CA2160871A1 - Human erythroid progenitor cell population - Google Patents

Abstract

The invention provides a method of preparing from hematopoietic cells a population of cells enriched for erythroid progenitor cells (BFU-E) in which up to 100% of the colony-forming units are BFU-E. The method entails separating hematopoietic progenitor cells on the basis of binding to a specific cell surface antigen such as CD34, and separation of erythroid progenitor cells based on binding to a lectin such as Ulex europaeus agglutinin.
After separation of the erythroid progenitors, the lectin may be eluted off the cells by incubation with excess sugar, such as fucose, which competes for the cell-binding site on the lectin. 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.

Description

~ WO94/25571 21~ O ~ 7 ~ PCT~S941041~

HUMAN ERYTHROID PROGENITOR CELL POPULATION

Technical Field of the Invention 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.

Background of the Invention 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.

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.
In addition, erythroid progenitors, namely burst forming unit--erythroid (BFU-E) and colony forming unit--erythroid (CFU-E), are depleted even though the proliferation and differentiation of other blood cell lineages continues normally.

WO94/25571 ~ - PCT~S94/041 Adult pure red cell aplasia (PRCA) served as the first human model of an autoimmune myelopathy. Although an infrequent disease, 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.
For example, 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. In other instances, erythroid suppression is mediated by large granular lymphocytes which have the CD8+ antigen.

Congenital hypoplastic anemia of infancy, otherwise known as Diamond-Blackfan anemia, is believed to be the result of erythroid progenitor cells being insensitive to erythropoietin. Transient erythroblastopenia of infancy (TEC) differs from Diamond-Blackfan anemia in that it lacks fetal erythropoiesis and is transient.

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 WO94/2~571 2 i 6 0 ~ 71 PCT~S94/041~

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.

After birth, 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. The BFU-E's, although usually few in number, have a great capacity to proliferate. CFU-E, on the other hand, are more differentiated and, accordingly, have limited proliferation capacity. Herein, the term "erythroid progenitor cell"
refers to BFU-E.

Erythroid differentiation is stimulated primarily by the action of erythropoietin tEpo). 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 predo~;n~ntly 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-l 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:BiotechnoloqY of Blood (Goldstein, J ~ ed), WO94/25S71 216 ~ 8 ~ ~ PCT~S941041~

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., et al, 1991 J Biomech Enqineerinq 113:171-177; Hows, J.M., et al., 1992 Lancet 340:73-76;
Ash, R.C., et al., 1981 58:309-316; Leary, A.G., et al., 1992 PNAS 89:4013-4017).

It is generally believed that 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%. 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~. (See Table 1-1, page 27, Blood: Textbook of Hematolo~Y, James H. Jandl, ed.
Little Brown and Co., Boston, 1987.) Morphologically, the first recognizable cell of the erythroid lineage is the pronormoblast. These 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 normoblast 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.

WO94/25571 216 0 8 71 PCT~S94/041~

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 chromatin, 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.
(See, for example, Figure 8, Loken et al., Blood 69(l):
255-263 (Jan. 1987).) During this progressive, morphologic differentiation, changes in the surface antigens, i.e., proteins, glycoproteins, glycolipids, and lipoproteins, of these cells can be observed. The presence or absence of these surface antigens can be used to distinguish these cells from other cells. For example, the multipotent stem cell and the early hematopoietic progenitor cell both express the CD34 antigen. The designation "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+. Although CFU-E
continue to be positive for the transferrin receptor and CD38, the CFU-E are negative for HLA-DR. As differentiation progresses beyond CFU-E, cell-surface expression of HLA-ABC decreases, as does the expression of the transferrin receptor and CD38. However, as the precursors progressively differentiate from blast cells to erythroblasts to basophilic normoblasts to polychromatophilic normoblasts to orthochromatic normoblasts to reticulocytes, the precursors gradually decrease their expression of the transferrin receptor.

WO94125571 2 L 6 o 8 7 ~ PCT~S94/041~

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.

These changes in cell-surface antigens can be utilized to separate cells at different stages of development. For example, 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 multivalent (such as divalent) or univalent may be used. The antibodies or fragments thereof may be used as probes in immunofluorescence techniques, 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. For example, one anti~ody 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 WO94/2~571 216 0 8 7 I PCT~S94/04124 cell sorter (FACS). 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.

Alternatively, the antibodies may be conjugated with biotin, which then can be removed with avidin or streptavidin bound to a support.

