compartment of the bone marrow.
  • erythroid progenitors namely burst forming unit—erythroid (BFU-E) and colony forming unit—erythroid (CFU-E)
  • BFU-E burst forming unit
  • CFU-E colony forming unit
  • PRCA Progenitors
  • PRCA may be secondary to a wide variety of drugs, infections and hemolytic anemias in which cases the disease is generally acute.
  • Chronic PRCA may be associated with thymic tumors and/or other chronic diseases.
  • All types of immunity may be involved in PRCA.
  • a humoral type of immunity is exemplified by IgG antibodies that react with the erythroblastic compartment and BFU-E and, in rare cases, with erythropoietin.
  • erythroid suppression is mediated by large granular lymphocytes which have the CD8+ antigen.
  • TEC Transient erythroblastopenia of infancy
  • Total erythroblastic aplasia may result from major ABO incompatibility between a donor and recipient of blood.
  • Erythropoiesis refers to the proliferation and differentiation of red blood cells. During prenatal development, erythropoiesis occurs sequentially in the yolk sac, liver, and bone marrow. Initially, these sites are seeded by primitive cells that migrate from the yolk sac. The mesoblastic phase, when erythropoiesis is taking place in the yolk sac, is characterized by the differentiation in the wall of the yolk sac of mesenchymal elements that are grouped in solid masses referred to as blood islands. The primitive blood cells are in the centers of these islands and differentiate into primitive erythroblasts.
  • the hepatic phase when erythropoiesis is taking place in the liver, is characterized by the differentiation of erythroblasts into megaloblasts and then normoblasts, including enucleated cells that are slightly larger than adult red cells.
  • the myeloid phase corresponds to the period when erythropoiesis is taking place in the bone marrow.
  • the differentiated cells are predominantly normoblasts.
  • pronormoblasts are the earliest recognizable erythroid cells in the bone marrow.
  • Multipotent stem cells differentiate into the committed progenitors (BFU-E) and then into BFU-E colonies (CFU-E) , which, in turn, differentiate into erythroblasts.
  • BFU-E committed progenitors
  • CFU-E BFU-E colonies
  • the BFU-E's although usually few in number, have a great capacity to proliferate.
  • CFU-E are more differentiated and, accordingly, have limited proliferation capacity.
  • the term "erythroid progenitor cell” refers to BFU-E.
  • Erythroid differentiation is stimulated primarily by the action of erythropoietin (Epo) .
  • Epo is a glycoprotein with a molecular weight of approximately 40,000 daltons.
  • the major source of Epo is the kidney, although a small amount of Epo is produced by the liver and by the macrophages. Epo predominantly acts upon BFU-E and CFU-E.
  • Erythroid progenitor cells BFU-E also respond to IL-3 and GM-CSF. In addition, these cells may respond to IL-4, G-CSF, and IL-1 via indirect action of accessory cells or synergism with other obligatory growth factors.
  • Literature reports of %BFU-E in normal bone marrow aspirates or blood range from 13.6% to 55.4% of the colony- forming cell population, which in turn comprises less than 0.01 - 0.1% of the leukocyte fraction of blood or bone marrow (Pierelli, L. , et al. 1991 Bone Marrow Transplantation 7:335-361; Ciavarella, E. 1991 In;Biotechnology of Blood (Goldstein, J., ed) , Boston:Butterworth-Heinemann, 317-349; Arnold, R. , et al., 1986 EXP Hematol 14:271-277; Sieff, C, et al., 1982 Blood 60:703; Naughton, B.A.
  • proerythroblasts comprise about 0.2-1.4% of bone marrow cells, with an average of 0.6%, whereas basophilic erythroblasts range from 0.7-3.7% with an average of 2.0%.
  • basophilic erythroblasts range from 0.7-3.7% with an average of 2.0%.
  • a range of 12.2-24.2% characterizes polychromatophilic erythroblasts with an average of 12.4%.
  • Orthochromatic erythroblasts range from 2.0-22.7% with an average of 6.5%.
  • the first recognizable cell of the erythroid lineage is the pronor oblast.
  • pronormoblasts give rise to basophilic normoblasts in two generations, early polychromatophilic normoblasts in the third generation, and late polychromatophilic normoblasts in the fourth generation.
  • the cells between the pronormoblast and the early polychromatophilic normobla ⁇ t are proliferative, whereas the late polychromatophilic normoblast is not.
