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Definitions

• the present invention provides methods for preparing therapeutic compositions of myeloid progenitor cells for reconstituting hematopoiesis in a mammalian host, comprising: a) culturing ex vivo a starting cell population including hematopoietic stem cells in a suitable culture medium to expand said cell population and increase the number of myeloid progenitor cells within said cell population; and b) resuspending said myeloid progenitor cells in a pharmaceutically acceptable medium suitable for administration to a mammalian host.
• the cytokine and growth factor mixture in its base composition has stem cell factor (SCF), FLT-3 ligand (FL), and thromobopoietin (TPO).
• the cytokine and growth factor mixture has an additional cytokine selected from IL-3, IL-6, IL-11 , G-CSF, GM-CSF, and combinations thereof, and particularly from IL-3, IL-6, IL-11 , and combinations thereof.
• the cytokine and growth factor mixture has the composition SCF, FL, TPO, and IL-3 while in another embodiment, the mixture has the composition SCF, FL, TPO, and IL-6.
• One combination of the additional cytokine is IL-6 and IL-11 such that the cytokine and growth factor mixture has the composition SCF, FL, TPO, IL-6 and IL-11.
• Forms of the cytokines and growth factors are their naturally occurring products, recombinant products, variants or analogs, or modified forms having similar biological activity to the naturally occurring forms.
• the cytokines are chosen to be active on the cells used for expansion, and thus their source will generally reflect the origin of the initial cells used for expansion, although this correspondence between the form of the cytokine and the origin of the cells need not be rigorous since cross-reactivity between forms is known for various cytokines and growth factors, and is readily testable.
• the expanded culture comprises a CMP population which is at least about 0.5%, at least about 1 %, at least about 2%, at least about 5%, and at least about 10% of the total cells in the culture.
• adjunctive treatments are therapeutic compounds that augment the differentiation of myeloid progenitor cells in the myeloid pathway. These adjunctive treatments have the effect of inducing differentiation and mobilization of myeloid progenitor cells that are endogenous, or administered to the subject as part of the therapy. In one embodiment, particularly for treating or preventing neutropenia, G-CSF or GM-CSF is administered concurrently with or subsequent to cell administration.
• FIG. 2 shows derivation of myeloid progenitors from HSC in culture.
• FIG. 7 show time to effective protection using myeloid progenitors in mice.
• FIG. 17 show the effect of IL-3 and IL-6, alone and in combination on human myeloid progenitors cells.
• FIG. 18 shows a results of a colony formation assay of the myeloid progenitors cultures with IL-3, IL-6 or in combination.
• FIG. 23 is FACS phenotype of the human culture-derived myeloid progenitors in NOD/SCID mice.
• HSCs present in the expanded cell population are principally short-term repopulating hematopoietic stem cells (ST-HSC), and are generally less than 10% of the expanded cells, more preferably less than 5%, and typically in the range of 2-5% of the expanded cell population.
• ST-HSC principally short-term repopulating hematopoietic stem cells
• Committed myeloid progenitor cell or “myeloid progenitor cell” refers to a multipotent or unipotent progenitor cell capable of ultimately developing into any of the terminally differentiated cells of the myeloid lineage, but which do not typically differentiate into cells of the lymphoid lineage.
• myeloid progenitor cell refers to any progenitor cell in the myeloid lineage.
• Committed progenitor cells of the myeloid lineage include oligopotent CMP, GMP, and MEP as defined herein, but also encompass unipotent erythroid progenitor, megakaryocyte progenitor, granulocyte progenitor, and macrophage progenitor cells. Different cell populations of myeloid progenitor cells are distinguishable from other cells by their differentiation potential, and the presence of a characteristic set of cell markers.
• CLP Common lymphoid progenitor cell
• BCP B-cell progenitors
• TCP T-cell progenitors
• NK cells NK cells
• dendritic cells dendritic cells
• substantially pure cell population refers to a population of cells having a specified cell marker characteristic and differentiation potential that is at least about 50%, preferably at least about 75-80 %, more preferably at least about 85-90%, and most preferably at least about 95% of the cells making up the total cell population.