Use also has been made of plant lectins and agglutinins.
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)). In surveying the lectins, either by selective agglutination of cells or by cell sorting after binding of the FITC-derivatives, it was determined that the lectins Pokeweed mitogen, soybean agglutinin, Helix Pomatia agglutinin, and peanut agglutinin reacted preferentially with myeloid CFUs.

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 tetraqonolobus 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. Monocytes and nucleated erythroid cells in the marrow bound to the lectin, in contrast to monocytes and non-nucleated red cells in the peripheral blood.

WO94/25571 ; 216 0~8 7 ~ PCT~S94/041~

In a separate study of human peripheral blood, the Lotus tetraqonolobus lectin bound granulocytes but not lymphocytes, erythrocytes or monocytes. The binding was competitively inhibited by the sugar e-L-fucose. (Morstyn et al., Blood 56(5): 798-805 (1980)). 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)).

Using a different approach, 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 n vitro (Sawada et al., The Journal of Clinical Investigation 80: 357-366 1987; Sawada et al., Journal of Cellular Physiology 142: 219-230 1990).

Lansdorp and Dragowska purified cells with a CD34+CD45RA CD71+ phenotype and reported expression of transferrin receptor after 14 days in culture, suggesting that this phenotype is typical of erythroid progenitor cells (Lansdorp, P.M., et al 1992 J. Exp Med 175:1501-1509).

Alternatively, molecules with an affinity for the lipid bilayer of the membrane were used. Such molecules include the membrane binding portion of mellitin and protamine.

W094/25571 216 0 8 7 ~ PCT~S94/04124 other very basic peptides that are capable of binding to phosphates in the lipid bilayer may be efficacious.
Peptides, which have been modified to prevent lysis, such as malaria peptides and proteins known to have affinity for the red cell membrane, also may be used.

There remains a need for a simplified method of isolating an enriched population of erythroid progenitor cells in a highly purified state. The erythroid progenitors should be free of toxins and other contaminants that would limit the utility of the cells in clinical applications.

Brief DescriPtion of the Fiqures 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.

SummarY of the Invention 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 WO94/2~571 216 0 87 1 PCT~S94/041~
~
to a lectin such as Ulex euroPaeus agglutinin. Also provided are 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.

Detailed DescriPtion of the Preferred Embodiments The present invention provides an enriched population of human erythroid progenitor cells and methods for obtaining same. Preferably, 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.
Although any source of human erythroid progenitor cells may be used in research applications, bone marrow, in particular autologous bone marrow as opposed to allogeneic bone marrow, and 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. Such 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 21~0~71 WO94/2~71 - ~ - PCT~S94/041~
~ 11 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.
Herein, the term "leukocyte" refers to any of the hematopoietic cells which do not contain hemoglobin.

Alternatively, 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 (white blood cells) before starting the method of the invention. Herein, 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. Herein, the term "nucleated cell" refers to all hematopoietic cells except mature erythrocytes, which are non-nucleated.

In a process known as "apheresis", leukocytes are separated from the patient's blood by centrifugal fractionation.
Alternatively, the patient's blood may be passed through a device containing ligands linked to a solid phase which binds the desired cell types. The advantage of such methods is that they allow extremely rare peripheral blood stem cells and progenitor cells to be harvested from a large volume of blood, sparing the donor the expense and pain of harvesting bone marrow and the associated risks of anesthesia, analgesia, blood transfusion, and infection.
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 WO94/25~71 2 1~ 0 8 7 ~ PCT~S94/04124 blood, thus greatly increasing the yield of desired stem cells.

The general principle of the instant invention applies to = 5 all sources of hematopoietic 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, CD45R0 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. Thus, the term "hematopoietic progenitor cells" refers to a cell population containing precursors of all hematopoietic lineages but which contains essentially no mature blood cells.

Preferably, a monoclonal antibody against CD34 is used to select hematopoietic progenitor cells. If desired, cells committed to differentiate along myeloid and lymphoid lines may be removed on the basis of their having surface markers specific for those lines. However, it will be appreciated by one who is skilled in the art that it is not necessary to remove any or every undesired class of lineage-committed cells from the final population enriched for erythroid progenitor cells.

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 ~ ;ze the retention of viability of the fraction of the cells to be collected.
The particular technique employed will, of course, depend WO94/2~571 216 0 8 71 PCT~S94/041~

upon the efficiency of separation, cytotoxicity of the method, the ease and speed of separation, and what equipment and/or technical skill is re~uired.

