WO1998023773A1

WO1998023773A1 - High purity mast cells and basophils and methods for in vitro differentiation thereof

Info

Publication number: WO1998023773A1
Application number: PCT/US1997/022162
Authority: WO
Inventors: Joshua A. Boyce; Frank K. Austen; Qian Yuan
Original assignee: Brigham & Women's Hospital
Priority date: 1996-11-27
Filing date: 1997-11-26
Publication date: 1998-06-04
Also published as: AU5370998A

Abstract

High purity populations of mast cells and basophils are obtained by culturing progenitor cells in the presence of a soluble basement membrane, e.g., Matrigel®, ECM Gel of Sigma BioSciences, or other such substances that mimic the extracellular matrix. Such populations of mast cells are obtained via culturing precursor cells, e.g., CD-34 bearing pleuripotent hematopoeitic precursor cells, in a medium supplemented with stem cell factor (SCF) and recombinant human interleukin-6 (rhIL-6) and, more preferably, also with recombinant human interleukin-10 (rhIL-10), in the presence of the soluble basement membrane. Such populations of basophils are obtained by culturing progenitor cells, e.g. cord blood mononuclear cells, in a medium supplemented with recombinant human interleukin-3 (rhIL-3) and transforming growth factor beta (TGF-beta), in the presence of a soluble basement membrane. Methods of identifying the effect of a factor (e.g., a pharmaceutical or growth factor) on development of human mast cells and basophils involve culturing populations, as described above, with and without the factor. Characteristics of the resulting populations are then compared to determine the effect, if any.

Description

HIGH PURITY MAST CELLS AND BASOPHILS AND METHODS FOR IN VITRO DIFFERENTIATION THEREOF

Sponsorship

The invention, or a portion thereof, may have resulted from work under NIH Grant/Contract Numbers AI31599 and DK-45656.

Priority Information

The present application claims the benefit of priority of U.S. Provisional Application Serial No. 60/031,881, filed November 27, 1996.

Background of the Invention The invention pertains to purified of biological substances and, more particularly, to high purity mast cells and basophils and methods for in vitro differentiation thereof.

Mast cells are bone marrow-derived resident tissue cells. They develop in situ from progenitor cells found in the peripheral blood that migrate into various tissues and differentiate into mature mast cells under the influence of microenvironmental factors. Additionally, they play a pivotal role in the pathophysiology of acute allergic reactions. Mast cells are also believed to participate in diseases causing fibrosis of various tissues, such as the lung, and are believed be important for the recruitment of neutrophils to tissue in response to bacterial invasion. When stimulated with antigen and specific IgE, mast cells respond with the release of potent pre-formed bioactive inflammatory mediators, including histamine, neutral proteases (tryptase, chymase), cytokines (tumor necrosis factor (TNF)-alpha, basic fibroblast growth factor), and interleukin (IL-4 and IL-5), and secrete other mediators (Leukotriene (LT) C₄, prostaglandin (PG) D2) that are synthesized de novo and implicated in many of the symptoms of allergy and asthma (wheezing, cough, congestion). Because of their diverse repertoire of potent inflammatory mediators, mast cells are implicated in the pathophysiology of numerous human diseases, including asthma, allergic rhinitis, anaphylaxis, urticaria and angioedema, and other allergic and non-allergic chronic inflammatory diseases, such as fibrotic lung disease and collagen vascular diseases. Thus, a thorough understanding of mast cell biology is essential to the therapy of these disorders.

Basophils share some biochemical and morphologic characteristics with mast cells. However, basophils arise from progenitors that mature terminally in the bone marrow and are thus found in the peripheral blood. Like mast cells, basophils respond to stimulation with specific IgE and allergen with the release of histamine. Basophils also secrete cytokines (IL-4 and IL-13) that are implicated in allergic reactions, and secrete LTC₄. Although their participation in allergic disease is less generally accepted than the mast cell, basophils and then- products are of potential importance in allergic rhinitis and in nasal and endobronchial late- phase responses to allergen. Thus, like mast cells, basophils are potential targets of therapeutic interventions in allergy.

Mast cells may be harvested from human lung or skin through a series of tissue digestions and a long (> 18 hr) isolation procedure. Limited cell numbers, limited number of donors, and changes in cell characteristics that are a function of the isolation procedure itself render studies of human mast cells expensive, time consuming, and limited in scope. Human mast cells may also be grown in vitro from hematopoietic progenitors found in bone marrow, peripheral blood, umbilical cord blood, and fetal liver , when maintained in liquid culture in the presence of recombinant human (rh) stem cell factor (SCF). However, these mast cells are cytologically immature, often lack surface IgE receptors, and are of low purity, with predominant contamination of these cultures with immature myeloid-lineage cells. More recently, relatively mature human mast cells of higher (99%) purity were developed in vitro from cord blood progenitors cultured in the presence of rhSCF, rhIL-6, and prostaglandin (PG) E2. These mast cells possessed a functional response to IgE and antigen and generated quantities of tryptase and histamine comparable to mast cells found in vivo. However, 8-14 weeks of culture were required to obtain these mast cells. Thus the length of culture necessary to obtain high-purity mature mast cells, the lack of a functional IgE receptor, or both, render large-scale functional mast cell studies difficult.

