CA2168348A1 - Novel stem cell marker - Google Patents

Abstract

A method for obtaining human hematopoietic stem cells is provided by separation of the stem cells from dedicated cells by virtue of a novel stem cell marker. The cells obtained are provided. The isolated cells demonstrate increased proliferative potential.

Description

NOVEL STEM CELL MARI<ER.

INTRODUCTION

Technical Field The field of this invention ls the isolation, of a population of hematopoietic cells.

Backqround M~mm~l ian hematopoietic cells provide a diverse range of physiological activities. These cells are divided into lymphoid, myeloid and erythroid lineages.
The lymphoid lineages, comprising B cells and T cells, provides for the production of antibodies, regulation of the cellular immune system, detection of foreign agents in the blood, detection of cells foreign to the host, and the like. The myeloid lineage, which includes monocytes, granulocytes, megakaryocytes as well as other cells, monitors for the presence of foreign bodies, provides protection against neoplastic cells, scavenges foreign materials, produces platelets, and the like. The erythroid lineage provides the red blood cells, which act as oxygen carriers.
All publications cited herein are hereby incorporated herein by reference in their entirety.
Despite the diversity of the nature, morphology, characteristics and function of hematopoietic cells, it is presently believed that these cells are derived from a single progenitor population, termed "stem 3~g WO95/03693 PCT~S94/08574 cells." Stem cells are capable of self-regeneration and may become lineage committed progenitors which are dedicated to differentiation and expansion into a specific lineage.
A highly purified population of stem cells is necessary for a variety of in vitro experiments and in vivo indications. For instance, a purified population of stem cells will allow for identification of growth factors associated with their self-regeneration. In - 10 addition, there may be as yet undiscovered growth factors associated with: (1) the early steps of dedication of the stem cell to a particular lineage; (2) the prevention of such dedication; and (3) the negative control of stem cell proliferation.
Stem cells find use in: (1) regenerating the hematopoietic system of a host deficient in stem cells;

(2) a host that is diseased and can be treated by removal of bone marrow, isolation of stem cells and treatment of individuals with drugs or irradiation prior to re-engraftment of stem cells; (3) producing various hematopoietic cells; (4) detecting and evaluating growth factors relevant to stem cell self-regeneration; and (5) the development of hematopoietic cell lineages and assaying for factors associated with hematopoietic development. Stem cells are also a target for gene therapy to endow blood cells with useful properties.
Highly purified stem cells are essential for hematopoietic engraftment including, but not limited to, that in cancer patients and transplantation of other organs in association with hematopoietic engraftment.
Stem cells are important targets for gene therapy, where the inserted genes promote the health of the individual into whom the stem cells are transplanted. In addition, the ability to isolate stem cells may serve in the treatment of lymphomas and leukemias, as well as other 2168~
W095/03693 PCT~S94/08574 neoplastic conditions. Thus, there have been world-wide efforts toward isolating the human hematopoietic stem cell in substantially pure or pure form.
Stem cells constitute only a small percentage of the total number of hematopoietic cells.
Hematopoietic cells are identifiable by the presence of a variety of cell surface protein "markers." Such markers may be either specific to a particular lineage or progenitor cell or be present on more than one cell type.
Currently, it is not known how many of the markers associated with differentiated cells are also present on stem cells. One marker which was previously indicated as present solely on stem cells, CD34, is also found on a significant number of lineage committed progenitors.
U.S. Pat. No. 4,714,680 describes a composition comprising human stem cells.
These markers are found on numerous lineage committed hematopoietic cells. In particular, 80-90~ of the CD34+ population is marked by other lineage specific and non-specific markers. Therefore, in view of the small proportion of the total number of cells in the bone marrow or peripheral blood which are stem cells, the uncertainty of the markers associated with the stem cell as distinct from more differentiated cells, and the general inability to assay for human stem cells biologically, the identification and purification of stem cells has been elusive. Characterizations and isolation of human hematopoietic stem cells are reported in: Baum et al. (1992) Proc. Natl. Acad. Sci. USA 89:2804-2808;
and Tsukamoto et al. U.S. Patent No. 5,061,620.
Decreased rhodamine 123 (rhol23) staining of hematopoietic cells is determined not by the initial dye accumulation but by an efflux process sensitive to P-glycoprotein (P-gp) inhibitors. Retention of several P-gp-transported fluorescent dyes, including rhol23, in W095/03693 216 8 ~ i PCT~S94/08574 human bone marrow cells was inversely correlated with the expression of P-gp. Bone marrow cells expressing physical and antigenic characteristics of pluripotent stem cells showed high levels of P-gp expression and fluorescent dye efflux. Fractions of human bone marrow cells isolated on the basis of either increased rhol23 afflux or P-gp expression contained practically all the primitive progenitor cells of human bone marrow, including LTC-initiating cells. Chaudhary and Roninson ~ 10 (1991) Cell 66:85-94.
It has been shown that rhol23 labeling of stem cells is extremely dependent on the labeling conditions.
Eor instance, cells stained for longer periods of time and/or at higher temperatures will take up more rhol23.
Characterization of cells with respect to their rhol23 staining must thus take into account the staining conditions.
Recently, the mouse stem cell has been obtained in at least highly concentrated, if not a purified form, where fewer than about 30 cells obtained from bone marrow were able to reconstitute all of the lineages of the - hematopoietic system of a lethally irradiated mouse.
Each assayed cell is multipotent for all hematopoietic lineages, while self-renewal is variable amongst these cells. Spangrude et al. (1988) Science 241:58-62; Smith et al. (1991) Proc. Natl. Acad. Sci. USA 88:2788-2792;
Uchida (1992) Ph.D. Thesis Stanford U.; and see also, EPA 89 304651.6 and the references cited therein which describe the isolation of mouse stem cells.
SUMMARY OF THE INVENTION
Methods resulting in the isolation of substantially homogeneous compositions of human hematopoietic stem cells are provided. The cells are designated hu-HCA+ for expressing the marker designated WOg5/03693 216 ~ ~ 4 ~ PCT~S94/08574 the human hematopoietic cell antigen (hu-HCA). The methods employ a separation regimen utilizing an antibody that recognizes the molecule that carries the epitope recognized by a monoclonal antibody termed F84.1. Such antibodies are designated ahu-HCA. The cells derived from the method are also provided and are designated hu-HCA+.

