WO1994008039A1

WO1994008039A1 - Method of enrichment for human hematopoietic stem cells using c-kit

Info

Publication number: WO1994008039A1
Application number: PCT/US1993/008772
Authority: WO
Inventors: Anne-Marie Buckle; Charles M. Baum
Original assignee: Systemix, Inc.
Priority date: 1992-10-02
Filing date: 1993-09-16
Publication date: 1994-04-14
Also published as: AU5143393A

Abstract

Methods to enrich for human hematopoietic stem cells by selection for cells which express c-kit protein are provided. A human stem cell gives rise to progeny in all defined hematopoietic lineages, and is capable of fully reconstituting an immunocompromised host in all blood cell types, including the pluripotent human stem cell with limiting numbers of cells. C-kit is expressed by a number of early hematopoietic progenitors, including the human stem cell. Selection for cells which express c-kit provides a method to enrich for human hematopoietic stem cells.

Description

METHOD OF ENRICHMENT FOR HUMAN HEMATOPOIETIC STEM CELLS

USING C-KIT

INTRODUCTION Technical Field

The field of this invention is the isolation of human hematopoietic stem cells.

Background

In mammals, there is a constant turnover of blood cells. To provide a source for these cells, it is believed that there is a single cell type, the hematopoietic stem cell, which is capable of giving rise to all the blood cell lineages. The stem cell divides to give rise to cells which are committed to a specific lineage, or to produce more stem cells by self-regeneration. The stem cell population constitutes only a small percentage of the total number of cells in bone marrow. They have been characterized by the absence or presence of markers on the cell surface. The phenotype for a highly enriched human stem cell fraction is reported as CD34+, Thy-1+ and lin-. The blood cell lineages include lymphoid, myeloid and erythroid cells. Cells of the lymphoid lineage, B cells and T cells, produce antibodies, regulate the cellular immune system and detect foreign antigens and cells. The myeloid lineage, which includes monocytes, granulocytes, megakaryocytes as well as other cells, monitors for the presence of foreign bodies in the blood stream, provides protection against neoplastic cells, scavenges foreign materials from the blood stream and produces platelets. The erythroid lineage provides red blood cells, which act as oxygen carriers.

There are a number of clinical uses for a purified stem cell population. Gene therapy may rely on transformation of a self-renewing population such as the stem cell. Bone marrow transplantation is currently used in conjunction with chemotherapy and radiation for the treatment of leukemia and other cancer patients. However, there is considerable difficulty in finding a suitably matched donor for bone marrow. Autologous transplants, with the patients own cells, avoid the danger of graft rejection, but have a high incidence of disease reappearance, due to the presence of tumor cells in the bone marrow. Methods for purifying stem cells away from other cell types are a benefit to such patients. While there are protocols for such separations, there is a constant need to provide alternative and improved purification techniques.

Defects in the normal regulation of hematopoiesis have been mapped to the dominant white spotting (W) and steel (SI) loci. Mutations at the SI locus result in microenvironmental hematopoietic abnormalities in mice. The Si locus has been shown to encode a stromal cell growth factor, termed Steel factor (SLF) . The product of the W locus has been identified as the receptor for SLF, and is the proto-oncogene c-kit. Data suggest that SLF is required for the normal development of hematopoietic cells. It is therefore of interest to determine whether its receptor, the c-kit protein, may serve as a useful marker in a separation scheme to enrich for human hematopoietic stem cells or other hematopoietic cell fractions of interest.

Relevant Literature U.S. Patent no. 5,061,620 describes the characterization of human stem cells. The phenotype of stem cells with rhodamine staining is discussed in Spangrude and Johnson (1990) P.N.A.S. 87:7433-7437. The expression of c-kit on mouse hematopoietic progenitor cells is discussed in Ikuta and Weissman (1992) P.N.A.S. 89:1502-1506; and Okada, et al. (1991) Blood 78:1706-1712. Analysis of c-kit expression on human hematopoietic progenitors cells can be found in Broudy, et al. (1992) Blood 79:338-346; Buhring, et al. (1991) Leukemia 5:854-860; and Andre, et al. (1989) Oncogene 4:1047-1049. Background on the c-kit gene can be found in Besmer, et al. (1986) Nature 320:415-421; and Chabot, et al. (1988) Nature 335:88-89.