Similar to the case with mast cells, basophils have been difficult to study. Human peripheral blood typically contains few basophils. Isolation of basophils from the peripheral blood requires a long procedure involving multiple steps that likely alter the biologic behavior of the cells, and purity is characteristically low. It is known that while basophils may be cultured from umbilical cord blood or bone marrow-derived hematopoietic progenitor cells in media supplemented with rhIL-3 or rhIL-3 with transforming growth factor beta, purity is typically low (>50%) due to contamination with eosinophils and monocytes, confounding interpretation of studies In view of the foregoing, an object of the invention is to provide high purity mast cells and basophils of sufficient quality and quantity to facilitate their study and medical use.

A related object of the invention is to provide improved methods of generating high purity mast cells and basophils.

Yet another object of the invention is to provide cytologically mature mast cells with functional responses at least comparable to naturally occurring cells.

Still another object is to provide such methods as can be used to provide mast cells and basophils quickly, reliably and at reasonable cost.

Another object of the invention is to provide the methods of facilitating the identification of factors involved in mast cell or basophil differentiation, including the identification of agents (such as pharmaceuticals) that alter the rate or extent of mast cells or basophil differentiation and proliferation.

Summary of the Invention

The foregoing objects are attained by the invention which provides a method for producing high purity populations of mast cells via differentiating them in vitro from biological tissues and/or fluids, e.g., cord blood, using a soluble basement membrane, e.g., Matrigel,^® ECM Gel of Sigma BioSciences, or other such substances that mimic the extracellular matrix.

In one aspect, the invention provides a high purity mast cell or basophil populations. These populations are characterized by expression of cell type-specific markers comparable to those of naturally occurring populations of human mast cells and basophils, respectively. For example, mast cell populations cultured according to the invention show expression of markers including c-kit, the high affinity IgE receptor, and/or carboxypeptidase A. In addition, they stain with toluidine blue (as expected for metachromatic granules). Moreover, cell populations obtained in accord with the invention comprise at least 80%, still more preferably at least about 90% , and most preferably 100%, mature mast cells. Preferred basophil populations of the present invention are characterized by metachromatic granules, expression of IgE receptor, and an active 5-lipoxygenase pathway. Preferably, these populations are at least about 70% pure, more preferably at least about 80% pure, and most preferably at least about 95 % pure. In another aspect, the invention provides a method for producing a high purity population of mast cells via culturing precursor cells, e.g., CD-34 bearing pleuripotent hematopoeitic precursor cells, in a medium supplemented with stem cell factor (SCF) and recombinant human interleukin-6 (rhIL-6) and, more preferably, also with recombinant human interleukin-10 (rhIL-10), in the presence of the soluble basement membrane. The precursor cells can be obtained, for example, via isolation from biological tissues and/or fluids, e.g., cord blood, cryopreserved human bone marrow, and other such sources of hematopoietic progenitor cells.

Further aspects of the invention provide methods as described above in which the precursor cells are so cultured for 3 - 7 weeks, more preferably, from 4 -6 weeks and, still more preferably, for about 5 weeks. Still further aspects of the invention provide methods as described above in which mast cell populations resulting from culture are further purified, for example, using a magnetic cell separator or a flow cytometer.

The invention provides, in yet another aspect, a method for producing a high purity population of basophils by culturing progenitor cells, e.g. cord blood mononuclear cells, in the presence of a soluble basement membrane of the type described above. Related aspects of the invention provide for so culturing the progenitor cells in a medium that is supplemented with recombinant human interleukin-3 (rhIL-3) and transforming growth factor beta (TGF-beta).

Still further aspects of the invention provide methods for producing a high purity population of basophils from progenitor cells isolated from bodily tissues and fluids, e.g., cord blood, cryopreserved human bone marrow, and other such sources of hematopoietic progenitor cells.

Yet still further aspects of the invention provide methods of identifying the affect of a factor

(e.g., a pharmaceutical or growth factor) on development of human mast cells. According to these methods, a first population of mast cells is cultured as described above without the presence of the factor, while a second population is cultured in the presence of that factor. One or more characteristics, such as mast cell yield, maturity, viability, purity and receptor activity, are compared as between the two populations. Based on such comparison, the affect (if any) of the factor is identified.

Yet still further aspects provide parallel methods of identifying the affect of a factor (e.g., a pharmaceutical or growth factor) on development of human basophils. These and other aspects of the invention are evident in the description that follow.

Brief Description of the Drawings

A more complete understanding of the invention may be attained by reference to the drawings, in which

Figure 1 shows the effect of Matrigel^® on stimulating differentiation and proliferation of mast cells according to the present invention.