BRIEF DESCRIPTION OF THE DRAWINGS
~ 10 Fiqure 1 depicts fluorescence activated cell sorter (FACS) analyses of various sources of stem cells.
A depicts the FACS analysis of FBM stained with the IgGl isotype control antibody and anti-CD34 antibodies (aCD3g). B depicts the FACS analysis of FBM stained with F84.1 and aCD34. C depicts the FACS analysis of adult bone marrow (ABM) stained with the IgGl isotype control antibody and aCD34. D depicts the FACS analysis of ABM
stained with F84.1 and aCD34. E depicts the FACS
analysis of mobilized peripheral blood (MPB) stained with an IgGl isotype control and aCD34. F depicts the FACS
analysis of MPB stained with F84.1 and aCD34.
Fiqure 2 depicts FACS analyses of FBM cells stained with rhol23 versus several different antibodies.
A depicts the FACS analysis of staining with aCD34 and rhol23, the gate is set for CD34+; Figures 2B-D are gated on the CD34+ population. B depicts the FACS analysis of staining with the IgG1 isotype control and rhol23. C
depicts the FACS analysis of staining with anti-Thy-1 antibody (aThy-1) and rhol23. D depicts the FACS
analysis of staining with F84.1 and rhol23.
Fiqure 3 depicts FACS analyses of ABM cells stained with rhol23 and a variety of antibodies. A
depicts the FACS analysis of staining with aCD34 and rhol23, the gate is set on CD34+ cells; Figures 3B-D are gated on the CD34+ population as shown in A. B depicts W095/03693 1 ~ PCT~S94/08~74 the FACS analysis of staining with the IgG1 isotype control and rhol23. C depicts the FACS analysis of staining with aThy-1 and rhol23. D depicts the FACS
analysis of staining with F84.1 and rhol23.
Fiqure 4 depicts FACS analyses of an early harvest of MPB cells stained with rhol23 versus a variety of antibodies. A depicts the FACS analysis of staining with ~CD34 and rhol23, the gate is set on CD34+ cells.
Analysis of staining with F84.1 and rhol23; Figures 4B-D
are gated on the CD34+ population. B depicts the FACS
analysis of staining with the IgG1 isotype control and rhol23. C depicts the FACS analysis of staining with ~Thy-1 and rhol23. D depicts the FACS of staining with F84.1 and rhol23.
Fiqure 5 depicts a late harvest of MPB cells stained with rhol23 versus a variety of antibodies. A
depicts the FACS analysis of staining with ~CD34 and rhol23, the gate is set on CD34+ cells; Figures SB-C are gated on the CD34+ population. B depicts the FACS
analysis of staining with the IgG1 isotype control and rhol23. C depicts the FACS analysis of staining with F84.1 and rhol23.
Fiq,ure 6 depicts a FACS analysis of rhol23 staining vs. hu-HCA (F84.1) staining of various populations of human hematopoietic cells. The cells were not gated on CD34+. A depicts the FACS analysis of FBM.
B depicts the FACS analysis of ABM. C depicts the FACS
analysis of mobilized peripheral blood.
Fiqure 7 is a graph depicting the proliferative potential of hu-HCA+rho+CD34+ cell populations on AC6 cells treated with LIF and IL-6. The closed squares represent cells that are hu-HCA+rhohi. The open squares represent cells that are hu-HCA+rhohi. The closed diamonds represent cells that are hu-HCA+rhomid. The open diamonds represent hu-HCA+rhol cells.

W095/03693 216 ~ 3 1~ PCT~S94/08574 DESCRIPTION OF THE SPECIFIC EMBODIMENTS
In one embodiment, the present invention provides enriched human cell compositions designated - hu-HCA+, for carrying the human marker designated hematopoietic cell antigen, "hu-HCA". hu-HCA is recognized by mAB F84.1 and antibodies which recognize the molecule that carries the epitope recognized by F84.1. Such antibodies are designated ~hu-HCA. A
substantially homogeneous composition may be obtained by selective isolation of cells that are hu-HCA+.
In another embodiment of the invention, a composition comprising hu-HCA+ stem cells is provided.
Such a composition has utility in reconstituting human hematopoietic systems and in studying various parameters of hematopoietic cells as described above.
The present invention provides for cells which are enriched for human hematopoietic cells. Cells that are hu-HCA+ and Lin~ are enriched for human hematopoietic stem cells. Lin~ cells generally refer to cells which lack markers associated with T cells (such as CD2, CD3 and 8), B cells (such as CD10, 19 and 20), myeloid cells (such as CD14, 15, 16 and 33), and RBC (such as glycophorin+). The cells may be further enriched for selecting for other stem cell specific markers. Such markers include but are not limited to Thy-1+, CD34+ and rhol. By appropriate selection with particular factors and the development of bioassays which allow for self regeneration of stem cells and screening of the stem cells as to their markers, an enriched viable human hematopoietic stem cell composition may be produced for a variety of purposes.
The stem cells may be used in hematopoietic engraftment, where the cells may be freed of neoplastic cells. Further, the use of purified stem cells will prevent graft-versus-host disease. In addition, the 21683~8 -` .
W095/03693 PCT~S94/08574 cells may be modified by appropriate gene transfer recombination, either homologous or non-homologous, to correct genetic defects or provide genetic capabilities naturally lacking in the stem cells, either as to the individual or as to stem cells generally. In addition, because the composition is substantially free of other cells, the stem cell composition may be used to isolate and define factors associated with regeneration and differentiation.
~ 10 The present invention further provides for cells which are substantially homogeneous in human hematopoietic stem cells. Thus, by appropriate selection with particular factors and the development of bioassays which allow for self regeneration of stem cells and screening of the stem cells as to their markers, a substantially homogeneous, viable, human hematopoietic stem cell composition may be produced for a variety of purposes. The stem cells may be used in hematopoietic engraftment, where the cells may be freed of neoplastic cells. Further, the use of purified stem cells will prevent graft-versus-host disease. In addition, the cells may be modified by appropriate recombination, either homologous or non-homologous, to correct genetic defects or provide genetic capabilities naturally lacking in the stem cells, either as to the individual or as to stem cells generally. In addition, because the composition is substantially free of other cells, the stem cell composition may be used to isolate and define factors associated with regeneration and differentiation.
F84.1 was obtained by immunizing a mouse with a rat cell line, XKM, a neuronal cell line. Prince et al.
(1992) Devel. Br. Res. 68:193-201. The rat neuronal protein recognized by F84.1 (the "F84.1 glycoprotein") has been purified from the clonal cell line B49 and subject to partial amino terminal amino acid sequencing.