  • the late polychromatophilic normoblast develops into a reticulocyte after extruding its nucleus and crossing the sinusoidal barrier of the bone marrow.
  • Pronormoblasts, normoblasts and reticulocytes will be referred to as "precursors.” Reticulocytes further develop into mature red cells. This maturation entails the loss of transferrin receptors and the reticulum. In addition, this progressive differentiation from blast cells to mature erythrocytes is accompanied by a progressive decrease in cell size. The steps in differentiation and maturation of erythroid cells are also reflected by biochemical changes of both nuclear and cytoplasmic components.
  • the developmental sequence of erythroid development can be characterized by progressive condensation of chro atin, reduction in the number of nucleoli, a decrease in the number of ribosomes and mitochondria, a gradual increase in the electron density of the cytoplasm, which corresponds to hemoglobin accumulation, and an increase in ferritin within the cytoplasm as aggregates or plasma membrane-bound particles.
  • CD34+ or “cluster designate-34" is used to describe a cell that has the particular cell surface glycoprotein 34, as defined by analysis of antibodies to this glycoprotein that were performed at international workshops.
  • BFU-E are CD34+, HLA-DR+, transferrin receptor-*- and CD38+.
  • CFU-E continue to be positive for the transferrin receptor and CD38, the CFU-E are negative for HLA-DR.
  • cell-surface expression of HLA-ABC decreases, as does the expression of the transferrin receptor and CD38.
  • the precursors gradually decrease their expression of the transferrin receptor.
  • Glycophorin expression begins at about the transition from blast cells to erythroblasts and continues through maturation to erythrocytes.
  • the progressive expression of Band 3 cell-surface antigen is similar to that of glycophorin. (See, for example, Figure 2, Sieff et al., Blood 60(3): 703-713 (Sept. 1982).) Similar changes are observed for cell-surface carbohydrate residues.
  • antibodies can be generated for a cell-surface antigen, even a cell-surface carbohydrate moiety, although carbohydrates are more difficult to use to generate antibodies.
  • Antibodies that recognize cell-surface constituents may be prepared by conventional techniques that use the membrane or purified constituents thereof as immunogens.
  • the antibodies may be polyclonal or monoclonal. Either an intact antibody or a specific binding fragment thereof, whether ultivalent (such as divalent) or univalent may be used.
  • the antibodies or fragments thereof may be used as probes in immunofluorescence technigues, for example.
  • the antibodies are labelled with fluorochromes and applied to cell suspensions.
  • Fluorochrome-labeled antibodies may be used in combination to detect simultaneously two cell-surface antigens, especially if the cell suspension is comprised of a mixed population of cell types.
  • one antibody that is specific for one cell-surface antigen could be labeled with the fluorochrome fluorescein, whereas another antibody that is specific for another cell-surface antigen could be labeled with a fluorochrome with distinct spectral properties, such as rhodamine or phycoerythrin.
  • Such cell suspensions labelled with two or more fluorochrome-labeled antibodies, can be sorted into populations of cell types with a fluorescence-activated cell sorter (FACS) .
  • FACS fluorescence-activated cell sorter
  • a FACS employs a plurality of light- scattering detection channels, and impedance channels, among other more sophisticated levels of detection, to separate or sort cells.
  • the antibodies may be conjugated with biotin, which then can be removed with avidin or streptavidin bound to a support.
  • Plant lectins recognize and bind cell-surface carbohydrate moieties but do not perform any enzymatic action on the bound residue.
  • Normal GM-CFUs have been enriched from adult and fetal mice using a FACS to sort lectin-labeled cells (Nicola et al., Journal of Cellular Physiology 103:217-237 (1980)).
  • a series of fluorescein-conjugated lectins also have been analyzed for their binding to human peripheral blood cells using a fluorescence-activated cell sorter (Nicola et al., Blood Cells 6: 563-579 (1980)). Most of the lectins surveyed demonstrated increased cell binding in the order of erythrocytes, lymphocytes, monocytes and then neutrophils. The Lotus tetragonolobus lectin appeared to bind only to neutrophils in the peripheral blood. Analysis of the binding of this particular lectin to human bone marrow cells indicated that the degree of binding increased with progressive differentiation within the granulocytic series.
  • Bone marrow cells increasingly bound the lectin in the order of lymphocytes, blast cells, promyelocytes and myelocytes, monocytes, and polymorphonuclear cells.