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 acrylamide 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 con~ugated 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.

WO94/25~71 216 0 ~ 71 PCT~S94/04124 The progenitor cells initially may be separated from other cells by the cell-surface expression of CD34. For example, 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. 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 CD34 selection. Debray, et al, described the red cell agglutinating properties of twelve lectins (Lebray, V., et al., 1981 Eur J Biochem 117:41-55). Subsequently, Craig, et al, reported the binding of various lectins to hematopoietic cells (Craig, W.H., 1992 J Hematother 1:55-64). Table 2 on page 57 of Craig, et al, (suPra) shows that various lectins are expected to bind with weak to strong affinity to lymphocytes, monocytes, and/or granulocytes. Unexpectedly, it was found that binding to a lectin could select for erythroid progenitor cells (see Figure 4 and Table 1 of this application).

The chosen lectin must be either nontoxic or completely removable from the final cell population. In practicing this invention, it is preferred to use the fucose-binding lectin Ulex euroPaeus agglutinin derived from the plant WO94/25571 2 1 6 0 ~-7-1 PCT~S94/041~

Ulex europaeus (Debray, et al., supra). Cells can be competed off this lectin by addition of excess fucose, the particular carbohydrate residue that is recognized by Ulex.

In an alternative embodiment of the invention, 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. A combination of a solid support, such as a magnetic bead, and flow cytometry, in either order, also may be used.

It is also possible to reverse the order of selection by first separating the cells which bind to lectin, followed by CD34+ selection.

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. Furthermore, 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 WO 9412S571 2 1 ~ jQ ~8 7 ~ ` PCT~S94/041~

elute the bound cells from the lectin. In a preferred embodiment of this invention, 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.

Alternatively, other substances which compete directly with the lectin for binding to fucose may be employed as elution agents. 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 o~ 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). Alternatively, purified or recombinantly synthesized blood group antigen OtH) may be used to compete the Ulex lectin off its fucose binding site on the erythroid progenitor cells (Sharon, et al 1991 Cytometry 12:545-549).

We turn now to assessment of the success of the methods of the invention. For use in therapy and diagnostics it is desirable to ascertain the extent of enrichment for erythroid progenitor cells in the population isolated according to the above description. It is also desirable to ascertain whether the patient's erythroid progenitor cells are normal or abnormal, and to estimate their percentage within the patient's tissue. In order to assess the number and quality of BFU-E in the enriched population, it is necessary to culture the isolated cell population for several days to allow time for the BFU-E to proliferate to form recognizable colonies.

Preferably, an aliquot of the isolated cell population enriched for erythroid progenitor cells is cultured in ~ W094/25571 216 0 ~ 7 i PCT~S94tO41~

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.
Herein, 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). Herein, the term "BFU-E colony" refers to a multicentric burst of hemoglobinized erythroid cells as depicted in Figure 2. Herein, "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. Alternatively, the cells may be cultured in liquid medium to proliferate and differentiate WO94/25571 216 0 8j 7 1- ` PCT~S94/041~

into more differentiated erythroid precursor cells or mature red cells. Preferably, the medium to be used in culture plates is one that is well-defined and enriched.
An example of a sui~able 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). Alternatively, 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.
It is preferred that 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 (Koller, M.R., 1993 Bio/Technoloqy 11:358-363).

At selected days during the culture period, 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.

., 13 ~ ~ ~ ,, .
WO94/25~71 ~16 ~ 8 71 PCT~S94/041~

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 1011 red blood cells (Erslev, A.J. 1990 IN: HematoloqY, 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 mature red blood cells. Thus, 2x 10 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 107 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 WO 94125~71 ~ ~ 216 0 8 7 1 PCT~S94/04124 preparation capable of meeting the requirements for several day's suppl~ of red blood cells.

The erythroid progenitor cells have the capacity to ~oth 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 lo 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 n 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 -21~ 0.87~
WO94/25571 - PCT~S94/041 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 andtor 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.
Typically, 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.

Alternatively, 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.

W094/25571 2 i 6 ff 8 7 ~ PCT~S941041~

The following examples serve to illustrate further the present invention but are not intended to limit the scope of the invention.

ExamPle l 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 Histopaque 1077 (Sigma, St. Louis, M0) 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 0C on melting ice.