W095/03693 2 1 6 ~ 3 4 ~ PCT~S94/08574 _9_ Prince et al. (1992). The F84.1 glycoprotein is a 90-105 kD cell surface glycoprotein with a nervous system distribution similar to that of a chicken antigen recognized by a mouse mAB designated BEN for bursal, epithelial, neural. A comparison of the partial sequence of the F84.1 glycoprotein and BEN indicates that they may belong to a family of adhesion proteins. Prince et al.
(1992). BEN also recognizes a glycoprotein of 95-100 kD
formed on bursal epithelial cells, chicken neurons and ~ 10 chicken hematopoietic cells. BEN recognizes certain chicken hematopoietic cells such as differentiating T
cells, CD4+ CD8+ T cells, and myeloid and erythroid progenitors, but not B cells. Pourquié et al. (1990) Devel. 109:743-752; and Corbel et al. (1992) Ex~. Cell Res. 203:91-99.
The results presented herein indicate that F84.1 recognizes and binds with high specificity to a cell surface antigen found on human hematopoietic cells, specifically including human hematopoietic stem cells.
This specificity can be used to isolate and purify hu-HCA+ stem cells.
As used herein, hu-HCA refers to the human protein that carries the epitope recognized by F84.1. As used herein, hu-HCA+ cells refer to those cells expressing a marker recognized by F84.1 or any antibody that binds to the molecule that carries the epitope recognized by F84.1. As used herein, ~hu-HCA means monoclonal antibody F84.1 and any monoclonal antibody and polyclonal antibody, that binds to the molecule that carries the epitope recognized by F84.1. This also includes any antibody having the same antigenic - specificity as F84.1.
The hu-HCA+ stem cells are characterized by the presence of hu-HCA and the absence or low expression of markers associated with lineage committed cells, 216~34~
WOg5/03693 ~ PCT~S94/08574 including but not limited to, T cells (such as CD2, CD3 or CD8); B cells (such as CD10, 19 or 20); myelomonocytic cells (such as CDl4, 15, 16); natural killer ("NK") cells (such as CD2) and red blood cells ("RBC") (such as glycophorin+) megakaryocytes, mast cells, eosinophils and basophils. The absence or low expression of such lineage specific markers is identified by the lack of binding of - antibodies specific to the cell specific markers, useful in so-called "negative selection". Analyses for hematopoietic progenitors have been reported by Whitlock and Witte (1982) Proc. Natl. Acad. Sci. USA 79:3608-3612;
and Whitlock et al. (1987) Cell 48:1009-1021.
Table 1 summarizes probable phenotypes of stem cells in fetal, adult, and mobilized peripheral blood.
In Table 1, myelomonocytic stands for myelomonocytic associated markers, NK stands for natural killer cells and AMPB stands for adult mobilized peripheral blood. As used herein, both infra, supra and in Table 1, the negative sign or, uppercase negative sign, (~) means that the level of the specified marker is undetectable above Ig isotype controls by FACS analysis, and includes cells with very low expression of the specified marker.

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PCT~S94/08574 Selection of stem cells need not be achieved with a marker specific for stem cells. By using a combination of negative selection (removal of lineage committed cells) and positive selection (isolation of cells), an enriched stem cell composition can be achieved.
If desired, a large proportion of differentiated cells may be removed by initially using a "relatively crude" separation. The source of the cells may be the bone marrow, fetal, neonate or adult or other hematopoietic cell source, e.g., fetal liver or peripheral blood. For example, magnetic bead separations may be used initially to remove large numbers of lineage committed cells. Desirably, at least about 80~, usually at least 70~ of the total hematopoietic cells will be removed.
At the initial stage, it is not essential to remove every dedicated cell class, particularly the minor population members. Usually, the platelets and erythrocytes will be removed prior to sorting. Since there will be positive selection in the protocol, the dedicated cells lacking the positively selected marker will be left behind. However, it is preferable that there be negative selection for all of the dedicated cell lineages, so that in the final positive selection, the number of dedicated cells present is minimized.
In addition to being hu-HCA+, the stem cells may be characterized by the following phenotypes in the case of fetal cells including, but not limited to:
CD34+, CD3-, CD7-, CD8-, CD10-, CD14-, CD15-, CDl9-, CD20-, and Thy-1+; and in the case of adult cells including, but not limited to: CD34+, CD3-, CD7-, CD8-, CD10-, CD14-, CD15-, CDl9-, CD20-, and Thy-11/+ or as represented in Table 1. Also, for human CD34+, rhol23 can divide the cells into high and low subsets ~"rhol"