SUMMARY OF THE INVENTION Methods resulting in the isolation of substantially enriched compositions of human hematopoietic stem cells are provided. The cell enrichment methods employ reagents which specifically recognize c-kit protein, in conjunction with other markers which discriminate between hematopoietic stem cells and other hematopoietic cells.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS Methods to enrich for human hematopoietic stem cells by employing a separation scheme which includes selection for cells which express c-kit protein are provided. C-kit is a surface membrane protein with tyrosine kinase activity. It is the receptor for the gene product of the Steel locus, SLF, or stem cell factor. Mutations in both the c- kit and Steel loci are known to cause abnormalities in hematopoietic function. Expression of c-kit may be regulated by the differentiation state of a cell, as well as the local microenvironment.

The stem cell may be distinguished from other progenitor cells by its ability to repopulate all hematopoietic lineages, that is, lymphoid, myeloid and erythroid cells. C-kit protein is expressed on a number of early hematopoietic progenitors, including highly enriched human stem cells, and so may be used as a selection marker. There is interest in the use of viable, functional, purified hematopoietic stem cells for bone marrow transplantation. Either a patient's own marrow or blood, or allogeneic bone marrow or blood may be used for stem cell isolation to reconstitute a severely immunocompromised patient-. Often such patients are being treated for leukemias and lymphomas by intense radiation or chemotherapy, followed by a transplant to restore hematopoietic function. In order to perform an autologous bone marrow transplant, the stem cell source must be at least substantially free of contaminating tumor cells.

A method for enrichment of substantially pure hematopoietic stem cells is provided. Such cells may serve as the progenitors for all human hematopoietic cell lineages. The stem cells are separated from other bone marrow, blood or other peripheral blood organ cells on the basis of specific markers which are identified with monoclonal antibodies. The method calls for the selective isolation of cells which are free of markers associated with differentiated cells, while displaying epitopic characteristics of stem cells.

The specific markers may be chosen to minimize the background contamination from specific cell types. In particular, it has been reported that c-kit is not expressed by the common lymphocytic leukemias C-ALL, B-ALL and T-ALL (Buhring, et al. (1991) Leukemia 5:854). Selection with this marker is therefore useful in preparing stem cells from patients with these diseases, and other blood diseases, where c-kit is not found on the diseased cells.

Human stem cells have been reported to have the phenotype CD34+, CD3-, CD7-, CD8-, CD10-, CD14-, CD15-, CD19-, CD20-, and Thy-1+ (see U.S. Patent 5,061,620). In addition, the cells may be CD38 dull. A negative designation indicates that the level of staining is at or below the brightness of an isotype matched negative control. A dull designation indicates that the level of staining may be near the level of a negative stain, but may also be brighter than an isotype matched control. An enriched population for stem cells is CD34+, more particularly in addition CD10-, CD19- and optionally CD33-, preferably in addition CD3- and CD8-. The combination of markers may be detected by staining with a lineage cocktail which combines antibodies against CD10, CD19 optionally CD33, and may also include antibody against CD3 and CD8. The CD10-, 19- and optionally 33- will be referred to as lin^". The CD10/19 markers are associated with B cells, while the CD33 cell marker is associated with myeloid cells. Also, rhodamine 123 staining can split the cells into high and low fluorescent subsets; stem cell activity is usually found in the rhodamine low fraction.

Stem cell activity has also been found in the c-kit positive fraction of bone marrow. The percentage of bone marrow monocytic cells that stains with c-kit is comparable to the number that stain with CD34. It is therefore possible to use c-kit instead of CD34 to enrich for stem cell activity. There is a technical advantage in the use of c-kit as a marker, because the population is sharply delineated when analyzed by fluorescence activated flow cytometry. This phenomenon may be attributed to a higher antigen density on the cell surface.

In order to obtain the subject stem cells it is necessary to isolate the rare pluripotent human stem cells from the other cells in the bone marrow or other hematopoietic source. Initially, bone marrow cells may be obtained from a source of bone, eg. tibiae, femora, spine or other bone cavities. Other sources of human hematopoietic stem cells include embryonic yolk sac, fetal liver, fetal and adult spleen and blood. For isolation of bone marrow from fetal bone or other bone source, an appropriate solution may be used to flush the bone. Such solution will be a balanced salt solution, conveniently supplemented with fetal calf serum or other naturally occurring factors, in conjunction with an acceptable buffer at low concentration, generally from 5- 25 mM. Convenient buffers include HEPES, phosphate buffers, lactate buffers, etc. Otherwise bone marrow may be aspirated from the bone in accordance with conventional methods.