  • the number of erythroid progenitor cells in a population was increased by depleting mature hematopoietic cells with monoclonal antibodies and positively selecting BFU-E and CFU-E with a murine monoclonal antibody that recognizes the transferrin receptor (Herrmann et al. Blut 56: 179-183 (1988)) .
  • the number of BFU-E in a population was increased by density centrifugation, sheep erythrocyte rosetting, surface immunoglobulin-positive cell depletion, adherence to plastic, and negative panning with monoclonal antibodies.
  • the BFU-E were cultured to generate CFU-E in vitro (Sawada et al., The Journal of Clinical Investigation 80: 357-366 1987; Sawada et al., Journal of Cellular Physiology 142: 219-230 1990).
  • molecules with an affinity for the lipid bilayer of the membrane were used.
  • Such molecules include the membrane binding portion of mellitin and protamine.
  • Other very basic peptides that are capable of binding to phosphates in the lipid bilayer may be efficacious.
  • the erythroid progenitors should be free of toxins and other contaminants that would limit the utility of the cells in clinical applications.
  • Figure 1 is a schematic depiction of the invention method for preparing a cell population enriched for human erythroid progenitor cells.
  • Figure 2 shows the morphological criteria for scoring hematopoietic colonies formed from selected cells after 14 days in methylcellulose culture.
  • Figure 3 shows the scatter plot results of FACS sorting of cells labeled with CD34 and Ulex as compared with labeling with CD13 and CD71.
  • Figure 4 depicts the number of colonies formed from CD34+ULEX+ cells after 14 days in methylcellulose culture.
  • the present invention provides a method of preparing from hematopoietic cells a population of cells enriched for erythroid progenitor cells.
  • the method entails separating hematopoietic progenitor cells on the basis of binding to a specific cell surface antigen such as CD34, followed by separation of erythroid progenitor cells based on binding to a lectin such as Ulex europaeus agglutinin.
  • cell populations enriched for erythroid progenitor cells in which at least about 60%, more preferably 80-100% of the colony-forming units are BFU-E.
  • the invention also provides a method of treatment for human patients having an erythroid disorder via administration of a cell population enriched for human erythroid progenitor cells. Also provided are diagnostic methods based on the enriched cell population.
  • the present invention provides an enriched population of human erythroid progenitor cells and methods for obtaining same.
  • the original source for the enriched population of human erythroid progenitor cells is bone marrow or peripheral blood, although other sources such as cord blood, yolk sac, and liver may be used.
  • bone marrow in particular autologous bone marrow as opposed to allogeneic bone marrow
  • peripheral blood cells are preferred sources of human erythroid progenitor cells for the therapeutic treatment of a human patient suffering from a disease that affects the erythroid lineage.
  • diseases may have been induced by a disease, drug, toxin, or radiation, or may be due to a genetic anemia or red cell aplasia.
  • Bone marrow cells may be obtained from a source of bone marrow, such as the iliac crest, tibia, femur, sternum, or another bone cavity. Bone marrow may be aspirated from the bone and processed in accordance with techniques that are well known to those who are skilled in the art.
  • the marrow may be harvested from a donor, in the case of an allogeneic transplant, or from the patient, himself, in the case of an autologous transplant.
  • the bone marrow aspirate is processed to separate out and discard hemoglobin-containing red cells, leaving a starting population of leukocytes.
  • leukocyte refers to any of the hematopoietic cells which do not contain hemoglobin.
  • the peripheral blood may serve as the original source of hematopoietic cells. It is not strictly necessary to separate the hemoglobin-containing erythrocyte fraction (red blood cells) from the leukocyte fraction
  • the term “population of hematopoietic cells” refers to any population of cells of blood-forming lineage, including stem cells and progenitor cells, and which may include mature blood cells of all types. It is essential that the starting population of hematopoietic cells contain nucleated cells, although not necessarily to the exclusion of non-nucleated cells.
  • the term “population of hematopoietic cells” refers to any population of cells of blood-forming lineage, including stem cells and progenitor cells, and which may include mature blood cells of all types. It is essential that the starting population of hematopoietic cells contain nucleated cells, although not necessarily to the exclusion of non-nucleated cells.
  • the term “population of hematopoietic cells” refers to any population of cells of blood-forming lineage, including stem cells and progenitor cells, and which may include mature blood cells of all types. It is essential that the starting population of hematopoietic cells contain nucleated cells, although not
  • nucleated cell refers to all hematopoietic cells except mature erythrocytes, which are non-nucleated.