• a “substantially pure cell population” refers to a population of cells that contain fewer than about 50%, preferably fewer than about 20-25%, more preferably fewer than about 10-15%, and most preferably fewer than about 5% of cells that do not display a specified marker characteristic and differentiation potential under designated assay conditions.
• “Syngeneic” refers to deriving from, originating in, or being members of the same species that are genetically identical, particularly with respect to antigens or immunological reactions. These include identical twins having matching MHC types.
• a “syngeneic transplant” refers to transfer of cells or organs from a donor to a recipient who is genetically identical to the donor.
• the cell types relevant to the present disclosure are those of the hematopoietic system, particularly hematopoieitc stem cells and cells of the myeloid lineage. Descriptions of cells herein will use those known to the skilled artisan, with the understanding that these descriptions reflect the current state of knowledge in the art and the invention is not limited thereby to only those phenotypic markers described herein.
• HSCs give rise to committed lymphoid or myeloid progenitor cells.
• committed myeloid progenitor cells refer to cell populations capable of differentiating into any of the terminally differentiated cells of the myeloid lineage.
• myeloid progenitor cells Encompassed within the myeloid progenitor cells are the common myeloid progenitor cells (CMP), a cell population characterized by limited or non-self- renewal capacity but which is capable of cell division to form granulocyte/macrophage progenitor cells (GMP) and megakaryocyte/erythroid progenitor cells (MEP).
• CMP common myeloid progenitor cells
• GFP granulocyte/macrophage progenitor cells
• MEP megakaryocyte/erythroid progenitor cells
• a "committed lymphoid progenitor cell” refers to a cell capable of differentiating into any of the terminally differentiated cells of the lymphoid lineage.
• the common lymphoid progenitor cells CLP
• NK cells a cell population characterized by limited or non-self-renewal capacity but which is capable of cell division to form T lymphocyte and B lymphocyte progenitor cells
• lymphoid dendritic cells The marker phenotypes useful for identifying CLPs will be those commonly known in the art.
• CLP cells of mouse the cell population is characterized by the presence of markers as described in Kondo, M.
• Lin2 CD3, CD4, CD5, CD8, B220, Gr-1 , CD90.1 , CD127. TER119
• Lin 1 CD2, CD3, CD7, CD8, CD10, CD11 b, CD14, CD19, CD56, CD235a
• Lin2a CD2, CD3, CD4, CD7, CD8, CD10, CDH b, CD14, CD19, CD20, CD56, CD235a
• Lin 2b CD10, CDH b 1 CD14, CD19, CD235a
• the initial cells for expansion are isolated cells. These include isolated HSCs, which under the presence of the indicated mixture of cytokines and growth factors, develop into CMPs that further expand into other progenitor cells of the myeloid lineage.
• the initial cells for expansion are CMPs with the characteristic differentiation potential and cell marker phenotypes as described above. CMPs may have limited self-renewal capacity, and thus can expand to generate additional CMPs for a limited number of cells divisions while also differentiating into GMPs and MEPs.
• the initial population of cells obtained above is expanded ex vivo in culture by contacting the cells with a medium having a cytokine and growth factor mixture permissive for expansion of myeloid progenitor cells.
• Cytokines in their natural context are typically proteins made by cells that modulate a cell's physiological state, whether the cell is another cell or the cell producing the cytokine. Cytokines made by lymphocytes are often described as lymphokines (IL), but are cytokines as defined herein. Cytokines typically act via cellular receptors on the cells modulated by the cytokine.
• growth factors in their natural context are also compounds typically made by cells, affecting the proliferation and differentiation of cells, whether the cell is another cell or the cell producing the growth factor.
• G-CSF is related to the class 1 cytokine family, as indicated by the presence of a four-alpha-helix bundle (Hill, C. et al., Proc Natl Acad Sc/ USA 90(11):5167-71 (1993); Lovejoy, B. et al., J MoI Biol. 234(3):640-53 (1993).