Figure 2 shows effects of Matrigel^® in promoting most cell differentiation according to the present invention.

Description of Certain Preferred Embodiments

In view of the aforementioned difficulties in the isolation of mast cells and basophils and their importance in the pathophysiology of numerous diseases, the present invention describes a method for the production of high purity mast cells and basophils.

The present invention encompasses the discovery that the use of a soluble basement membrane, preferably supplemented with specific growth factors, enables culturing of populations of high purity mast cells or basophils in relatively short periods of time while retaining functional characteristics, e.g. a functioning IgE receptor, comparable to those of naturally occurring cell populations.

Mast Cells

High purity populations of mast cells are prepared according to the invention by a method including steps of providing progenitor cells, and culturing them, in the presence of a soluble basement membrane such as Matrigel^®, e.g., for four to six weeks. In other embodiments, a method according to the invention further includes a step of purifying mast cells from the culture. Progenitor cells useful for differentiating mast cells according to the present invention are preferably prepared or derived from biological tissues or fluids, such as for example, cord blood, cryopreserved human bone marrow, peripheral blood hematopoietic progenitor cells, and other such sources of CD34⁺ pluripotent hematopoietic progenitor cells. The preferred source of CD34⁺ progenitor cells is cord blood. Where especially large numbers of differentiated mast cells are desired, it is preferable to culture the cord blood mononuclear cells directly. On the other hand, as will be understood by those of ordinary skill the in art, it may be desirable to purify CD34⁺ progenitors, e.g., by fluorescence-activated cell sorting (FACS) immunomagnetic bead purification, or other available method, when exceptionally high purity mast cells are desired, e.g., for studies of mast cell development.

Once isolated, progenitor cells are cultured in the presence of the soluble basement membrane (preferably, Matrigel^® available from Cellaborative Biotech, Burlington, MA on

November 25, 1997) according to known techniques. Those skilled in the art will appreciate art that other soluble basement membranes may be used, such as ECM Gel of Sigma BioSciences and other such substances that mimic the extracellular matrix.

In addition to the soluble basement membrane, the progenitor cells are cultured in an enriched culture medium. For example, in one particular embodiment, the culture medium comprises RPMI supplemented with 100 U/ml penicillin G, 100 μg/ml streptomycin, 10 μg gentamycin, 1 mM-L glutamine, 0.1 mM non-essential amino acids, 10 % fetal calf serum (FCS) and 50 μM β-mercaptoethanol.

It will be appreciated that other enriched media can be used according to the method of the present invention, thus providing the necessary nutrients for cell proliferation. Furthermore, it will be appreciated that the foregoing and other concentrations of the aforementioned components of the medium may be used. For example, ranges of 10 to 100 μg/ml streptomycin; 1 to 100 μg gentamycin; preferably 0.1 to 10 mM-L, more preferably 0.5 to 2 mM-L, and still more preferably about 1 mM-L glutamine; preferably 0.01 mM to 1.0 mM, more preferably 0.5 to 2.0 mM, and still more preferably about 0.1 mM non-essential amino acids; and preferably 5 to 500 μM, more preferably 20 to 70 μM, and still more preferably about 50 μM β-mercaptoethanol may be used. In preferred embodiments, the culture medium is supplemented with rhSCF and rhIL-6 and, still more preferably, also with rhIL-10. In particularly preferred embodiments, the concentration of the supplements is 80 ng/ml rhSCF, 50 ng/ml rhIL-6 and 5 ng/ml rhIL-10. Those of ordinary skill in the art will appreciate that these and other concentrations of the aforementioned supplements may be used. More particularly, ranges of 20 to 120 ng/ml and more preferably 60 to 100 ng/ml and, still more preferably about 80 ng/ml rhSCF; preferably 10 to 100 ng/ml, more preferably 30 to 70 ng/ml, and still more preferably about 50 ng/ml rhlL- 6; and preferably 0.5 to 10 ng/ml, more preferably 3 to 7 ng/ml and still more preferably about 5 ng/ml rhIL-10 can be used. According to the present invention, the progenitor cells are cultured for at least 3 to 7 weeks,

4 to 6 weeks and preferably about 5 weeks. Confirmation of cell lineage for cells differentiated according to the above method can be accomplished via any method that detects a mast cell- diagnostic marker. For example, expression of tryptase, carboxypeptidase A, c-kit, and/or the high affinity IgE receptor can be detected by known methods such as northern blot, primer extension, polymerase chain reactor, western blot, or immunostaining. Also, cells containing metachromatic granules can be quantified using toluidine blue. Alternatively or additionally, ultrastructural analysis can be performed to identify class with mature granule crystalloids and scrolls; and/or functional assays (e.g., testing LTC₄ generation in response to an ant-hlgE receptor antibody) can be performed. Those of ordinary skill in the art will appreciate that any of the variety of other diagnostic techniques can additionally or alternatively be employed.