  • leukocytes are separated from the patient's blood by centrifugal fractionation.
  • the patient's blood may be passed through a device containing ligands linked to a solid phase which binds the desired cell types.
  • ligands linked to a solid phase which binds the desired cell types.
  • the numbers of stem cells present in peripheral blood may be increased by pretreatment of the patient with cytokines which "mobilize" stem cells from the bone marrow to the blood, thus greatly increasing the yield of desired stem cells.
  • Hematopoietic progenitor cells are separated from the general hematopoietic population on the basis of their having specific cell surface antigens such as CD34, CD71, CD45RO or c-kit receptor. These antigens are believed to be present on progenitors of B-lymphocytes and myeloid lineages, as well as on BFU-E, but not present on mature blood cells.
  • hematopoietic progenitor cells refers to a cell population containing precursors of all hematopoietic lineages but which contains essentially no mature blood cells.
  • a monoclonal antibody against CD34 is used to select hematopoietic progenitor cells.
  • cells committed to differentiate along myeloid and lymphoid lines may be removed on the basis of their having surface markers specific for those lines.
  • Various techniques may be used to separate the hematopoietic progenitor cells from mature blood cells. For relatively crude separations, i.e., separations where up to 20%, usually not more than about 5%, generally not more than about 1%, of the total cells present have the positively selected marker, e.g., CD34, various techniques of differing efficacy may be employed.
  • the separation techniques employed should maximize the retention of viability of the fraction of the cells to be collected. The particular technique employed will, of course, depend upon the efficiency of separation, cytotoxicity of the method, the ease and speed of separation, and what equipment and/or technical skill is required.
  • Separation procedures may include magnetic separation, using antibody-coated magnetic beads, affinity chromatography, cytotoxic agents, either joined to a monoclonal antibody or used in conjunction with complement, and "panning", which utilizes a monoclonal antibody attached to a solid matrix, or another convenient technique.
  • Antibodies attached to magnetic beads and other solid matrices such as agarose beads or acryla ide beads, polystyrene beads, hollow fiber membranes and plastic petri dishes, allow for direct separation. Cells that are bound by the antibody can be removed from the cell suspension by simply physically separating the solid support from the cell suspension. The exact conditions and duration of incubation of the cells with the solid phase-linked antibodies will depend upon several factors specific to the system employed. The selection of appropriate conditions, however, is well within the skill in the art.
  • the unbound cells then can be eluted or washed away with physiologic buffer after sufficient time has been allowed for the CD34+ cells to bind to the solid-phase linked antibodies.
  • the bound cells are then separated from the solid phase by any appropriate method, depending mainly upon the nature of the solid phase and the antibody employed.
  • Antibodies may be conjugated to biotin, which then can be removed with avidin or streptavidin bound to a support, or fluorochromes, which can be used with a fluorescence activated cell sorter (FACS) , to enable cell separation. Any technique may be employed as long as it is not detrimental to the viability of the desired cells.
  • the progenitor cells initially may be separated from other cells by the cell-surface expression of CD34.
  • CD34+ cells may be positively selected by magnetic bead separation, wherein magnetic beads are coated with CD34- reactive monoclonal antibody. The CD34+ cells then may be removed from the magnetic beads.
  • CD34+ cells Release of the CD34+ cells may be checked with a FACSCAN® flow cytometer (Becton Dickinson, San Jose, CA) , for example, if so desired. Selection of CD34+ cells is preferred to minimize the starting volume for subsequent lectin selection and to enrich the progenitor cell population while removing unwanted accessory cells, both alive and dead or dying, which may produce factors that affect the subsequent proliferation and differentiation of the selected cells in culture. However, the enriched CD34+ population of cells does not necessarily have to be pure.
  • the resulting enriched population of CD34+ cells is then contacted with a lectin essentially as described above for
  • the chosen lectin must be either nontoxic or completely removable from the final cell population.
  • the CD34 and Ulex may be labeled and isolation of the erythroid progenitor cells may be performed by flow cytometry as described in Example 2 below.
  • the antibody and lectin ligands are removed before therapeutic use.