W095/03693 21 6 8 3 4~ PCT~S94/08574 and "rhohi"). See Spangrude (1989) ImmunoloqY Today I0:344-350, for a description of the use of rhol23 with mouse stem cells. Preferably the cells are rhol.
In order to obtain hu-HCA+ stem cells, it is necessary to isolate the rare pluripotent human stem cell from the other cells in bone marrow or other hematopoietic source. Initially, bone marrow cells may be obtained from a source of bone marrow, including but not limited to, ilium (e.g. from the hip bone via the iliac crest), tibiae, femora, spine, or other bone cavities. Other sources of human hematopoietic stem cells include, but are not limited to, embryonic yolk sac, fetal liver, fetal and adult spleen, blood and umbilical cord blood.
For isolation of bone marrow from fetal bone or other bone source, an appropriate solution may be used to flush the bone, including, but not limited to, salt solution, conveniently supplemented with fetal calf serum (FCS) or other naturally occurring factors, in conjunction with an acceptable buffer at low concentration, generally from about 5-25 mM. Convenient buffers include, but are not limited to, Hepes, phosphate buffers and lactate buffers. Otherwise bone marrow may be aspirated from the bone in accordance with conventional ways.
Various techniques may be employed to separate the cells by initially removing cells of dedicated lineage. Monoclonal antibodies are particularly useful for identifying markers associated with particular cell lineages and/or stages of differentiation. The antibodies may be attached to a solid support to allow - for crude separation. The separation techniques employed should maximize the retention of viability of the fraction to be collected. Various techniques of different efficacy may be employed to obtain "relatively WOg5/03693 PCT~S94/08574 crude" separations. Such separations are where up to 10~, usually not more than about 5~, preferably not more than about 1%, of the total cells present not having the marker may remain with the cell population to be retained. The particular technique employed will depend upon efficiency of separation, cytotoxicity of the methodology, ease and speed of performance, and necessity for sophisticated equipment and/or technical skill.
Procedures for separation may include, but are not limited to, magnetic separation, using antibody-coated magnetic beads, affinity chromatography, cytotoxic agents joined to a monoclonal antibody or used in conjunction with a monoclonal antibody, including, but not limited to, complement and cytotoxins, and "panning"
with antibody attached to a solid matrix, e.g., plate, elutriation or any other convenient technique.
The use of separation techniques include, but are not limited to, those based on differences in physical (density gradient centrifugation and counter-flow centrifugal elutriation), cell surface (lectin and antibody affinity), and vital staining properties (mitochondria-binding dye rhol23 and DNA-binding dye Hoechst 33342).
Techniques providing accurate separation include, but are not limited to, FACS, which can have varying degrees of sophistication, e.g., a plurality of color channels, low angle and obtuse light scattering detecting channels, impedance channels, etc.
One procedure which may be used is first incubating the cells for a short period of time at reduced temperatures, generally about 4C, with saturating levels of antibodies specific for a particular committed cell type, including, but not limited to, CD3 and 8 for T cell determinants, and then washing the cells with a FCS cushion. The cells may then be suspended in a WO95/03693 2 1 6 `~ ~ ~ 8 PCT~S94/08574 buffer medium as described above and separated on the basis of the antibodies for the particular determinants, using various protein(s) specific for the antibodies or antibody-antigen complex.
Conveniently, the antibodies may be conjugated with markers, including, but not limited to, magnetic beads, which allow for direct separation, biotin, which can be removed with avidin or streptavidin bound to a support, fluorochromes, which can be used with a FACS, or the like, to allow for ease of separation of the particular cell type. Any technique may be employed which is not unduly detrimental to the viability of the re~aining cells.
Conveniently, after substantial enrichment of the cells lacking the mature cell markers, generally by at least about 50~, preferably at least about 70%, the cells may then be separated by a FACS or other methodology having high specificity. Multi-color analyses may be employed, by a variety of methods including, but not limited to, ~ACS. The cells may be separated on the basis of the level of staining for the particular antigens.
In a first separation, typically starting with about 1 x 108-9, preferably at about 5 x 108-9 cells, the antibody for CD34 or hu-HCA may be labeled with one fluorochrome, while the antibodies for the various dedicated lineages may be conjugated to a different fluorochrome. Fluorochromes which may find use in a multi-color analysis include, but are not limited to, phycobiliproteins, e.g., phycoerythrin and allophycocyanins; fluorescein and Texas red.
While each of the lineages may be separated in a separate step, desirably the lineages are separated at the same time as one is positively selecting for hu-HCA
and/or Thy and/or c/kit and/or CD34. Generally, the 216834~
W095/036g3 PCT~S94/08574 number of cells obtained after the dual selection will be fewer than about 1% of the original cells, generally fewer than about 0.5% and may be as low as 0.2% or less.
The cells may be then further separated by positively selecting for Thy+ or rhol. The mitochondrial-binding dye rhol23 has been useful in separating bone marrow progenitor populations. Low levels of rhol23 staining of hematopoietic cells correlate with its export due to expression of P-gp.
The separated cells will generally be fewer than 0.5% of the original cells, generally in the range of 0.01-0.05%. The cells may be selected against dead cells, by employing dyes associated with dead cells (including but not limited to, propidium iodide (PI)).
Preferably, the cells are collected in a medium comprising 2% FCS.
Other techniques for positive selection may be employed, which permit accurate separation, such as affinity columns, and the like. The method should permit the removal to a residual amount of less than about 20~, preferably less than about 5%, of the non-stem cell populations.
The hu-HCA+Lin~; and hu-HCA+Thy+Lin~;
CD34+Lin~; and the CD34+Lin~Thy-1+ purified cells have low side scatter and low to medium forward scatter profiles by FACS analysis. Cytospin preparations show the enriched stem cells to have a size between mature lymphoid cells and mature granulocytes. Cells may be selected based on light-scatter properties as well as their expression of various cell surface antigens.
While it is believed that the particular order of separation is not critical to this invention, the order indicated is preferred. Preferably, cells are initially separated by a coarse separation, followed by a fine separation, with positive selection of a marker W095/03693 2 1 6 8 3 4 8 PCT~S94/08574 associated with stem cells and negative selection for markers associated with lineage committed cells. This separation is followed by selection for a cellular composition having multi-lineage potential and enhanced self-regeneration capability.
- Compositions having greater than 90%, usually greater than about 95~ of hu-HCA+ stem cells may be achieved in this manner. The desired stem cells are identified by being hu-HCA+Lin~Thy-1+; and/or CD34+, and Lin~; and preferably rhol, or combinations of these markers as listed in Table 2, and being able to provide for cell regeneration and development of members of all of the various hematopoietic lineages.
Ultimately, a single cell could be obtained from such a stem cell composition and be used for long term reconstitution of a human. Note that the blank spaces in Table 2 do not mean that the cells are negative for the specified marker; they simply mean the marker is not used.