In preferred embodiments of the invention, additional purification of differentiated mast cells can be performed, for example, using MACS-magnetic cell separation column, which yields at least 80 % purity, or FAC sorting, which yields 92-100 % purity. Other cell separation techniques known in the art can be employed instead of or in addition to these aforementioned techniques.

Basophils

High purity populations of basophils according to the invention are also prepared by culturing the CD34⁺ bearing pluripotent hematopoietic progenitor cells on a soluble basement membrane, such as Matrigel^®, in culture medium that is preferably supplemented with rhIL-3 and rhTGF-β.

As above, progenitor cells useful for differentiating basophils according to the present invention are preferably prepared or derived from biological tissues or fluids, such as for example, cord blood. Once isolated, progenitor cells are cultured in the presence of the soluble basement membrane. As above, that soluble basement membrane is preferably, Matrigel^®, though other soluble basement membranes may be used, such as ECM Gel of Sigma BioSciences and other such substances that mimic the extracellular matrix. In addition to the soluble basement membrane, the progenitor cells are cultured in an enriched culture medium, e.g., of the type described above.

The rhIL-3 used for culture of is preferably utilized at concentrations in the range of preferably 250 to 500 pM, more preferably 300 to 400 pM, and still more preferably about

350 pM rhIL-3. The rhTGF-β used for culture is preferably utilized at concentrations of preferably 1 to 100 ng/ml, more preferably 2 to 20 ng/ml, and still more preferably about 10 ng/ml rhTGF-β.

According to the method of the present invention, the cells are cultured preferably for 5 to 28 days and most preferably for about 10 to 14 days.

Cultured basophils prepared according to the present invention can be characterized, for example, by analysis of metachromatic granules (e.g., by toluidine blue staining), detection of markers such as IgE receptor, and assays of functional activities such as histamine release in response to passive sensitization or production of LTC₄ in response to calcium ionophore stimulation. Ultrastructural analysis may also be performed.

As described above, the present invention provides systems for preparing high purity mast cells or basophils in vitro. These systems offer advantages over prior art systems in that, for example, cell differentiation occurs more rapidly and cell proliferation is enhanced.

In addition to the culture of high purity cell populations, the systems described herein may also be utilized (i) to prepare modified mast cells and basophils; (ii) to identify biological factors involved in mast cells or basophil differentiation (and/or proliferation); and (iii) to identify modulatory agents that alter the extent or rate of mast cell or basophil differentiation or proliferation. For example, with respect to preparing modified mast cells and basophils. the present invention provides large numbers of high purity cells that can readily be generically manipulated, e.g., by transformation, transfection or infection (using, for example, retroviral vectors or adeno- or adeno-associated viruses), transduction, electropuration, etc. (see for example, Sombrook et al., Molecular Cloning: A Laboratorv Manual. Cold Spring Harbor Press, NY, 1989, incorporated here by reference).

With respect to identifying factors that regulate most cell and basophil development, the present in vitro system, or components thereof, can be fractionated according to known procedures, and individual fractions can be tested for their effects on cell development and/or proliferation, as detected according to methods described herein.

These identified factors, or other exogenously added factors, may also be tested for their effects on modulating (i.e., stimulating or inhibiting) mast cell or basophil development and/or proliferation. For example, agent may be added to the system at any time during culturing and their effects on the system quantified using assays described herein. Preferred agents exert at least a two-fold effect.

Examples Example 1 : Method of Production of High Purity Mast Cells

Whole blood was drawn into 50 ml heparinized sterile syringes from the umbilical vein of the placenta at uncomplicated cesarean section deliveries following clamping and severing of the cord. A 1 :5 volume of 4.5% dextran in phosphate buffered saline (PBS), pH 7.2, was added to the blood and the red blood cells were allowed to sediment at 37 degrees for 1 hour. The buffy coat was layered onto a cushion of Ficoll-Paque (1.77 g/ml) and centrifuged at 300 x g for 30 minutes at room temperature. The mononuclear cell interface was harvested and washed x 2 in PBS containing 5 % fetal calf serum (FCS). Contaminating red blood cells were removed using hypotonic lysis. The resultant mononuclear cells were suspended in enriched medium (RPMI 1640 supplemented with 100 U/ml of penicillin G, 100 μg/ml of streptomycin,

10 μg /ml of gentamycin, 2 mM L-glutamine, 0.1 mM non-essential amino acids, 10% fetal calf serum (FCS) [Sigma Chemical Co., St. Louis, MO] and 50 μM β-mercaptoethanol) supplemented with 80 ng/ml of rhSCF, 50 ng/ml rhIL-6, and 350 pM rhIL-10 (all from R & D systems, Minneapolis, Minn.). The cells were maintained for 5-6 weeks of culture in 25 ml plastic tissue culture flasks (Falcon) coated with an 0.5 mm layer of Matrigel^®. Fresh media and cytokines were replaced every 7 d, at which time glass slides were prepared by cytocentrifugation and stained with toluidine blue to quantify cells containing metachromatic granules. Confirmation of mast cell lineage was obtained using immunostaining for tryptase and the receptor for stem cell factor, c-kit. Alternatively, confirmation has been achieved using standard flow cytometry, which confirms 100 %> c-kit positive after 5 weeks of culture. In some experiments, replicate cells were cultured in flasks not coated with Matrigel.