Exam~le 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 (8Gl2 (Baxter Immunotherapy Div., Irvine, CA) or QBEND lO
(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 CDl3 (MY7) or CD71 (HTR) in combination with biotin-QBENDlO and phycoerythrin-ULEX. The biotin QBENDlO
was counterstained with allophycocyanin-avidin.

The flow cytometers used included a Becton Dickinson two-laser FACStar Plus and a FACScan. Flow cytometry wasconducted essentially as described by Bender, et al (Blood ~16~8~:71 WO94/25571 -- PCT~S94/04124 77:2591-2596, 1991). Briefly, fluorescence attributable to FITC and phycoerythrin was determined using excitation by an argon laser operating at 488 nm and adjusted to 0.3 W.
Emission from fluorescein and phycoerythrin was measured using short band pass filters of 530 +/- 15 nm and 575 +/-15 nm, respectively. 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:

WO 94/25571 ~ 2~16 0 8 7 1 PCT/US94/04124 5 1 oellh~ell sorted, inner 60 wells cf 3--~6well pbtes.

CD34+
T~tal oolonie~f Cloning % Erythroid ~reloid E~throd Mi,~ed 180ceUs B~iderx~ . '~ . ~s,~total Expt 1 ;6 14 0 30 16.? 46./
6~2 ~ 8 1 2~3 1~8 34.8 ~p~ 3 ~- Z3 37 20 6 622 Av_.ci~e :~ 16.7 47.9 CD34~/U l~x~
Tatal c~ i~/ Cloning % Erythrold Myeloid Erytludd Mi~ cells E~cienc~ colonies/~l r 1 16 ~4 1 81 4~ 79 2 13 25 1 39 2.- 64.1 3 4 5~ 62 ~
~ 4 Q 19 19 1C~.6 100 Ave~ 0.3 ~7.9 842 CD34~/Ulex-Total oo onlesl C~oning % Erythrold Erythroid Mi:c~d 180 ceDs E~aer~ cohni~s/to~al ~F~ 1~ 8 o . 31 t, 2 2~.8 Ex~r 27 ~. o 19 10.6 10 2 17 9.4 29.
Averaç,~ 17.4 2 ~ WO9412~571 ~ 216 O ~ 7 1 PCT~S94/04124 This example describes the sorting of cells according to phenotype into colony assays.

Cells were sorted using an Automatic Cell Deposition Unit (ACDU) on the FACStar Plus. Before each sort, the accuracy of the ACDU was validated by sorting single fluorescent beads into a 96 well plate and ~x~mi ning the wells with a fluorescent microscope. Single cells of a given phenotype were deposited into 200 ~l of colony assay media, which is comprised of methyl cellulose containing Iscoves'IMDM
(Sigma), 30% FBS (Sigma), BSA, and optimal concentrations of rIL-3 (150 U/ml), GM-CSF (200 U/ml), G-CSF (150 U/ml), rIL-6 (160 U/ml), and erythropoietin (lO U/ml; Amgen), in each of the 60 central wells of a 96 well microtiter plate.
The outer wells of the 96 well plate were filled with sterile water to maintain humidity. Alternatively, multiple cells were deposited into l ml of colony assay media in a 6 well plate. The plates were then incubated at 37C and scored after 14 days according to the morphological criteria shown in Figure 2, as described in the Detailed DescriPtion of the Invention above.

WO94/25571 216 0 8 71 PCT~S94/041~

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 lx10 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). The 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. Essentially, CD34+ cells were positively selected as described by Strauss et al., Am. J. Ped.
Hematol. Oncol. 13:217 (l991), with slight modification.
The magnetic beads and cells were rotated for 30 minutes at 4OC at 2.5 rpm at a bead:cell ratio of 1:1 or 5:1.

Following CD34 selection, 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 ~y 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 37C.
The cells that were released from the beads were evaluated for CD34 purity by staining with the monoclonal antibody to CD34, namely FITC-8Gl2 (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 -2160~71 WO94125~71 r ' i ~,, t `. PCT~S94/04124 resolved as a population having FITC fluorescence and low side scatter.

The level of purity of CD34+ cells obtained in accordance with this procedure averaged 66 + 16% (mean + l S.D., n=7) with a range from about 40% to about 93% CD34+.

This example describes an alternative method of enriching a population of Ulex+ cells from a population of CD34+
cells by positive selection.

CD34+ cells could be contacted with magnetic beads coated with Ulex euroPaeus I lectin. The 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 4C at 2.5 rpm and a bead:cell ratio of l:l or 5:1.
Following Ulex selection, 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.