  • the present invention offers many advantages over previous methods. Previous approaches have used monoclonal antibodies to identify primarily protein antigens on the surfaces of cells. Carbohydrate structures on cell surfaces are only weakly antigenic so it is difficult to make highly specific monoclonal antibodies to carbohydrate antigens, especially high affinity IgG antibodies. The use of a plant lectin allows specific carbohydrate structures on cell surfaces to be identified with high affinity both efficiently and without toxicity to the progenitor cells. Some approaches have used negative selection or non-specific methods to enrich erythroid progenitor cells. However, these methods are inefficient and potentially toxic.
  • the plant lectin can be easily tagged with a fluorescent molecule or other probe that allows for its detection or it can be cross-linked to a solid surface.
  • Cells selected with plant lectins can, in turn, be purified from the lectin because the specific sugar(s) that are recognized by the lectin can be used to elute the bound cells from the lectin.
  • the lectin is Ulex europaeus agglutinin, which binds fucose moieties on cell surface glycoproteins. Once the erythroid progenitor cells have been selected by binding to this lectin, the lectin can be competed off the cells using an excess of fucose.
  • Such substances may include oligosaccharides and glycoproteins having fucose moieties in an orientation which allows their successful competition for binding to the fucose binding site on the lectin.
  • Examples of such competing oligosaccharides are Glycopeptide III, Glycopeptide I, and Fucoside 1 (Debray, et al 1981 Eur J Biochem 117:41-55, see Fig 6 and Table 6).
  • purified or recombinantly synthesized blood group antigen 0(H) may be used to compete the Ulex lectin off its fucose binding site on the erythroid progenitor cells (Sharon, et al 1991 Cvto etry 12:545-549).
  • an aliquot of the isolated cell population enriched for erythroid progenitor cells is cultured in methylcellulose and the cells are allowed to proliferate and differentiate in vitro for 14 days. After 14 days in methycellulose culture, the colonies are counted and scored using microscopic examination (see Figure 2) . In conducting this assay, the total number of colony forming cells which were present in the originally isolated population is assessed according to the number of colonies of all types present in the 14-day methylcellulose culture.
  • a "colony" is defined as a cluster of greater than 50 cells which, because of their physical proximity and morphological relationship, are presumed to have a common parent cell.
  • Each colony is morphologically identified as myeloid (CFU-GM colony) , erythroid (BFU-E colony, also known in the art as CFU-E) , or mixed (CFU-mix colony) ( Figure 2) .
  • BFU-E colony refers to a multicentric burst of hemoglobinized erythroid cells as depicted in Figure 2.
  • percentage of BFU-E colonies is calculated as the number of BFU-E colonies divided by the number of total colonies of all types. The percentage of BFU-E colonies is then used to extrapolate back to the percentage of parent BFU-E which were present 14 days previously in the enriched population obtained by the invention method.
  • the methods of the present invention yield an enriched cell population in which preferably greater than 60%, more preferably greater than 80%, most preferably essentially all of the colony-forming cells are BFU-E. It will be understood by one of skill in the art that the presence of non-colony forming cells is immaterial to the instant invention.
  • the resulting enriched population of erythroid progenitor cells may be used immediately for therapeutic and/or diagnostic purposes.
  • the cells may be cultured in liquid medium to proliferate and differentiate into more differentiated erythroid precursor cells or mature red cells.
  • the medium to be used in culture plates is one that is well-defined and enriched.
  • An example of a suitable medium is McCoy's 5A culture medium (Sigma, St. Louis, MO) , which additionally contains fetal bovine serum (Hyclone, Logan, UT) , horse serum (Hyclone) , hydrocortisone (Sigma), ⁇ -thioglycerol, and gentamicin (Gibco) .
  • the medium may be serum-free.
  • the culture medium may further comprise hematopoietic growth factors, such as erythropoietin, IL3, and stem cell factor. It will be appreciated by one who is skilled in the art that other suitable culture media may be used as well as other suitable hematopoietic growth factors in various combinations.
  • the culture medium not be replaced during the period of culturing, although the cultures should be fed at weekly intervals. In some cases, it may be desirable to change the culture medium from time to time, at least about once or twice per week. Alternatively, continuous perfusion of media and growth factors could be employed (Roller, M.R. , 1993 Bio/Technology 11:358-363).
  • cell aliquots may be removed and labeled with fluorescence-conjugated monoclonal antibodies for sorting in a flow cytometer, based on expression of cell-surface antigens.
  • Cells sorted according to cell-surface antigen expression may be additionally characterized according to morphology and potential for colony-forming units. Cells may be characterized by morphology as pronormoblasts, normoblasts, reticulocytes, and erythrocytes.