At 5-6 weeks of culture, the non-adherent cells were harvested from the flasks coated with Matrigel. The proportion of toluidine blue-positive cells ranged from 51 -86% (mean 68%, n=3) of the total cell population (compared with 22-54% among replicate cells cultured under identical conditions but without Matrigel, n=2). Most of the non-metachromatic cells had the appearance of neutrophils. When subjected to flow cytometry, these cell preparations had two populations as defined by right-angle light scatter properties. Mast cells were further purified to >80%o purity using a magnetic cell separation (MACS) column following incubation with a magnetic bead bound to a mouse monoclonal anti-human antibody directed against CD 16 (a low affinity IgE receptor found on neutrophils but not on mast cells). Following purification, over 80%) of the cells were mast cells as evidenced by metachromasia with toluidine blue, and immunostaining for tryptase and c-Kit. Alternately, a population of 92-100%) purity was obtained using FAC sorting with enrichment of the population having a greater degree of right angle scatter. RNA from these mast cells contained strong signals for tryptase as determined by northern blot analysis. The mast cells had a functional response to cross-linking of their IgE receptor as evidenced by the generation of 18 ng of LTC₄ in response to an activating mouse anti-human IgE receptor alpha chain antibody. Ultrastructural analysis revealed that the mast cells grown on Matrigel had mature-appearing granule crystalloids and scrolls. Thus, a technique for the development of authentically mature, functional human mast cells has been developed. Advantages over current methodology include more rapid cell differentiation (5-6 weeks vs 8-14 weeks), greater cell number, more fully developed granule scrolls and crystalloids, and a functional IgE receptor. Example 2: Method of Production of High Purity Basophils: Figure 2 depicts a method for producing high purity basophils according to the invention. With reference to the drawing, the method includes the steps of isolating cord blood mononuclear cells, purifying CD-34⁺ progenitor cells and culturing them in the presence of an enriched medium supplemented with recombinant human interleukin-3, and transforming growth factor beta (TGF-beta), in the presence of Matrigel, for ten to fourteen days. More particularly, one example of how high purity basophils are produced is described below.

Cord blood mononuclear cells were isolated as above. A positive selection procedure to purify CD34-bearing hematopoietic progenitor cells from cord blood (31) was used in order to eliminate contaminating myelomoncytic cells. The mononuclear cells were washed two times in phosphate buffered saline (PBS), pH 7.2, containing 5 mM EDTA. After the cells were pelleted by centrifugation at 200 x g at 4° C for 10 minutes, they were re-suspended in PBS containing 10% FCS and 5 mM EDTA at 3.3 x 10⁸ cells/ml. The cells were incubated at 4°C with immunomagnetic beads conjugated to a rabbit anti-human CD34 antibody (Miltenyi Biotech, Sunnyvale, CA) according to the manufacturer's protocol. The CD34-bearing hematopoietic progenitor cells were collected by elution after their adhesion to a magnetic cell separation (MACS) column (Miltenyi) (31). The purified progenitor cells were then cultured in EM supplemented with 350 pM rhIL-3 and 10 ng/ml rhTGF-beta (both from Genzyme, Inc., Cambridge, MA) in Matrigel-coated flasks at an initial cell concentration of 1 x 10⁴ cells/ml. Cytocentrifuge-generated slides were stained at 14 days with Wright's and Geimsa stains, or with Toluidine blue stain to quantify the fraction of basophils.

At 10-14 d of culture, 77-98%) (mean purity 84%, n=3) of the nonadherent cells were strongly metachromatic when stained with toluidine blue and had the morphologic characteristics of basophils. Most of the remaining cells were primitive-appearing mononuclear cells that lacked distinct cytoplasmic granules. There was a 5-10 fold increase in total cell numbers compared with the starting population. In contrast, replicate cells cultured in IL-3 alone with Matrigel were only 23 %> basophils, with eosinophils comprising the majority of the remaining cells (n= 1). An IgE receptor was present on the basophils as evidenced by a positive signal for the human IgE receptor alpha chain on a western blot, as well as a positive signal on a northern blot, and histamine release in response to passive sensitization with IgE and antigen. These basophils derived in vitro had a fully active 5-lipoxygenase (5-LO) pathway as evidenced by their production of 136 ng of LTC₄ in response to stimulation with 20 μM calcium ionophore. Ultras,tructural analysis revealed fully developed reticular granules characteristic of mature basophils.