• Amino acid and nucleic acid sequences for G-CSF have been identified for, among others, murine (Tsuchiya, M. et al., Proc Natl Acad Sci USA. 83(20):7633-7 (1986); rat (Han, S.W.
• the growth factors for purposes of expansion are selected from stem cell factor (SCF or SF), FLT-3 ligand (FL), thrombopoietin (TPO), erythropoietin (EPO), and analogs thereof.
• SCF stem cell factor
• FL FLT-3 ligand
• TPO thrombopoietin
• EPO erythropoietin
• growth factor forms are either naturally occurring products or are recombinant forms having similar biological activity as the naturally occurring factors.
• the growth factors are recombinant human rhuSCF, rhuFL, rhuTPO, rhuEPO, and analogs thereof.
• SCF also known as c-kit ligand, mast cell growth factor, or Steel factor, acts on multiple levels of the hematopoietic hierarchy to promote cell survival, proliferation, differentiation, adhesion and functional activation in combination with other cytokines. It is of particular importance in the myeloid lineages, particularly on the development of mast cells, but also acts on multipotent stem and progenitor cells, megakaryocytes, and a subset of lymphoid progenitor (Broudy, V.C., Blood 90(4):1345-1364 (1997)). SCF exerts its biological effects by binding to its receptor, C-KIT.
• Amino acid and nucleic acid sequences for thrombopoietin are known for, among others, murine (Lok.S., Nature 369(6481 ):565-568 (1994)); rat (Ogami, K. et al., Gene 158(2):309-10 (1995)); and human (Foster, D.C. et al., Proc. Natl. Acad. Sci. USA. 91(26):13023- 13027 (1994); Bartley, T.D. et al., Ce// 77 (7):1117-1124 (1994)).
• Homologous amino acids may be classified based on the size of the side chain and degree of polarization, including, small non-polar (e.g., cysteine, proline, alanine, threonine); small polar (e.g., serine, glycine, aspartate, asparagine); intermediate polarity (e.g., tyrosine, histidine, tryptophan); large non-polar (e.g., phenylalanine, methionine, leucine, isoleucine, valine).
• small non-polar e.g., cysteine, proline, alanine, threonine
• small polar e.g., serine, glycine, aspartate, asparagine
• intermediate polarity e.g., tyrosine, histidine, tryptophan
• large non-polar e.g., phenylalanine, methionine, leucine, iso
• Homologous amino acid may also be grouped as follows: uncharged polar R groups (e.g., glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine); acidic amino acids (e.g., aspartic acid, glutamic acid); and basic amino acids (lysine, arginine, and histidine).
• uncharged polar R groups e.g., glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine
• acidic amino acids e.g., aspartic acid, glutamic acid
• basic amino acids lysine, arginine, and histidine
• Deletions range from about 1 to about 20 residues, although in some cases, deletions may be much larger, particularly when the cytokine or growth factor has physically separable structural and/or functional domains.
• a variant of FL is the cleaved extracellular domain, which, as discussed above, retains biological activity when separated from the sequences containing the transmembrane and cytoplamic domains.
• amino acids may be added to the amino or carboxy terminus, or in the amino acid sequences joining structural domains, such as a peptide region joining alpha helixes or beta sheets present in the cytokine or growth factor. Variants for each of the cytokines and growth factors will be apparent to the skilled artisan, exemplary references of which are given above.
• the cytokines G-CSF or GM-CSF is added to the foregoing cytokine mixtures. This may be done after an initial period of growth in media lacking G-CSF or GM-CSF to permit expansion of more primitive myeloid progenitor cells prior to promoting differentiation into the progenitor cells that are further along in the myeloid lineage.
• different cytokine and growth factor mixtures above may be used to favor expansion of specified progenitor cells.
• IL-1 is used at an amount sufficient to support expansion, generally in the amount of at least about 1 to about 100 ng/ml, and preferably at about 10 to about 50 ng/ml.
• IL-3 is used at an amount sufficient to support expansion, generally in the amount of at least about 1 to about 100 ng/ml, and preferably at about 10 to about 50 ng/ml.