  • Colony assays may be conducted in methyl cellulose containing other media components and growth factors to determine the existence of BFU-E and BFU-E colonies.
  • the erythroid progenitor cells may be administered to the patient alone or in combination with other cell types, such as stem cells or other lineage-uncommitted cells prepared according to the method of US Patent Nos 4,965,204; 5,035,994; 5,130,144.
  • the erythroid progenitor cells may also be administered together with more differentiated erythroid precursor cells or mature red cells.
  • the more differentiated erythroid precursor cells and mature red cells may be isolated from bone marrow, cord blood, or peripheral blood or may be derived from the erythroid progenitor cells in vitro. Precise, effective quantities can be readily determined by those who are skilled in the art and will depend, of course, upon the exact condition being treated by the particular therapy being employed.
  • the erythroid progenitor cells may be used to treat human patients who have reduced levels of erythroid progenitor/precursor cells and/or mature red blood cells. Such populations of cells could be used to supplement existing reduced populations of erythroid cells in the patient to bring the cell population numbers to within a normal healthy range.
  • a survey of published reports indicates that the daily circulating red blood cell requirement for an average 70 kg adult is 2 x 10 11 red blood cells (Erslev, A.J. 1990 IN: Hematology. ed: Williams, W.J. McGraw-Hill, New York, p. 389) . Assuming that a typical BFU-E divides 10 times in vivo, one BFU-E would give rise to 10 3 mature red blood cells.
  • 2x 108 BFU-E would be required for a one day supply of red blood cells.
  • Practicing the present invention it is possible to obtain greater than 10 BFU-E from a single patient's leukapheresis product.
  • the in vitro doubling time of a BFU-E is estimated to be 18-24 hours. By allowing the in vitro expansion of BFU-E, it is thus possible to obtain a cell preparation capable of meeting the requirements for several day's supply of red blood cells.
  • the erythroid progenitor cells have the capacity to both proliferate and differentiate in culture and may remain viable for an extended period of time. Accordingly, it should be possible to isolate an initial quantity of cells from the in vitro culture of erythroid progenitor cells and to administer that quantity of cells to the patient. After a period of time, one or more additional aliquots of viable erythroid progenitor cells and/or mature red cells may be isolated from the culture and administered to the same patient.
  • the erythroid progenitor cells also may find use in the treatment of red cell aplasia or anemia, whether induced by a disease, drug, toxin or radiation, as well as genetic or congenital defects. Allogeneic therapy may be useful in diseases such as aplastic anemia and the purified erythroid progenitor population may eliminate problems associated with GVHD.
  • the erythroid progenitor cells are expected to have utility in the diagnostic assessment of erythroid progenitors in pathologic states of anemia or polycythemia or for the evaluation of stem cell harvests for transplantation.
  • the method also could be used to deplete CD34+ cells of erythroid progenitors or malignant red cell progenitors for in vitro culture purposes, which may be useful in treating disorders such as polycythemia vera.
  • the present invention is also expected to have utility in the research of erythroid lineage cells, such as with regard to proliferation and differentiation. For example, factors associated with proliferation and differentiation, such as hematopoietic growth factors, may be evaluated. In addition, cytokine combinations and extracellular conditions may be evaluated. Similarly, the cells, themselves, may be used to evaluate particular media and fluids for cell proliferative and/or differentiative activity, and the like.
  • the erythroid precursor cells possibly may be frozen in liquid nitrogen for long periods of storage. The cells then may be thawed and used as needed. Cryoprotective agents and optimal cooling rates can protect against cell injury. Cryoprotection by solute addition is believed to occur by penetration into the cell, thereby reducing the amount of ice formed, which injures the plasma membrane and results in osmotic dehydration of the cell, or by decreasing the rate of water flow out of the cell in response to a decreased vapor pressure of external ice.
  • Cryoprotective agents that can be used include but are not limited to DMSO, metastarch, glycerol, polyvinylpyrrolidone, polyethylene glycol, albumin, dextran, sucrose, ethylene glycol, i-erythritol, D-ribitol, D-mannitol, D-sorbitol, i-inositol, D-lactose, choline chloride, amino acids, methanol, acetamide, glycerol monoacetate, and inorganic salts.
  • the cells may be stored in 10% DMSO, 50% FCS, and 40% RPMI 1640 medium. Once thawed, the cells may be induced to proliferate and further differentiate by the introduction of the appropriate hematopoietic growth factors.