Summary

Described herein are methods and cell populations meeting the objects set forth above. Those skilled in the art will appreciate that the above-described embodiments are set forth by way of example and that other embodiments, incorporating modifications thereto, fall within the scope of the invention. By way of example, it will be appreciated that the methods described above can be practiced with naturally occurring agents and factors, such as SCF, IL-6, IL-10, and TGF-β, as well as with the recombinant (and other) forms thereof, e.g., rhSCH, rhIL-6, rhIL-10, and rhTGF-β.

In view of the foregoing, what we claim is:

Claims

1. A method of stimulating development of a high purity population of human mast cells, comprising culturing precursor cells in the presence of a soluble basement membrane.

2. A method according to claim 1, wherein the precursor cells comprise CD-34 bearing pleuripotent hematopoeitic precursor cells.

3. A method according to claim 1, wherein the culturing step comprises culturing the precursor cells in a medium comprising recombinant human stem cell factor (rhSCF) and in the presence of the soluble basement membrane.

4. A method according to claim 3, wherein the culturing step comprises culturing the precursor cells in a medium comprising rhSCF in a concentration of 20 to 120 ng/ml.

5. A method according to claim 3, wherein the culturing step comprises culturing the precursor cells in a medium comprising rhSCF in a concentration of 60 to 100 ng/ml.

6. A method according to claim 3, wherein the culturing step comprises culturing the precursor cells in a medium comprising rhSCF in a concentration of substantially 80 ng/ml.

7. A method according to claim 3, wherein the culturing step comprises culturing the precursor cells in a medium additionally comprising recombinant human interleukin-6 (rhIL-6).

8. A method according to claim 7, wherein the culturing step comprises culturing the precursor cells in a medium comprising rhIL-6 in a concentration of 10 to 100 ng/ml.

9. A method according to claim 7, wherein the culturing step comprises culturing the precursor cells in a medium comprising rhIL-6 in a concentration of 30 to 70 ng/ml.

10. A method according to claim 7, wherein the culturing step comprises culturing the precursor cells in a medium comprising rhIL-6 in a concentration of substantially 50 ng/ml.

11. A method according to claim 4, wherein the culturing step comprises culturing the precursor cells in a medium additionally comprising recombinant human interleukin- 10

(rhIL-10).

12. A method according to claim 11, wherein the culturing step comprises culturing the precursor cells in a medium comprising rhIL-10 in a concentration of 0.5 to 10 ng/ml.

13. A method according to claim 11, wherein the culturing step comprises culturing the precursor cells in a medium comprising rhIL-10 in a concentration of 3 to 7 ng/ml.

14. A method according to claim 11, wherein the culturing step comprises culturing the precursor cells in a medium comprising rhIL-10 in a concentration of substantially 5 ng/ml.

15. A method of culturing a high purity population of human mast cells, comprising culturing precursor cells in a medium comprising rhSCF, rhIL-6 and rhIL-10 in the presence of a soluble basement membrane.

16. A method according to claim 15, wherein the soluble basement membrane is selected from the group consisting of Matrigel, ECM Gel, and other substances that mimic human extracellular matrix.

17. A method according to claim 15, comprising the step of isolating CD-34 bearing pluripotent hematopoeitic precursor cells from any of bodily tissues and fluids.

18. A method according to claim 15, wherein the culturing step comprises culturing the precursor cells in a medium comprising rhSCF in a concentration of 20 to 120 ng/m; rhIL-6 in a concentration of 10 to 100 ng/ml; and rhIL-10 in a concentration of 0.5 to 10 ng/ml.

19. A method according to claim 15, wherein the culturing step comprises culturing the precursor cells in a medium comprising rhSCF in a concentration of 60 to 100 ng/m; rhIL-6 in a concentration of 30 to 70 ng/ml; and rhIL-10 in a concentration of 3 to 7 ng/ml.

20. A method according to claim 15, wherein the culturing step comprises culturing the precursor cells in a medium comprising rhSCF in a concentration of substantially

80 ng/m; rhIL-6 in a concentration of substantially 50 ng/ml; and rhIL-10 in a concentration of substantially 5 ng/ml.

21. A method according to claim 15, wherein the culturing step comprises culturing the precursor cells in a medium comprising one or more of an antibiotic, glutamine, non- essential amino acids, calf serum and mercaptoethanol.

22. A method of stimulating development of a high purity population of human mast cells, comprising culturing precursor cells for a period of substantially between 21-49 days in a medium comprising rhSCF, rhIL-6 and rhIL-10 in the presence of a soluble basement membrane.

23. A method according to claim 22, wherein the culturing step comprises culturing the precursor cells for a period of substantially between 28 - 40 days.

24. A method according to claim 22, wherein the culturing step comprises culturing the precursor cells for a period of substantially 35 days.

25. A method of obtaining a high purity population of human mast cells, comprising culturing precursor cells in a medium comprising rhSCF, rhIL-6 and rhIL-10 in the

presence of a soluble basement membrane, and purifying a population of mast cells resulting from the culturing step.