Various techniques may be employed to separate the cells by initially removing cells dedicated to particular lineages and/or to levels of differentiation. Monoclonal antibodies are particularly useful for identifying markers (surface membrane proteins) 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 particular technique employed will depend on the 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 magnetic separation, using antibody-coated magnetic beads, affinity chromatography, cytotoxic agents joined to a monoclonal antibody or used in conjunction with a monoclonal antibody, eg. complement and cytotoxins, and "panning" with antibody attached to a solid matrix, eg. plate, or other convenient technique. Techniques providing accurate separation include fluorescence activated cell sorters, which can have varying degrees of sophistication, such as multiple color channels, low angle and obtuse light scattering detecting channels, impedance channels, etc. The cells may be selected against dead cells by employing dyes associated with dead cells (propidium iodide, LDS) . Red blood cells may be removed by elutriation. Other techniques for positive selection may be employed, which permit accurate separation, such as affinity columns, and the like. Any technique may be employed which is not unduly detrimental to the viability of the selected cells.

One procedure which may be used in a first step is to incubate the cells from the bone marrow for a short period of time at reduced temperatures, generally about 4°C, with saturating levels of antibodies specific for a particular cell type. The cells may then be washed with a fetal calf serum (FCS) cushion, resuspended in a buffer medium as described above and separated by means of the antibodies for the particular determinants.

Conveniently, these antibodies may be conjugated with markers, such as 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 fluorescence activated cell sorter, or the like, to allow for ease of separation of the particular cell type. Multi-color analyses may be employed with the FACS. Fluorochromes which find use in a multi-color analysis include phycobiliproteins, e.g. phycoerythrin and allophycocyanins, fluorescein and Texas red. While each of the lineages may be separated in a separate step, desirably a plurality of lineages may be separated at the same time as one is positively selecting for c-kit and/or CD34. Generally, the number of cells obtained will be fewer than 1% of the original cells, generally fewer than 0.5% and may be as low as 0.2% or less.

The purified stem cell fraction has low side scatter and low forward scatter profiles by FACS analysis. Cytospin preparations show the stem cell has 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 markers. Compositions highly enriched for human stem cells may be achieved in this manner. The stem cells will be at or about 80% or more of the cell composition, and preferably be at or about 90% or more of the cell composition. The desired stem cells are identified by being c-kit+ and at least one of thy-l+, HLA-DR+, CD34+, CD38 dull, rhodamine low and lin-, and being able to provide for development of members of all the various hematopoietic lineages. Ultimately, a single engrafted cell could be obtained from a stem cell composition and be used for long term reconstitution of an immunodeficient human.

Once stem cells have been isolated, they may be propagated by growing in conditioned medium from stromal cells, coculturing with such stromal cells, or in medium comprising maintenance factors supporting the proliferation of stem cells. Under the appropriate conditions the subject compositions are found to provide for production of myeloid cells and lymphoid cells in such cultures. In each of the cultures, mouse or human stromal cells are provided. Stromal cells may be a primary culture from bone marrow, or a cell line derived from mouse or human bone marrow by selection for the ability to maintain human stem cells. The medium employed for culturing cells is conveniently a defined enriched medium, such as IMDM or a mixture of IMDM and RPMI, and will generally be composed of salts, amino acids, vitamins, 5X10^"5M 2-ME, streptomycin/penicillin and 10% fetal calf serum, and may be changed from time to time, generally at least once to twice per week. By demonstrating the production of members of the different lineages in the cultures, the presence of the stem cell and its maintenance is supported. In this manner, one may identify the production of both myeloid and B cells. T cell production may be identified as described in U.S. Patent No. 5,061,620.

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. By providing for maturation, proliferation and differentiation into one or more selected lineages by employing a variety of factors the stem cells may be used as a source of committed cells. Such factors as erythropoietin, colony stimulating factors, e.g. GM-CSF, G-CSF or M-CSF, interleukins e.g. IL-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, etc., or the like, or stromal cells may be used which are associated with the stem cells becoming committed to a particular lineage, or with their proliferation, maturation or differentiation. The stem cells may also be used in the isolation and evaluation of factors associated with the differentiation and maturation of hematopoietic cells. Thus, the stem cells may be used in assays to determine the activity of media, such as conditioned media, evaluate fluids for growth factor activity, involvement with dedication of lineages, or the like.