• IL-11 is used at an amount sufficient to support expansion, generally in the amount of at least about 1 to about 100 ng/ml, and preferably at about 10 to about 50 ng/ml.
• G-CSF is used at an amount sufficient to support expansion, generally in the amount of at least about 1 to about 1000 ng/ml, and preferably at about 10 to about 100 ng/ml.
• Expansion of myeloid progenitor cells is carried out in a basal medium, which is supplemented with the mixture of cytokines and growth factors described above, sufficient to support expansion of myeloid progenitor cells.
• the basal medium will comprise amino acids, carbon sources (e.g., pyruvate, glucose, etc.), vitamins, serum proteins (e.g., albumin), inorganic salts, divalent cations, antibiotics, buffers, and other preferably defined components that support expansion of myeloid progenitor cells.
• the initial population of cells are contacted with the mixture of cytokines and growth factors in the basal medium, and cultured to expand the population of myeloid progenitor cells. Expansion is done for from about 2 days to about 14 days, preferably from about 4 days to 10 days, more preferably about 4 days to 8 days and/or until the indicated fold expansion and the characteristic cell populations are obtained.
• the final cell culture preparation is characterized by a GMP cell population that is expanded at least about 10 fold, about 20 fold, about 40 fold, and preferably at least about 80 fold.
• the myeloid cell population will comprise GMPs which are at least about 10%, at least about 20%, at least about 30%, and preferably at least about 50% of total cells in the culture.
• the cell populations are expanded to preferentially enrich for GMP cells.
• the final cell culture preparations are characterized by a MEP cell population that is expanded at least about 0.1 fold, about 1 fold, about 2 fold, about 5 fold, and preferably about 10 fold.
• the myeloid cell population will comprise MEPs which are least about 0.5%, about 1%, about 2%, and preferably at least about 5% of total cells in the culture.
• the expansion of cells with HSC characteristics will be limited to less than about 25 fold, preferably less that about 15 fold, and more preferably less than about 10 fold, and most preferably less than about 5 fold.
• the number of HSC cells will be less than the total number of myeloid progenitor cells (i.e., CMP, GMP, and MEP) in culture.
• the HSC and progenitor cells are weakly adherent or non-adherent under these culture conditions, which permits washing of the expanded cells away from the endothelial cells.
• the cytokine and growth factor genes introduced into the immortalized cells will reflect the combinations sufficient to support expansion of the committed myeloid progenitor cells. Accordingly, in one embodiment, the cytokine genes are selected from those encoding IL-1 ⁇ i.e., IL-1£), IL-3, IL-6, IL-11 , G-CSF, or CM-CSF.
• the growth factor genes are selected from those encoding SCF, FL, TPO, and EPO.
• expression vectors comprising genes encoding SCF, FL, and TPO are introduced into the feeder cells.
• expression vector comprising genes coding for an additional cytokine, including IL-3, IL-6, or IL-11 , or combinations thereof, are used with the growth factor combination.
• the genes introduced into the feeder cells encode SCF, FL, TPO, and IL-3.
• the genes introduced into the cells encode SCF, FL, TPO, and IL-6.
• the genes encode SCF, FL, TPO, IL-6, and IL-11.
• Cells expanded by the methods above are used without further purification, or are isolated into different cell populations by various techniques known in the art, such as immunoaffinity chromatography, immunoadsorption, FACS sorting, or other procedures as described above.
• FACS sorting or immunoadsorption is used.
• a FACS gating strategy has an initial selection for live cells based on characteristic forward scatter (cell size) and side scatter (cell density) parameters, and a second selection for expression of cell markers for myeloid progenitor cells or non-myeloid cells (e.g., Sca-1 neg c-kit hl ).
• the cell populations are preferably a mixture of allogeneic myeloid progenitor cells obtained from a plurality of allogeneic donors.
• the present therapy is meant to provide temporary protection as opposed to more permanent protection afforded by reconstitution of hematopoiesis by HSCs.