26. A method according to claim 25, wherein the purifying step comprises purifying the population of mast cells resulting from the culturing step by any of magnetic cell separation and flow cytometry.

27. A method according to claim 25, wherein the culturing step comprises culturing the precursor cells for a period of substantially between 21 - 49 days

28. A method according to claim 22, wherein the culturing step comprises culturing the precursor cells for a period of substantially between 28 - 40 days.

29. A method according to claim 22, wherein the culturing step comprises culturing the precursor cells for a period of substantially 35 days.

30. A method of identifying a regulatory factor affecting the development of human mast cells, comprising stimulating development of a first population of mast cells by culturing precursor cells in the presence of a soluble basement membrane and without the presence of the factor, stimulating development of a second population of mast cells by culturing precursor cells in the presence of a soluble basement membrane and in the presence of the factor, comparing a characteristic of the first population with that of the second population to identify an effect, if any, on development of human mast cells therein.

31. A method according to claim 30, wherein the comparing step comprises comparing a characteristic selected from a group consisting of mast cell maturity, mast cell viability, purity of mast cell population, IgE receptor activity, and other receptor activity.

32. A method according to any of claims 30 and 31 , wherein the stimulating steps comprise culturing the respective populations in a medium comprising rhSCF, rhIL-6 and rhIL-10 and in the presence of the soluble basement membrane.

33. A method according to claim 32, wherein the soluble basement membrane is selected from the group consisting of Matrigel, ECM Gel, and other substances that mimic human extracellular matrix.

34. A method according to claim 32, comprising the step of isolating CD-34 bearing pleuripotent hematopoeitic precursor cells from any of bodily tissues and fluids.

35. A method according to claim 32, wherein the culturing step comprises culturing the precursor cells in a medium comprising rhSCF in a concentration of 20 to 120 ng/m; rhIL-6 in a concentration of 10 to 100 ng/ml; and rhIL-10 in a concentration of 0.5 to 10 ng/ml.

36. A method according to claim 32, wherein the culturing step comprises culturing the precursor cells in a medium comprising rhSCF in a concentration of 60 to 100 ng/m; rhIL-6 in a concentration of 30 to 70 ng/ml; and rhIL-10 in a concentration of 3 to 7 ng/ml.

37. A method according to claim 32, wherein the culturing step comprises culturing the precursor cells in a medium comprising rhSCF in a concentration of substantially 80 ng/m; rhIL-6 in a concentration of substantially 50 ng/ml; and rhIL-10 in a concentration of substantially 5 ng/ml.

38. A method according to claim 32, wherein the culturing step comprises culturing the precursor cells in a medium comprising one or more of an antibiotic, glutamine, non-essential amino acids, calf serum and β-mercaptoethanol.

39. A method of stimulating development of a high purity population of human basophil cells, comprising culturing precursor cells in the presence of a soluble basement membrane.

40. A method according to claim 39, wherein the precursor cells comprise CD-34 bearing pluripotent hematopoeitic precursor cells.

41. A method according to claim 39, wherein the culturing step comprises culturing the precursor cells in a medium comprising recombinant interleukin-3 (rhIL-3) and in the presence of a the soluble basement membrane.

42. A method according to claim 41, wherein the culturing step comprises culturing the precursor cells in a medium comprising rhIL-3 in a concentration of 250 to 500 pM.

43. A method according to claim 41, wherein the culturing step comprises culturing the precursor cells in a medium comprising rhIL-3 in a concentration of 300 to 400 pM.

44. A method according to claim 41, wherein the culturing step comprises culturing the precursor cells in a medium comprising rhIL-3 in a concentration of substantially 350 pM.

45. A method according to claim 41, wherein the culturing step comprises culturing the precursor cells in a medium additionally comprising recombinant human TGF-β.

46. A method according to claim 41, wherein the culturing step comprises culturing the precursor cells in a medium comprising rhTGF-β in a concentration of 1 to 100 ng/ml.

47. A method according to claim 41, wherein the culturing step comprises culturing the precursor cells in a medium comprising rhTGF-β in a concentration of 2 to 20 ng/ml.

48. A method according to claim 41, wherein the culturing step comprises culturing the precursor cells in a medium comprising rhTGF-β in a concentration of substantially 10 ng/ml.

49. A culturing of a high purity population of human basophil cells, comprising culturing precursor cells in a medium comprising rhIL-3 and rhTGF-β in the presence of a soluble basement membrane.

50. A method according to claim 49, wherein the soluble basement membrane is selected from the group consisting of Matrigel, ECM Gel, and other substances that mimic human extracellular matrix.

51. A method according to claim 49, comprising the step of isolating CD-34 bearing pluripotent hematopoeitic precursor cells from any of bodily tissues and fluids.

52. A method according to claim 49, wherein the culturing step comprises culturing the precursor cells in a medium comprising rhIL-3 in a concentration of 250 to 500 pM and rhTGF-β in a concentration of 1 to 100 ng/ml.