The stem cells may be used for the treatment of genetic diseases. Genetic diseases associated with hematopoietic cells may be treated by genetic modification of autologous or allogeneic stem cells to correct a genetic defect or treat to protect against disease, e.g. HIV. For example, diseases such as β-thalassemia, sickle cell anemia, adenosine deaminase deficiency, recombinase deficiency, recombinase regulatory gene deficiency, etc. may be corrected by introduction of the wild-type gene into the human stem cell, either by homologous or random recombination. Alternatively, normal allogeneic stem cells may be transplanted. Other methods of gene therapy are the introduction of drug resistance genes to enable normal stem cells to have an advantage and be subject to selective pressure, e.g. the multiple drug resistance gene (MDR) .

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 such as hormone, enzyme, interferon, factor, or the like. DNA constructs may be introduced into the stem cells to provide for the synthesis and regulation of the desired product.

The cells may be 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.

A pluripotent human stem cell may be defined as a cell which gives rise to progeny in all defined hematopoietic lineages, and where limiting numbers of cells are capable of fully reconstituting an immunocompromised human host in all blood cell types, including the pluripotent human stem cell. Based on these standards, selection for cells which express c-kit will enrich for human hematopoietic stem cells.

The following examples are offered by way of illustration and not by way of limitation.

EXPERIMENTAL

Materials and Methods

Antibodies. Antibodies to CD3, 4, 8, 14, 15, 16, 19, 20 and 34 were obtained from Becton-Dickinson. The CD3, 4, 8, 14, 15, 16, 19, and 20 were purchased as FITC conjugates. The c-kit antibody was purchased from AMAC, and detected using the appropriate anti-Ig conjugated to fluorescein, phycoerythrin or Texas Red (Caltag) .

Fluorescence Activated Cell Sorter Analysis and Sorting. A Becton-Dickinson FACS modified as previously described (Parks and Herzenberg (1984) Meth. Enzym. 108:197) 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 (propidium iodide) staining or by scattering alone in 4 color analyses. Compensation for spatial overlaps of fluorescein and propidium iodide were adjusted electronically as described (Parks and Herzenberg (1984) supra) . Four color stains were performed using several reagents conjugated to different fluorochromes to ensure that the results were consistent regardless of the various spatial overlaps of the fluorochromes. In addition, the results of 4 color analyses were calibrated by comparison with data from 2 and 3 color analyses.

For cell sorting, the stained samples were maintained at 4°C throughout the sorting procedure. Sorted drops were collected in RPMI 1640 containing 10% fetal calf serum (Hazelton Biologies Inc., Lenexa, KS) . Two color sorts employed Texas Red to label CD34, phycoerythrin to label c-kit, and fluorescein to label lin cells, with propidium iodide to label dead cells, with both signals being detected and excluded in a single FACS channel. Following isolation of a cell population by FACS, the sample was diluted 1:1 in HBSS, centrifuged for 10 minutes at a RCF of 200 and resuspended in 50 or 100 μl of HBSS for hemocytometer counting. The culture assays were performed as follows:

Various murine stromal cells were employed, three of which are described in Whitlock, et al. (1987) Cell 48:1009. Confluent stromal cell layers were maintained for up to 3-4 weeks without passage by changing of tissue culture medium every 5-7 days. To passage, the stromal cells were washed 3 times with serum-free medium, then overlayed with 2.5 ml (T-25 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 37°C, 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 pipet, then cultured directly at l/5th to l/50th the original cell concentration. Cell suspensions from human fetal bone marrow were prepared from long bones of fetuses from 16-20 weeks gestation. The bones are split lengthwise and the medullary cavity is scraped with a scalpel blade. The bones are then placed in a 1 mg/ml solution of dispase- collagenase in RPMI-1640. The bones are incubated for 30 minutes at 37°C, after which time the medullary cavity is flushed with media (RPMI-1640 with Pen/Strep, 2-ME and 5% FCS) to remove hematopoietic cells. Alternatively, bone marrow may be flushed from the marrow cavity without dispase-collagenase treatment. Cultures were grown in a 50:50 mixture of RPMI-1640 and IMDM containing 50 μg/ml penicillin/50 μg/ml streptomycin, 1 mM sodium pyruvate, 1 mM glutamine, 5x10^"5 2- mercaptoethanol and 10% fetal calf serum. Limiting dilution cultures were prepared using 96 well plates containing mouse stromal cells as confluent layers. The human cells were titered into the plates at progressively lower concentrations with at least 24 wells plated at each concentration. The plates were then examined to determine the percentage of positive wells at each cell number. The data is then plotted graphically. One can determine the frequency of cells in the starting population which grow under the above defined conditions. The frequency is determined by the cell number at which 37% of the wells show no growth. In one study, 1/480 of the c-kit⁺ fraction, and <l/900 of the c-kit" fraction respond.