Claims (30)

What is claimed is:

1. A method of preparing a population of cells enriched for erythroid progenitor cells, which method comprises:
(a) providing a first population of hematopoietic cells, said first population containing nucleated cells, (b) contacting said first population with a ligand which binds specifically to hematopoietic progenitor cells, (c) separating said hematopoietic progenitor cells from said first population to form a second population comprising hematopoietic progenitor cells, (d) contacting said second population with a lectin which binds to erythroid progenitor cells, and (e) separating said erythroid progenitor cells from said second population to form a third population enriched for erythroid progenitor cells.

2. The method of claim 1 wherein said first population of hematopoietic cells is obtained from a source selected from the group consisting of bone marrow, cord blood, peripheral blood, yolk sac, and liver.

3. The method of claim 1 wherein said first population consists essentially of nucleated cells.

4. The method of claim 1 wherein said ligand binds to a human cell-surface antigen selected from the group consisting of CD34, CD71, CD45RO, and c-kit ligand receptor.

5. The method of claim 4 wherein said ligand is an antibody.

6. The method of claim 5 wherein said antibody is specific for the cell-surface antigen CD34.

7. The method of claim 1, wherein said lectin binds to a sugar on said erythroid progenitor cells.

8. The method of claim 7, wherein said sugar is fucose.

9. The method of claim 1, wherein said lectin is derived from a plant.

10. The method of claim 9, wherein said plant is Ulex europaeus.

11. The method of claim 10, wherein said lectin is Ulex europaeus agglutinin.

12. The method of claim 8 further comprising eluting said lectin from said erythroid progenitor cells using a substance which competes directly with lectin for binding to fucose.

13. The method of claim 12 wherein said substance is fucose.

14. A method of preparing a population of cells enriched for erythroid progenitor cells, which method comprises:
(a) providing a first population of hematopoietic cells, said first population containing nucleated cells, (b) contacting said first population with a lectin which binds erythroid progenitor cells and other hematopoietic cells, (c) separating lectin-bound cells from said first population to form a second population comprising erythroid progenitor cells and other hematopoietic cells, (d) contacting said second population with a ligand which binds specifically to hematopoietic progenitor cells, and (e) separating said hematopoietic progenitor cells from said second population to form a third population enriched for erythroid progenitor cells.

15. The population of cells enriched for erythroid progenitor cells obtained by the methods of any one of claims 1 - 14.

16. A human cell population enriched for erythroid progenitor cells wherein at least about 60% of the colony-forming cells are BFU-E.

17. The human cell population of claim 16 wherein over 80% of the colony-forming cells are BFU-E.

18. A human cell population wherein essentially all of the colony forming cells are BFU-E.

19. A human cell population enriched for erythroid progenitor cells which is suitable for clinical applications.

20. The cell population of claim 18, further comprising additional cell types selected from the group consisting of pronormoblasts, normoblasts, reticulocytes and erythrocytes.

21. The cell population of claim 20, wherein said additional cell types are the progeny of said BFU-E which have proliferated and differentiated in vitro.

22. A method of treating a human patient having a reduced number of erythrocytes or an abnormal form of erythrocytes, which method comprises administering to said patient a cell population enriched for human erythroid progenitor cells.

23. The method of claim 22 wherein at least about 60%
of the colony-forming units within said cell population are BFU-E.

24. The method of claim 22, wherein said patient is suffering from anemia or selective red cell aplasia.

25. The method of claim 22, wherein said cell population is administered intravenously.

26. The method of claim 23, wherein said cell population additionally comprises one or more cell types selected from the group consisting of pronormoblasts, normoblasts, reticulocytes and erythrocytes.

27. The method of claim 26, wherein said additional cell types are the progeny of said BFU-E which have proliferated and differentiated in vitro.

28. The method of claim 22 further comprising co-administering a second population of lineage-uncommitted cells.

29. The method of claim 28 wherein said second population contains stem cells.

30. A method for evaluating the status of erythroid progenitors in a patient, comprising (a) providing a cell population containing leukocytes from said patient, (b) contacting said leukocytes with a ligand which binds specifically to hematopoietic progenitor cells, (c) contacting said hematopoietic progenitor cells with a lectin which binds to erythroid progenitor cells, thereby identifying erythroid progenitor cells, and (d) assessing the percentage of erythroid progenitor cells within said leukocyte population.