  • the erythroid progenitor cells could be allowed to immediately proliferate and differentiate into mature red blood cells in culture, by providing the appropriate growth factors.
  • the following examples serve to illustrate further the present invention but are not intended to limit the scope of the invention.
  • This example describes the preparation of leukocytes.
  • Human peripheral blood was obtained from patients whose CD34+ cells were mobilized to the blood by pretreatment with cytokines.
  • Bone marrow was obtained from normal adult volunteers.
  • the cell suspension was layered onto Histopague 1077 (Sigma, St. Louis, MO) and centrifuged at 300 x g for 20 minutes. The interface was removed and washed with phosphate-buffered saline solution containing 0.1% sodium azide and 0.5% bovine serum albumin (PAB) . All procedures were carried out at 0°C on melting ice.
  • PAB bovine serum albumin
  • Example 2 This example describes the isolation of erythroid precursor cells using labeled CD34 antibody and Ulex europaeus I lectin.
  • Biotinylated and FITC-conjugated anti-CD34 antibody (8G12 (Baxter Immunotherapy Div. , Irvine, CA) or QBEND 10 (Quantum Biosystems, England)) were used to isolate CD34+ cells by flow cytometry. The CD34+ cells were then stained with phycoerythrin-conjugated Ulex europaeus I lectin to identify a population of erythroid progenitor cells. Some cell preparations were also stained with FITC-conjugated antibodies to CD13 (MY7) or CD71 (HTR) in combination with biotin-QBENDIO and phycoerythrin-ULEX. The biotin QBEND10 was counterstained with allophycocyanin-avidin.
  • Compensation levels were set by gating on the lymphocyte population identified by forward versus side scatter and aligning mean channels of the single-stained positive populations for each color with the corresponding unstained control. Compensation levels were typically set at 1% PE from FITC subtraction and 16% FITC from PE subtraction. PMT voltages were typically set at 580 V for FITC and 610 V for PE. A minimum of 30,000 events was analyzed on each sample.
  • the identity of the cells as erythroid progenitors was confirmed by analysis of the distribution of colony forming cells in accordance with the method described in Example 3 below.
  • the colonies were scored as CFU-GM, CFU-Mix, or BFU-E as shown in Figure 2.
  • the BFU-E colonies were also identified by their red color due to the formation of hemoglobin.
  • the CD34+ cells that stained with Ulex were also CD13- and CD71+ ( Figure 3), which indicates that the cells are not of the granulocyte/macrophage lineage, but rather express high levels of the transferrin receptor, which is characteristic of erythroid progenitor cells.
  • the CD34+Ulex+CD13- cells contained only erythroid progenitors. Results are shown in Table 1 below:
  • This example describes the sorting of cells according to phenotype into colony assays.
  • rIL-3 150 U/ml
  • GM-CSF 200 U/ml
  • G-CSF 150 U/ml
  • rIL-6 160 U/ml
  • erythropoietin 10 U/ml; Amgen
  • Example 4 This example describes an alternative method of enriching a population of CD34+ cells by positive enrichment.
  • Mononuclear cells were isolated from bone marrow on 1.077 g/dl Histopaque (Sigma) . The cells were washed in calcium/magnesium-free phosphate-buffered saline (CMF-PBS, Gibco, Grand Island, NY) and were resuspended in Iscoves- modified Dulbecco's Medium (IMDM, Sigma) containing 2% fetal bovine serum (Hyclone, Logan, UT) to a concentration of lxlO 7 cells/ml, then labeled with a mouse monoclonal anti-CD34 antibody (1 ⁇ g/10 cells of the anti-CD34 monoclonal antibody QBEND 10 from Quantum Biosystems) .
  • CMF-PBS calcium/magnesium-free phosphate-buffered saline
  • IMDM Iscoves- modified Dulbecco's Medium
  • IMDM Iscoves- modified Dulbecco's Medium
  • IMDM
  • CD34+ cells were then contacted with magnetic beads, which were coated with sheep anti-mouse IgG antibodies (Dynal, Oslo, Norway) and used to capture CD34+ cells from the cell suspension.
  • CD34+ cells were positively selected as described by Strauss et al., Am. J. Ped. Hematol. Oncol. 13:217 (1991), with slight modification.
  • the magnetic beads and cells were rotated for 30 minutes at 4°C at 2.5 rpm at a bead:cell ratio of 1:1 or 5:1.