53. A method according to claim 49, wherein the culturing step comprises culturing the precursor cells in a medium comprising rhIL-3 in a concentration of 300 to 400 pM and rhTGF-β in a concentration of 2 to 20 ng/ml.

54. A method according to claim 49, wherein the culturing step comprises culturing the precursor cells in a medium comprising rhIL-3 in a concentration of substantially 350 pM and rhTGF-β in a concentration of substantially 10 ng/ml.

55. A method of stimulating development of a high purity population of human mast cells, comprising culturing precursor cells for a period of substantially between 5 to 28 days in a medium comprising rhIL-3 and rhTGF-β in the presence of a soluble basement membrane.

56. A method according to claim 55, wherein the culturing step comprises culturing the precursor cells for a period of substantially between 10 to 14 days.

57. A method of obtaining a high purity population of human basophil cells, comprising: culturing precursor cells in a medium comprising rhIL-3 and rhTGF-β in the presence of a soluble basement membrane, and purifying a population of mast cells resulting from the culturing step.

58. A method according to claim 57, wherein the purifying step comprises purifying the population of mast cells resulting from the culturing step by any of magnetic cell separation and flow cytommetry.

59. A method according to claim 57, wherein the culturing step comprises culturing the precursor cells for a period of substantially between 5 to 28 days.

60. A method according to claim 57, wherein the culturing step comprises culturing the precursor cells for a period of substantially between 10 to 14 days.

61. A method of identifying a regulatory factor, responsible for the development of human mast cells comprising: stimulating development of a first population of mast cells by culturing precursor cells in the presence of a soluble basement membrane and without the presence of the factor, stimulating development of a second population of mast cells by culturing precursor cells in the presence of a soluble basement membrane and in the presence of the factor, comparing a characteristic of the first population with that of the second population to identify an effect, if any, on development of human mast cells therein.

62. A method according to claim 61, wherein the comparing step comprises comparing a characteristic selected from a group consisting of basophil cell maturity, basophil cell viability, purity of basophil cell population, IgE receptor activity, and other receptor activity.

63. A method according to any of claims 61 and 62, wherein the stimulating steps comprise culturing the respective populations in a medium comprising rhIL-3 and rhTGF-b and in the presence of the soluble basement membrane.

64. A method according to claim 63, wherein the soluble basement membrane is selected from the group consisting of Matrigel, ECM Gel, and other substances that mimic human extracellular matrix.

65. A method according to claim 63, comprising the step of isolating CD-34 bearing pluripotent hematopoetitic precursor cells from any of bodily tissues and fluids.

66. A method according to claim 63, wherein the culturing step comprises culturing the precursor cells in a medium comprising rhIL-3 in a concentration of 250 to 500 pM and rhTGF-β in a concentration of 1 to 100 ng/ml.

67. A method according to claim 63, wherein the culturing step comprises culturing the precursor cells in a medium comprising rhIL-3 in a concentration of 300 to 400 pM and rhTGF-β in a concentration of 2 to 20 ng/ml.

68. A method according to claim 63, wherein the culturing step comprises culturing the precursor cells in a medium comprising rhIL-3 in a concentration of substantially 250 pM and rhTGF-β in a concentration of substantially 10 ng/ml.

69. The regulatory factor identified by the method of any of claims 30 and 61.

70. A method of identifying drugs which regulate the effects of human mast cells comprising: stimulating development of a first population of mast cells by culturing precursor cells in the presence of a soluble basement membrane and without the presence of the factor, stimulating development of a second population of mast cells by culturing precursor cells in the presence of a soluble basement membrane and without the presence of the factor, and comparing a characteristic of the first population with that of the second population to identify an effect, if any, on the development of human mast cells therein.

71. A method of identifying drugs which regulate the effects of human basophil cells comprising: stimulating development of a first population of basophil cells by culturing precursor cells in the presence of a soluble basement membrane and without the presence of the drug, stimulating development of a second population of cells by culturing precursor cells in the presence of a soluble basement membrane and without the presence of the drug, and comparing a characteristic of the first population with that of the second population to identify an effect, if any, on the development of human basophil cells therein.

72. The method according to any of claims 70 and 71, wherein the comparing step comprises comparing a characteristic selected from a group consisting of mast cell purity, mast cell viability, purity of mast cell population, IgE receptor activity, and other receptor activity.

73. The method according to claim 70, wherein the stimulating steps comprise culturing the respective populations in a medium comprising rhSCF, rhIL-6 and rhIL-10 and in the presence of a soluble basement membrane.

74. The method according to claim 71, wherein the stimulating steps comprise culturing the respective populations in a medium comprising rhIL-3 and rhTGF-β and in the presence of a soluble basement membrane.

75. The drug identified by the method of claim 70.

76. The drug identified by the method of claim 71.

77. The population of human mast cells produced according to claim 15.

78. The populatinon of human basophil cells produced according to claim 57.