21683 la WO95/03693 PCT~S94/08574 Table 2 Possible Combinations of Selections for Stem Cell Populations 5 hu-HCA+ CD34+ Thy+ Lin~ rhol + + + + +

+ + + +

+ + +

+ +

+ + +

+ + +
+ + + +

+ + +

+ + + +

+ + +

+ + +

The compositions comprising hu-HCA+ stem cells are found to provide for production of myeloid cells and lymphoid cells in appropriate cultures, cultures providing hydrocortisone for production of myeloid cells (associated with Dexter-type cultures) and B lymphocytes in cultures lacking hydrocortisone, (associated with Whitlock-Witte type cultures). In each of the cultures, mouse or human stromal cells are provided, which may come from various strains, including, but not limited to, AC3 or AC6, stromal cells derived from mouse or human FBM by 2 1 6 ~
W095/036g3 PCT~S94/08574 selection for the ability to maintain human stem cells, and the like.
The medium employed for the culturing of the - cells is conveniently a defined enriched medium, including, but not limited to, IMDM (Iscove's Modified Dulbecco's Medium), a 50:50 mixture of IMDM and RPMI, and will generally be composed of salts, amino acids, vitamins, 5 x 10-5 M ~-mercaptoethanol (~-ME), streptomycin/penicillin and 10% FCS, and may be changed ~ 10 from time to time, generally at least about once to twice per week. Particularly, by transferring cells from one culture with hydrocortisone, to another culture without hydrocortisone, and demonstrating the production of members of the different lineages in the different cultures, the presence of the stem cell and its maintenance is supported. In this manner, one may identify the production of both myeloid cells and B
cells.
In identifying myeloid and B cell capability, conveniently, the population to be tested is introduced first into a hydrocortisone containing culture and - allowed to grow for six weeks in such culture. The medium employed will comprise a 50:50 mixture of RPMI
1640 and IMDM containing 10% FCS, 10% horse serum, streptomycin/penicillin, glutamine and 5 x 10-7 M - 1 x 10-6 M hydrocortisone. During the six week period, it would be anticipated that in the absence of progenitor cells, all of the mature cells would die. If at the end of six weeks, myeloid cells are still observed, one may conclude that there is a progenitor cell which is providing for the continuous differentiation to myeloid cells. At this time, one may then change the medium, so that the medium now lacks hydrocortisone, to encourage the growth of B cells. By waiting 4-8 weeks and demonstrating the presence of B cells by FACS analysis, 216834~
W095/03693 PCT~S94/08574 one may conclude that the progenitor cells which previously were capable of producing myeloid cells are also capable of producing B cells. Human hematopoietic cells grown in the presence of hydrocortisone can be maintained for at least 1 month, sometimes greater than 4 months. Similarly, human hematopoietic cells grown in the absence of hydrocortisone contain B lymphocytes (CDl9+), as well as myelomonocytic cells for at least four months. From these cultures, one may sort for ~ 10 hu-HCA+Lin~, which should provide a composition substantially concentrated in the progenitor hematopoietic stem cell.
By separating hu-HCA+Lin~ cells from human hematopoietic sources, the long-term culture activity is enriched in the hu-HCA+ fraction compared to hu-HCA-.
Moreover, the hu-HCA+ cells will generate both B and myeloid cells in long-term cultures. In further enrichments of the hu-HCA+ cells using antibodies to Thyl and/or any of the combinations specified in Table 2 and/or c-kit, the frequency can be further increased.
The cells generated from hu-HCA+Lin cells and obtained from these cultures can give rise to B cells, T
cells and myelomonocytic cells in the in vivo assays described below.
To demonstrate differentiation to T cells, fetal thymus is isolated and cultured from 4-7 days at about 25C, so as to deplete substantially the lymphoid population. The cells to be tested for T cell activity are then microinjected into the thymus tissue, where the HLA of the population which is injected is mismatched with the HLA of the thymus cells. The thymus tissue may then be transplanted into a scid/scid mouse as described in US Patent No. 5,147,784, particularly transplanting under the kidney capsule.