• Allogeneic cells include, allogeneic mixtures of myeloid progenitor cells, mixtures of isolated CMPs, mixtures of isolated GMPs, mixtures of isolated MEPs, or combinations thereof.
• Cells in the mixture may be completely matched allogeneic, partially mismatched allogeneic, and/or fully mismatched allogeneic cells with respect to the MHC of the transplant recipient, and may be from related donors, usually siblings with the same parental alleles, or unrelated donors. Determining the degree of MHC mismatch will employ standard tests known and used in the art.
• MHC genes there are at least six major categories of MHC genes in humans, identified as being important in transplant biology.
• HLA-A, HLA-B, HLA-C encode the HLA class I proteins while HLA-DR, HLA-DQ, and HLA-DP encode the HLA class Il proteins.
• Genes within each of these groups are highly polymorphic, as reflected in the numerous HLA alleles or variants found in the human population, and differences in these groups between individuals is associated with the strength of the immune response against transplanted cells. Standard methods for determing the degree of MHC match examine alleles within HLA-B and HLA-DR, or HLA-A, HLA-B and HLA-DR groups. Thus, tests are made of at least 4, and preferably at least 6 MHC antigens within the two or three HLA groups, respectively.
• antibodies directed against each HLA antigen type are reacted with cells from one subject (e.g., donor) to determine the presence or absence of certain MHC antigens that react with the antibodies. This is compared to the reactivity profile of the other subject (e.g., recipient).
• Reaction of the antibody with an MHC antigen is typically determined by incubating the antibody with cells, and then adding complement to induce cell lysis (i.e., lymphocytotoxicity testing). The reaction is examined and graded according to the amount of cells lysed in the reaction (Mickelson, E. and Petersdorf, E.W., Hematopoietic Cell Transplantation, Thomas, E.D. et al. eds., pg 28-37, Blackwell Scientific, Maiden, MA (1999).
• Other cell-based assays include flow cytometry using labeled antibodies or enzyme linked immuno assays (ELISA).
• Molecular methods for determining MHC type generally employ synthetic probes and/or primers to detect specific gene sequences that encode the HLA protein. Synthetic oligonucleotides may be used as hybridization probes to detect restriction fragment length polymorphisms associated with particular HLA types (Vaughn, R.W., Methods in Molecular Biology: MHC Protocols 210:45-60 (2002)).
• primers may be used for amplifying the HLA sequences (e.g., by polymerase chain reaction or ligation chain reaction), the products of which can be further examined by direct DNA sequencing, restriction fragment polymorphism analysis (RFLP), or hydridization with a series of sequence specific oligonucleotide primers (SSOP) (Petersdorf, E.W. et al., Blood 92(10):3515-20 (1998); Morishima ,Y. et al., S/ooc/ 99(11):4200-6 (2002); and Middleton, D. and Williams, F., Methods in Molecular Biology: MHC Protocols 210:67-112 (2002)).
• RFLP restriction fragment polymorphism analysis
• SSOP sequence specific oligonucleotide primers
• MHCs for various animal species. These include, by way of example and not limitation, mouse, rat (Gill, T.J. et al., Transplant Proc. 27(2): 1495-500 (1995)), cow (Lewin, H.A, et al., Immunol Rev. 167:145-58 (1999), canine (Wagner, J. L. et al., J. Hered. 90(1 ):35-8 (1999)), feline (O'Brien, SJ. and Yuhki, N., Immunol Rev.
• Allogeneic mixtures of myeloid progenitor cells may have varying degrees of match in the MHC.
• progenitor cells that undergo temporary engraftment and differentiation may be isolated from a donor having a higher degree of MHC match with the recipient than cells intended to provide a more immediate therapeutic benefit.
• CMPs may be from a donor having a complete or partial match with the MHC of the recipient, while GMPs and MEPs may be from mismatched donors. Other combinations will be apparent to those skilled in the art.
• allogeneic myeloid progenitor cells are effective in protecting mammalian subjects with compromised hematopoiesis from potentially lethal neutropenic and/or thrombocytopenic conditions.