Generation of Erythroid Cells

The methylcellulose assay for BFU-e generation is performed as follows. Fetal bone marrow is prepared as described above, and fractionated into c-kit^+/", lin⁺" and thy-^1+/" populations. The cells are plated at concentrations ranging from 10² to 10⁶ in 24 well plates containing 200 μl RPMI-1640 media with 2% methylcellulose, 20% fetal calf serum and 100 ng/ml of the growth factors IL-1, IL-3, IL-6 and erythropoietin. The plates are then incubated at 37°C, 5% C0₂ for 7-14 days. After incubation the plates are examined microscopically and scored for the presence of erythroid colonies to determine the frequency of erythroid progenitors in each cell fraction. Generation of T Cells

The thymus assay for T cell generation is performed as follows. Fetal thymus fragments are obtained of about 1 mm³ in size. The fragments are cultured in a thymus organ culture system at 25° C for 3-7 days to stimulate the in vitro receptivity of the thymus for precursor cells. Fetal bone marrow prepared as described above, and sorted into c-kit^+/", lin^+/" and thy-^1+/" fractions. The cell composition comprising about 10²-10⁴ fractionated cells in a FCS containing balanced salt solution is injected at a volume of 1 μl using a glass micropipet linked to an oil- filled micrometric screw-operated syringe. Twenty-four hours after injection, the in vitro colonized thymus fragments are implanted under the kidney capsule of SCID mice. The injected cells are HLA mismatched with the thymus. At intervals, recipient animals are sacrificed and the grafts harvested. Cell suspensions are analyzed in a two-color im unofluorescence assay for the presence of donor derived T lymphocytes in each cell fraction..

AC3 and AC6 cocultures

Cocultures established with the mouse bone marrow stromal cell lines, AC3 or AC6, have served successfully as feeder layers for human cultures and have inhibited fibroblast overgrowth at low cell densities. Cell suspensions from human fetal bone marrow samples have been cocultured for up to 7 weeks with the continuous production of hematopoietic cells during this time, indicating that early human progenitors or stem cells have been established in these cultures.

Cultures show small to medium sized human bone marrow cells attached to the mouse stromal cells and proliferation occurs over the first one to three weeks of culture, thereafter they remain fairly stable. Cells form loose aggregates consisting of non-adherent and adherent cells overlying stromal cells, which in turn overlie small to intermediate cells (pseudo-emperiopoiesis) . Overall, the appearance of the cultures is similar to mouse long- term cultures.

FACS analysis shows maintenance of human hematolymphoid cells. In the absence of hydrocortisone, cultures are a mixture of myeloid, monocytoid and lymphoid lineages. The majority of cells are myeloid and vary from myeloblasts to mature polymorphonuclear cells. From 15-40% of the cells are mononuclear, and many of these cells have a lymphoid morphology. Approximately 0-20% of the cells stain with the B lineage marker CD19, indicating a significant number of B cells. The presence of CD19⁺ positive cells after >4 weeks in culture indicates that early B cells are arising from committed progenitors. Furthermore, cultures initiated after depletion of B cells (CD20, CD19) by cell sorting show B cell development within one week of culture initiation. This data would further substantiate the presence of an active progenitor cell in this coculture system. The following tables indicate the results for the in vitro culture assays. Table 1 shows the frequency of progenitor cell activity from fetal bone marrow which was sorted by FACS into CD34^+" and c-kit^+/" fractions. The sorted cell fractions were prepared, plated at limiting dilution onto stromal cell cocultures, and progenitor cell frequency determined, as described above.