WO95/03693 21 fi ~ 3 4 ~ PCT~S94/08574 Specifically, the sorted population of hu-HCA+Lin~ can be microinjected into HLA mismatched thymus fragments. After 6-10 weeks, assays of the thymus fragments injected with hu-HCA+Lin~ cells can be performed and assessed for donor derived T cells. One - expects to find that thymus fragments injected with the hu-HCA+Lin~ fraction generates and sustains CD3+, 4+, and 8+ T cells along with their progenitors. This would be distinct from what is observed in the fragments injected ~ 10 with the hu-HCA~Lin~ fractions. Subfractionation of the hu-HCA+Lin~ fraction based on Thy+ and/or CD34+ and/or c-kit and/or rhol23 should demonstrate enrichment of activity.
Further demonstration of the sustained ability of the various cell populations might be accomplished by the detection of continued myeloid and B-lymphoid cell production in the SCID-hu bone model. Kyoizumi et al.
(1992) Blood 79:1704. To analyze this, one may isolate human fetal bone and transfer a longitudinally sliced portion of this bone under the skin of a SCID animal:
the bone cavity is depleted of endogenous cells by incubation in vivo for about 8 weeks. Prior to injection of the test donor population, the host mouse is irradiated. The HLA of the population which is injected is mismatched with the HLA of the bone cells.
By separating hu-HCA+Lin~ cells from human hematopoietic sources, or from long-term in vitro cultures, the hu-HCA+Lin~ fraction may be able to sustain B lymphopoiesis and myelopoiesis. In addition, activity of the various cell populations may be found by harvesting the cells from this primary recipient and transferring the cells to a secondary host.
For RBCs, one may use conventional techniques to identify BFU-E units, for example methylcellulose 3S culture demonstrating that the cells are capable of 216~3~8 W095/036g3 ;' PCT~S94/08574 developing the erythroid lineage. Metcalf (1977) In:
Recent Results in Cancer Research 61. Springer-Verlag, Berlin, pp. 1-227 A pluripotent human stem cell may be defined as follows: (1) gives rise to progeny in all defined hematolymphoid lineages; and (2) limiting numbers of cells are capable of fully reconstituting a seriously immunocompromised human host in all blood cell types and their progenitors, including the pluripotent hematopoietic stem cell by cell renewal.
Once stem cells have been isolated, they may be propagated by growing in conditioned medium from stromal cells, such as stromal cells that can be obtained from bone marrow, fetal thymus or fetal liver, and are known to provide for the secretion of growth factors associated with stem cell maintenance, co-culturing with such stromal cells, or in medium comprising maintenance factors supporting the proliferation of stem cells, where the stromal cells may be allogeneic or xenogeneic.
Before using in the co-culture, the mixed stromal cell preparations may be freed of hematopoietic cells employing appropriate monoclonal antibodies for removal of the undesired cells, e.g., with antibody-toxin conjugates, antibody and complement, etc. Alternatively, cloned stromal cell lines may be used where the stromal lines may be allogeneic or xenogeneic.
The subject cell compositions may find use in a variety of ways. Since the cells are naive, they can be used to fully reconstitute an irradiated host and/or a host subject to chemotherapy; or as a source of cells for specific lineages, by providing for their maturation, proliferation and differentiation into one or more selected lineages by employing a variety of factors, including, but not limited to, erythropoietin, colony stimulating factors, e.g., GM-CSF, G-CSF, or M-CSF, 216~3~
WOg5/03693 PCT~S94/08574 interleukins, e.g., IL-1, -2, -3, -4, -5, -6, -7, -8, etc., or the like, or stromal cells associated with the stem cells becoming committed to a particular lineage, or with their proliferation, maturation and differentiation.
- 5 The hu-HCA+ stem cells may also be used in the isolation and evaluation of factors associated with the differentiation and maturation of hematopoietic cells.
Thus, the hu-HCA+ stem cells may be used in assays to ~ determine the activity of media, such as conditioned media, evaluate fluids for cell growth activity, involve-ment with dedication of particular lineages, or the like.
The hu-HCA+ stem cells may be used for the treatment of genetic diseases. Genetic diseases associ-ated with hematopoietic cells may be treated by genetic modification of autologous or allogeneic stem cells to correct the genetic defect. ~or example, diseases including, but not limited to, ~-thalassemia, sickle cell anemia, adenosine de~m;n~.ce deficiency, recombinase deficiency, recombinase regulatory gene deficiency, etc.
may be corrected by introduction of a wild-type gene into the hu-HCA+ stem cells, either by homologous or random recombination. Other indications of gene therapy are introduction of drug resistance genes to enable normal stem cells to have an advantage and be subject to selective pressure during chemotherapy. Suitable drug resistance genes include, but are not limited to, the gene encoding the multidrug resistance (MDR) protein.
Diseases other than those associated with hematopoietic cells may also be treated, where the disease is related to the lack of a particular secreted product including, but not limited to, hormones, enzymes, interferon, growth factors, or the like. By employing an appropriate regulatory initiation region, inducible production of the deficient protein may be achieved, so that production of the protein will parallel natural 216$3~8 W095/03693 PCT~S94/08574 production, even though production will be in a different cell type from the cell type that normally produces such protein. It is also possible to insert a ribozyme, antisense or other message to inhibit particular gene products or susceptibility to diseases, particularly hematolymphotropic diseases.
Alternatively, one may wish to ~e---ove a particular variable region of a T-cell receptor from the T-cell repertoire. By employing homologous recombination, or antisense or ribozyme sequence which prevents expression, the expression of the particular T-cell receptor may be inhibited. For hematotrophic pathogens, such as HIV, HTLV-I and II, etc. the stem cells could be genetically modified to introduce an antisense sequence or ribozyme which would prevent the proliferation of the pathogen in the stem cell or cells differentiated from the stem cells.
Methods for recombination in m~m~l ian cells may be found in Molecular Cloning, A Laboratory Manual (1989) Sambrook, Fritsch and Maniatis, Cold Spring Harbor, NY.
A composition comprising substantially homogeneous hu-HCA+ cells is also provided. The cells obtained as described above may be used immediately or frozen at liquid nitrogen temperatures and stored for long periods of time, being thawed and capable of being reused. The cells will usually be stored in 10% DMSO, 50% FCS, 40% RPMI 1640 medium. Once thawed, the cells may be expanded by use of growth factors or stromal cells associated with stem cell proliferation and differentiation.
In another embodiment, polyclonal and/or monoclonal antibodies capable of specifically binding to a protein(s) or fragments thereof are provided. The term antibody is used to refer both to a homogeneous molecular 216~3~
W095/03693 PCT~S94/08574 entity, or a mixture such as a serum product made up of a plurality of different molecular entities. Monoclonal or polyclonal antibodies specifically reacting with the protein(s) may be made by methods known in the art. See e.~., Harlow and Lane, (1988) Antibodies: A LaboratorY
Manual, CSH Laboratories; Goding (1986) Monoclonal Anti-bodies: Princi~les and Practice, 2d ed, Academic Press, New York; and Ausubel et al. (1987). Also, recombinant immunoglobulins may be produced by methods known in the art, including, but not limited to, the methods described in U.S. Patent No. 4,816,567. Monoclonal antibodies with affinities of 108 M 1 preferably 109 to 101 or more are preferred.
Antibodies specific for the protein may be useful in purifying stem cells. Such antibodies, whether monoclonal or polyclonal, can be made by methods known in the art utilizing as the immunogen the purified human protein recognized by F84.1. The purified hu-HCA may be obtained from native sources. Alternatively, the antibodies can be obtained in the same manner as described in the art. Pourquié et al. (1990); and Prince et al. (1992). Frequently, the polypeptides and antibodies will be labeled by joining, either covalently or noncovalently, a substance which provides a detectable signal. Suitable labels include, but are not limited to, radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent agents, chemiluminescent agents, magnetic particles and the like. United States Patents describing the use of such labels include, but are not 30 limited to, Nos. 3,817,837; 3,850,752; 3,939,350;
3,996,345; 4,277,437; 4,275,149; and 4,366,241.
The following examples are offered by way of illustration and not by way of limitation.