  • the bead-cell complexes were isolated using a magnetic tube holder (Fenwal Div. , Irvine, CA) . After a series of washes in CMF-PBS, the CD34+ cells were released from the beads by adding 50 U/ml of Chymodiactin® (Boots Pharmaceutical Co., Lincolnshire, IL) in RPMI 1640 (Sigma) and incubating for 15 minutes in a water bath at 37°C.
  • Chymodiactin® Boots Pharmaceutical Co., Lincolnshire, IL
  • the cells that were released from the beads were evaluated for CD34 purity by staining with the monoclonal antibody to CD34, namely FITC-8G12 (Fenwal), for 15 minutes on ice and quantitating the stained cells with a FACSCAN® flow cytometer (Becton-Dickinson) using a side scatter vs. fluorescence display.
  • the CD34+ cell population was resolved as a population having FITC fluorescence and low side scatter.
  • EXAMPLE 5 This example describes an alternative method of enriching a population of Ulex-t- cells from a population of CD34+ cells by positive selection.
  • CD34+ cells could be contacted with magnetic beads coated with Ulex europaeus I lectin.
  • Ulex lectin is expected to capture those CD34+ cells from the cell suspension that express fucose residues on their cell surfaces.
  • the magnetic beads and cells could then be rotated for 30 minutes at 4°C at 2.5 rpm and a bead:cell ratio of 1:1 or 5:1.
  • the bead-cell complexes could be isolated using a magnetic tube holder (Fenwal) . After a series of washes in CMF-PBS, the CD34+ cells could be released from the beads by adding excess fucose. The resulting population of CD34+Ulex+ cells is expected to comprise a population enriched for erythroid progenitor cells.

Abstract

L'invention concerne un procédé de préparation, à partir de cellules hématopoïétiques, d'une population de cellules enrichie en cellules souches érythroïdes (BFU-E), dans laquelle jusqu'à 100 % des unités formant la colonie sont des BFU-E. Le procédé comprend la séparation de cellules souches hématopoïétiques sur la base de la liaison avec un antigène à surface cellulaire spécifique tel que CD34, et la séparation des cellules souches érythroïdes basée sur la liaison avec une lectine telle que l'agglutinine Ulex europaeus. Après séparation des souches érythroïdes, la lectine peut être éluée des cellules par incubation avec un excès de sucre, tel que du fucose, qui remplit l'emplacement de liaison avec la cellule sur la lectine. L'invention concerne également un procédé de traitement de patients humains ayant une maladie érythroïde via l'administration d'une population de cellules enrichie en cellules souches érythroïdes humaines. L'invention concerne également les procédés de diagnostic basés sur la population de cellules enrichie.
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AU2333999A (en) * 1998-01-23 1999-08-09 Prolifaron, Inc. Methods and compositions for the identification of growth factor mimetics, growth factors and inhibitors
WO2007010941A1 (fr) * 2005-07-20 2007-01-25 Kaneka Corporation Antigene et anticorps monoclonal capable de reconnaitre l’antigene
JP2010516239A (ja) 2007-01-18 2010-05-20 スオメン プナイネン リスティ，ヴェリパルベル 細胞の産生に対する新規方法および試薬
WO2012048275A2 (fr) 2010-10-08 2012-04-12 Caridianbct, Inc. Procédés et systèmes configurables pour la culture et la récolte de cellules dans un système de bioréacteur à fibres creuses
JP6633522B2 (ja) 2013-11-16 2020-01-22 テルモ ビーシーティー、インコーポレーテッド バイオリアクターにおける細胞増殖
WO2015148704A1 (fr) 2014-03-25 2015-10-01 Terumo Bct, Inc. Remplacement passif de milieu
JP6830059B2 (ja) 2014-09-26 2021-02-17 テルモ ビーシーティー、インコーポレーテッド スケジュール化された細胞フィーディング
WO2017004592A1 (fr) 2015-07-02 2017-01-05 Terumo Bct, Inc. Croissance cellulaire à l'aide de stimuli mécaniques
WO2017205667A1 (fr) 2016-05-25 2017-11-30 Terumo Bct, Inc. Expansion cellulaire
US11104874B2 (en) 2016-06-07 2021-08-31 Terumo Bct, Inc. Coating a bioreactor
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US11629332B2 (en) 2017-03-31 2023-04-18 Terumo Bct, Inc. Cell expansion
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