Table 1

It can be seen from these results that the progenitor cell activity resides in the c-kit⁺ fraction of bone marrow. Further experiments were performed to analyze the phenotype of cells which grew from limiting dilution cultures of sorted c-kit^+/" cells. Fetal bone marrow from the K898 sample above was sorted by FACS into CD34^+/", c-kit^+/-, lineage^+/" and rhodamine^high/low fractions. The antibodies used for the lineage markers were CD3, 4, 8, 19, 20, 14, 15 and 16. The cells were plated at limiting dilition as decribed above. After 7 weeks of culture the cells which grew out were analyzed for the presence of myeloid and B lineage cells by staining for the presence of CD19 (B cells) and CD33 (myeloid) . The limiting dilution data is shown in Table 2, and the phenotype data in Table 3.

Table 2 Limiting Dilution Analysis/Percent Positive Wells

It-cells 34+ 34- 34+ W1+ 34+ M1- 34+/Mt+ 34+W1+ 34+kll+ 34+Jrit-f w ll lin- lin+ lin-R"**¹" lin-Rh¹"

100% 12.5% 100% 16%

91 % 4% 100% 0%

This data shows that the ability to grow at limiting dilution in the described culture conditions resides in the cells which are c-kit⁺, CD34⁺, lin^" and rhodamine^lo .

The cells which grew in the limiting dilution cultures were analyzed after 7 weeks for the presence of B lineage and myeloid lineage markers. The data is shown in Table 3.

Table 3

Sorted Cell Population CD34⁺

CD34⁺lin^'

CD34⁺lin⁺

CD34⁺c-kit⁺

CD34⁺c-kit⁺lin^" CD34 ⁺c-kit⁺lin-Rh^,ow

It can be seen that the cell populations which are CD34⁺, c-kit⁺, lin" and rhodamine^lo are capable of giving rise to both B and myeloid lineages.

It is evident from the above results that the subject invention provides for a method of enrichment for human hematopoietic stem cells by selection for cells which express c-kit. A substantially enriched stem population may be produced for a variety of purposes. The stem cells may be used in bone marrow transplants, where is it necessary for the cells to be substantially free of contaminating tumor cells. In addition, the cells may be modified by genetic recombination to provide capabilities naturally lacking in the cells, either as to the individual or to stem cells generally.

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

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 readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method of enrichment for human hematopoietic stem cells, the method comprising: combining reagents which specifically recognize c-kit and at least one of Thy-1 and a lineage cocktail, said lineage cocktail comprising reagents which specifically recognize CD10, CD19, and optionally CD33 and CD3, to a mixed population of human cells comprising hematopoietic stem cells; and selecting for those cells which are c-kit⁺ and at least one of lin^" and Thy-1⁺; wherein said stem cells are characterized by the ability to reconstitute all hematopoietic lineages.

2. A method according to claim 1, further comprising: combining said population of human cells with reagents which specifically recognize at least one of CD34 and CD38; and selecting for those cells which are CD34⁺ and CD38^duU.

3. A method according to claim 1, further comprising: binding said population of human cells with rhodamine

123; and selecting for those cells which are rhodamine¹⁰".

4. A method according to claim 1, wherein said reagents are antibodies.

5. A method according to claim 4, wherein at least one of said antibodies is fluorochrome conjugated.

6. A method according to claim 5, wherein said selecting with said fluorochrome conjugated antibodies is by flow cytometry.

7. A method according to claim 1, wherein at least one of said antibodies is conjugated to magnetic beads and said selecting is by magnetic selection.

8. A method for enrichment of human hematopoietic stem cells, the method comprising: combining fluorochrome conjugated antibodies directed against c-kit and at least one of Thy-1 and a lineage cocktail, said lineage cocktail comprising reagents which specifically recognize CD10, CD19, and optionally CD33 and CD3, to a mixed population of human cells comprising hematopoietic stem cells; and separating those cells which are c-kit⁺ and at least one of lin" and Thy-1⁺ by flow cytometry to provide an enriched population of human hematopoietic stem cells; wherein said stem cells are characterized by the ability to reconstitute all hematopoietic lineages.

9. A method for enrichment of human hematopoietic stem cells, the method comprising: combining fluorochrome conjugated antibodies directed against c-kit and CD38 to a mixed population of human cells comprising hematopoietic stem cells; and separating those cells which are c-kit⁺ and ^CD38du11 by flow cytometry to provide an enriched population of human hematopoietic stem cells; wherein said stem cells are characterized by the ability to reconstitute all hematopoietic lineages.

10. A method according to Claim 9, further comprising: combining said population of human cells with reagents which specifically recognize Thy-1; and selecting for those cells which are Thy-1⁺.