216834~
WOg5/03693 ~ PCT~S94/08574 Materials and Methods.
Antibodies. The antibodies to the various markers were obtained as follows: CD3, 8, 10, 14, 15, 19, 20 and 33 (Becton-Dickinson). A Thy-l antibody like that described in Dalchau and Fabre (1979) J. Ex~. Med.
149:576 was used. ~CD34 (Tuk3) from Dr. S. Ziegler has also been used. The antibodies were detected using the appropriate anti-Ig conjugated to fluorescein or ~ 10 phycoerythrin or Texas red (Caltag). The Thy-l antibody was a fluorescein, phycoerythrin or biotin conjugate, where the biotin conjugate was detected with Texas Red-avidin (Caltag). The CD34 antibody was detected using anti-mouse IgG3 conjugated to Texas red (Southern Biotech). The Thy-l and F84.1 antibodies were detected using anti-mouse IgGl conjugated to phycoerythrin (Caltag).

FACS AnalYsis and Sortinq A Becton-Dickinson FACStar Plus was employed.
The dual laser instrument allows four fluorescent parameters and two light scatter parameters to be recorded for each analyzed cell. Residual erythrocytes and dead cells and debris were excluded from analysis by light scattering gating and PI staining or by scattering alone in 4-color analyses. Compensation for spatial overlaps of fluorescein and phycoerythrin, and fluorescein and PI was adjusted electronically as described by Parks and Herzenberg (1984? Met. EnzYmol.
108:197. Four color stains were performed using several combinations of the same reagents conjugated to different fluorochromes to assure that the results were consistent regardless of the various spatial overlaps of the fluorochromes. In addition, the results of 4-color 21fi834~
WOg5/03693 PCT~S94/08574 analyses were calibrated by comparison with data from 2-and 3- color analyses.
For cell sorting, the stained samples were maintained at 4C throughout the sorting procedure.
Sorted drops were collected in RPMI 1640 or HBSS
containing 2% FCS (Hyclone, Inc.) and 10 mM Hepes pH 7.4.
Two or three color sorts employed Texas red to label CD34, phycoerythrin to label Thy-1 or F84.1, and rhol23 in the fluorescein channel, with PI to label dead cells, ~ 10 with both signals being detected and excluded in a single FACS channel. Following isolation of a cell population by FACS, the sample was pelleted and resuspended in HBSS
for hemocytomer counting.
Rhol23 staining was performed as described previously by Bertoncello et al. (1988) Ex~. Hematol.
16:24S-249; and Spangrude and Johnson (1990) Proc. Natl.
Acad. Sci. USA 87:7433-7437. Briefly, l mg/ml stock solution of rhol23 (Molecular Probes, Eugene, OR) was prepared. Human cells were resuspended at 1-5 x 106/ml or less in buffer (HBSS, RPMI with HEPES or PBS) containing 2% FCS, and incubated with 0.1 ~g/ml of rhol23 for 30 min at 37C. The cells were washed, and incubated at 37C for 30 to 120 min, typically, 40 min with buffer without rhol23 dye. The cells were washed again, and stained with antibodies.
The fluorescence intensity of rhol23 was analyzed in the FITC channel. The gate for rhohi versus rhol was set just below the staining of the major population of either total cells or C~34+ cells. Note, as discussed above, the incubation time and temperature critically define the relative percent of rhol versus rhohi cells.
Examples of the staining of CD34+ cells with Thy-1 or F84.1 antibodies are given in Figures 1-5. The FACS analyses shown in Figure 1 depict the co-staining of 216~34~
W095/03693 PCT~S94/08574 cells with ~CD34 and F84.1. In addition, the results presented in Figure 1 indicate that F84.1 stains a subset of hematopoietic cells which are CD34- and therefore probably lineage-committed. The FACS analyses shown in Figure 2 show that, for FBM, F84.1 subdivides the CD34+
cells into two distinct populations, F84.1+ and F84.1-.
Moreover, most or all the rhol23 low-staining cells are F84.1+. The FACS analyses shown in Figure 3 show that, for ABM, F84.1 subdivides the CD34+ cells into two ~ 10 distinct populations, F84.1+ and F84.1-. Moreover, all the rhol23 low-staining cells are F84.1+. The FACS
analyses shown in Figure 4, show that, for ABM, F84.1 subdivides the CD34+ cells into two distinct populations, F84.1+ and F84.1-. Moreover, all the rhol23 low-staining cells are F84.1+. The FACS analyses shown in Figure 5 show that, for MPB, F84.1 subdivides the CD34+ cells into two distinct populations, F84.1+ and F84.1-. Moreover, all the rhol23 low-staining cells are F84.1+.
FACS analysis of FBM, ABM or MPB was performed dividing fractions into CD34+1in~ cells and further analyzing this fraction for Thy+, F84.1+ and/or rhol23 staining. Based on this analysis it is possible to subdivide the CD34+ fraction or alternately whole bone marrow or MPB and grow them continuously in culture for eight weeks and screen for the presence of myeloid cells and B cells.
FBM, ABM, and MPB can be separated by FACS into hu-HCA+rholThy+ or other combinations of hu-HCA, rhol23, Thy, CD34, and lin~ and the cells can be grown in co-cultures. (See Table 2) By employing limited dilutionanalysis, one can determine which populations are able to be maintained in the co-culture for greater than six weeks and be differentiated into mature myeloid and B
cells.

WOg5/03693 216 8 3 ~ ~3 PCT~S94/08574 The sorted cell populations as well as the unsorted cells are analyzed at the time of separation (t=0) as well as twenty-one days (t=21) later.