• the cells Prior to freezing, the cells may be portioned into several separate containers to create a cell bank.
• the cells may be stored, for example, in a glass or plastic vial or tube or a bag.
• a portion of the cryopreserved cells (from one or more containers) may be selected from the cell bank, thawed and used.
• the cells may be used prophylactically to reduce the occurrence of neutropenia and thrombocytopenia, and their associated complications, particularly to lessen infection by opportunistic pathogens in patients that have been treated with myeloablative agents or have undergone HSCT.
• myeloid cells can be used concurrently or subsequent to stem cell transplantation until the recipients own HSCs or transplanted HSCs begin to restore hematopoiesis and raise neutrophil and platelet levels sufficiently.
• Infusion of myeloid progenitor cells increases the number of neutrophils and megakaryocytes in the treated subject, thereby providing temporary but needed protection during the neutropenic or thrombocytopenic period.
• Use of myeloid progenitor cell populations, as opposed to more differentiated neutrophils and platelets, provides for longer lasting protection because of the temporary engraftment of myeloid progenitors and their differentiation in vivo.
• the cells are in a pharmaceutically acceptable carrier at about 1 x10 9 to about 5 x10 9 cells.
• Cells are administered in one infusion, or through successive infusions over a defined time period sufficient to generate a therapeutic effect. Different populations of cells may be infused when treatment involves successive infusions.
• a pharmaceutically acceptable carrier as further described below, may be used for infusion of the cells into the patient. These will typically comprise, for example, buffered saline (e.g., phosphate buffered saline) or unsupplemented basal cell culture medium, or medium as known in the art.
• adjunctive treatments may be used with the cells, expanded or unexpanded, described above.
• the expanded cells may be used in combination with other agents and compounds that enhance the therapeutic effect of the infused cells or treat complications arising from neutropenia.
• the adjunctive treatments include, among others, anti-fungal agents, anti-bacterial agents, and anti-viral agents. Use of these agents is also suitable for thrombocytopenia, either as prophylaxis to reduce the occurrence of infections or address any ongoing infections that lead to destruction of platelets.
• the adjunctively administered agent is an anti-fungal agent.
• Fungal infections are one of the major causes of mortality in patients suffering from neutropenia, being a significant problem in patients who have undergone myeloablative therapy and HSCT. Recipients with delayed engraftment and patients who develop GVHD typically have prolonged neutropenia, and thus are at high risk for fungal infections.
• Types of fungal infections are varied, and include, among others, candidiasis (e.g., with Candida krusei, Candida glabrata, Candida albicans, Candida tropicalis), aspergillosis (e.g., with aspergillus fumigatus, aspergillus flavus), mucormycosis (e.g., with rhizobium arrhizus, absidia corymbifera, rhizomucor pusillus), cryptococcosis, histoplasma capsulatum, and coccidioides immitis.
• candidiasis e.g., with Candida krusei, Candida glabrata, Candida albicans, Candida tropicalis
• aspergillosis e.g., with aspergillus fumigatus, aspergillus flavus
• mucormycosis e.g., with rhizobium arrhizus, absidia
• Another antifungal agent is flucytosine, a fluorinated pyrimidine. Deamination of flucytosine by the fungus generates 5-flurouracil, an anti-metabolite and DNA synthesis inhibitor. Flucytosine is typically used for infections of cryptococcus and candiadosis. Although used alone, flycytosine is generally used in combination with amphotericin B.
• Imidazoles and triazoles represent a broad class of azole based antifungal agents. It is believed that imidazoles and triazoles inhibit sterol 14-D-demethylase, resulting in impaired biosynthesis of ergosterol and disruption of cell membrane based activities, such as electron transport.
• Therapeutics directed against retroviruses include, among others, nucleoside reverse transcriptatse inhibitors (e.g., zidovudine, didanosine, stavudine, zalcitabine, lamividudine), non- nucleoside reverse transcriptase inhibitors (e.g., nevirapine, efavirenz, delvirudine), and protease inhibitors (e.g., saquinivir, indinavir, ritonavir, nelfinavir, amprenavir, and lopinavir).