Characterization of hu-HCA+ cells In order to determine the ability of hu-HCA+
cells to repopulate the hematopoietic system, the following experiments were performed. First, the expression of HCA and rhol23 were monitored on a variety of human hematopoietic cells. Second the long-term proliferative potential of ABM cells was determined in cell cultures assay.
For both experiments, the cells were obtained by methods known in the art. CD34+ cells were obtained from these crude cell preparations using the CellPro column according to the manufacturer's instructions.
Staining and separation with rhol23, ~CD34 and F84.1 were as described in Example 2. Figure 6 shows the results of FACS analyses of the various cell types. The results show that in both adult and fetal bone marrow, a small percentage of rhol cells are also hu-HCA+ whereas in mobilized peripheral blood, a larger proportion of rhol cells are also hu-HCA+. The rhohi populations in each case are approximately half hu-HCA+.
In order to determine long-term proliferative potential of the various sorted populations, culture assays were performed as follows.
A murine stromal cell line, AC6, described in Whitlock et al. (1987) Cell 48:1009-1021, serves as the supportive environment. Confluent stromal cell layers were maintained for up to 7-8 weeks without passage by changing of the tissue culture medium every 5-7 days. To passage, the stromal cell layers were washed 3 times with serum-free medium, then overlayed with 2.5 ml (T-25 21683~g WO95/036g3 PCT~S94/08S74 flask) of 0.5 mg/ml collagenase-dispase (Boehringer-Mannheim, Indianapolis, IN) in serum-free medium. The cultures were allowed to incubate 15-30 minutes at 37C; then the cells in the enzyme-containing medium were collected and RPMI-1640 medium with serum added. The stromal cells were suspended by pipetting with a Pasteur pipette, then cultured directly at 1/5th to l/50th the original cell concentration. In general, confluent stromal layers subcultured at 1:10 reached confluency again after 5-7 days. Subclones were obtained by limiting dilution culture from 30 to 0.3 cells per well.
Adult bone marrow cells were sorted for CD34 expression as described above and divided into hu-HCA-rhohi, hu-HCA+rhohi, hu-HCA+rhomid and hu-HCA+rhol and were placed by automated deposition directly onto 96-well microtiter plates containing the pre-established mouse stromal cell lay at 1, 3, 5 or 15 cells per well with at least 96 wells plated at each concentration.
The cells were cultured on the stromal monolayer in medium containing IL-6 (lO ng/ml) and leukemia inhibitory factor (LIF) (50 ng/ml) (Sandoz Pharma). Half the cytokine-containing medium was replaced weekly. The cultures were read starting at 3 weeks, with positive cells being harvested at 6-8 weeks for phenotypic analyses. Phenotypic analyses using antibodies to CDl9, CD33 and CD34 showed that progenitor, B and myeloid cells were present. The results from the two experiments were averaged and the long term culture initiating cell (LTC-IC) frequencies were arrived at by a linear regression analysis. The results obtained are shown in Figure 7. These results indicate that the CD34+hu-HCA+rhol subset is highly enriched for LTC-IC.

wo gs/03693 2 1 6 8 3 ~ ~ PCT~S94/08574 Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent to those skilled in the art that certain changes and modifications may be practiced. Therefore, the description and examples should not be construed as limiting the scope of the invention which is delineated by the appended claims.
.

Claims (34)

What is claimed is:

1. A method for obtaining a substantially homogeneous composition comprising human hematopoietic stem cells, comprising:
separating a mixture of human hematopoietic cells into an enriched fraction comprising cells recognized by an antibody that recognizes the molecule that carries the epitope recognized by monoclonal antibody F84.1.

2. The method according to claim 1, wherein the hu-HCA+ cells are further characterized by having other stem cell specific markers.

3. The method according to claim 1, wherein the hu-HCA+ cells are further characterized by being CD34+.

4. The method according to claim 1, wherein the hu-HCA+ cells are further characterized by being Thy-1+.

5. The method according to claim 1, wherein the hu-HCA+ cells are further characterized by being rho10.

6. The method according to claim 1, wherein the hu-HCA+ cells are further separated from cells expressing lineage specific markers.

7. The method according to claim 1, wherein the hu-HCA+ cells are further characterized by having other stem cell specific markers.

8. The method according to claim 1, wherein the hu-HCA+ cells are further characterized by being CD34+.

9. The method according to claim 8 wherein the hu-HCA+ cells are further characterized by being lin.

10. The method according to claim 8, wherein the hu-HCA+ cells are further characterized by being Thy-1+.

11. The method according to claim 8, wherein the hu-HCA+ cells are further characterized by being rho?.

12. The method according to claim 8, wherein the separation comprises:
combining the mixture with an antibody to hu-HCA conjugated to fluorescent labels and antibodies to lineage specific markers wherein said .alpha.hu-HCA antibodies have a different fluorescent label from the other antibodies; and separating a cell fraction characterized by being hu-HCA+Lin~ by means of said fluorescent labels.

13. A composition comprising a substantially pure population of hematopoietic cells obtained by the method according to claim 1.

14. The composition according to claim 13, wherein the hu-HCA+ cells are further characterized by having other stem cell specific markers.

15. The composition according to claim 13, wherein the hu-HCA+ cells are further characterized by being CD34+.

16. The composition according to claim 13, wherein the hu-HCA+ cells are further characterized by being Thy-l+.

17. The composition according to claim 13, wherein the hu-HCA+ cells are further characterized by being rho?.

18. A composition comprising a substantially pure population of hematopoietic stem cells obtained by the method according to claim 8.

19. The composition according to claim 18, wherein the hu-HCA+ cells are further characterized by having other stem cell specific markers.

20. The composition according to claim 18, wherein the hu-HCA+ cells are further characterized by being CD34+.

21. The composition according to claim 18, wherein the hu-HCA+ cells are further characterized by being Lin.

22. The composition according to claim 18, wherein the hu-HCA+ cells are further characterized by being Thy-1+.

23. The composition according to claim 18, wherein the hu-HCA+ cells are further characterized by being rho10.

24. A composition comprising human hematopoietic cells recognized by an antibody which recognizes hu-HCA.

25. The composition according to claim 24, wherein the hu-HCA+ cells are further characterized by having other stem cell specific markers.

26. The composition according to claim 24, wherein the hu-HCA+ cells are further characterized by being CD34+.

27. The composition according to claim 24, wherein the hu-HCA+ cells are further characterized by being Thy-1+.

28. The composition according to claim 24, wherein the hu-HCA+ cells are further characterized by being rho10.

29. A composition comprising human hematopoietic stem cells recognized by an antibody which recognizes hu-HCA.

30. The composition according to claim 29, wherein the hu-HCA+ cells are further characterized by having other stem cell specific markers.

31. The composition according to claim 29, wherein the hu-HCA+ cells are further characterized by being CD34+.

32. The composition according to claim 29, wherein the hu-HCA+ cells are further characterized by being Lin-.

33. The composition according to claim 29, wherein the hu-HCA+ cells are further characterized by being Thy-1+.

34.- The composition according to claim 29, wherein the hu-HCA+ cells are further characterized by being rho10.