• nucleoside reverse transcriptatse inhibitors e.g., zidovudine, didanosine, stavudine, zalcitabine, lamividudine
• non- nucleoside reverse transcriptase inhibitors e.g., nevirapine, efavirenz, delvirudine
• protease inhibitors e.g., saquin
• the pharmaceutical compositions will generally comprise a pharmaceutically acceptable carrier and a pharmacologically effective amount of the compounds, or mixture of thereof, or suitable salts thereof.
• the pharmaceutical composition may be formulated as powders, granules, solutions, suspensions, aerosols, solids, pills, tablets, capsules, gels, topical cremes, suppositories, transdermal patches, and other formulations known in the art.
• the pharmaceutical compositions will often further comprise one or more buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants (e.g., ascorbic acid, sodium metabisulfite, butylated hydroxytoluene, butylated hydroxyanisole, etc.), bacteriostats, chelating agents such as EDTA or glutathione, solutes that render the formulation isotonic, hypotonic or weakly hypertonic with the blood of a recipient, suspending agents, thickening agents, preservatives, flavoring agents, sweetening agents, and coloring compounds as appropriate.
• buffers e.g., neutral buffered saline or phosphate buffered saline
• carbohydrates e.g., glucose, mannose, sucrose or dextrans
• compositions may be formulated for any appropriate manner of administration, including for example, oral, nasal, mucosal, rectal, vaginal, topical, intravenous, intraperitoneal, intradermal, subcutaneous, and intramuscular administration.
• a pharmaceutical composition comprising the subject expanded myeloid progenitor cells cryopreserved in a suitable cryopreservation medium, which can then be thawed and resuspended as needed for administration to a patient.
• the cells are plated at about 10,000 cells/well in 24 well plates.
• the cells are cultured for 7 days to obtain MP 0 (culture-derived MP).
• Cells are fed with 500ul/well on day 2, and on day 4 half of the media is replaced with fresh media.
• cells are transferred to 6 well plates with an addition of 1 ml fresh media.
• the cultured cells are collected and two small aliquots are removed for analysis.
• the aliquots are mixed with 30,000 beads and stained to analyze for MP (CMP/GMP/MEP) and HSC content.
• cell are stained with trypan blue and counted on a haemocytometer.
• the analysis data provides information for calculating fold expansion and total cell numbers of HSC and MP (CMP/GMP/MEP).
• CMPs are sorted based on lineage ne9/lo c-kit pos Sca-1 neg CD34 pos 2.4G2 low ;
• GMPs are sorted based on lineage ⁇ eg/lo c-kit pos Sca- 1 ne9 CD34 pos 2.4G2 pos ;
• MEPs are sorted based on iineage ⁇ eg/lo c-kit pos Sca-1 ne9 CD34 low 2.4G2 low .
• the solution is dark from the spores and can contain up to 10 8 conidia per ml. Spore stocks are stored at 4 0 C. To titrate the spores, serial dilutions are made in PBS/Tween 80 and plated on SDA plates. Following an overnight incubation, the plates are examined for number of colonies. For long-term storage of spores, one volume of the harvested stock spores is mixed with a one volume of 50% glycerol and stored at -80 0 C
• FIG. 12 are photographs of cells from human MP cultures and treated with growth factors.
• Donor: 1319 cells were cultured in flasks for 8 days (Xvivo15+PenStrep, Glutamax, 100ng/ml KITL, 100ng/ml FLT3L, 50ng/ml TPO and 10ng/ml IL-3, switched to T25 flasks with different growth factors (100ng/ml KITL, 20ng/ml IL-3 and 300ng/ml G-CSF).
• FIG. 12 shows cells at different timepoints after transfer (4 to 19 days). The presence of granulocytes peaks at day 12, by day 19 only macrophages are seen. Human MP cultures can differentiate into morphologically mature neutrophils and macrophages, as well as megakaryocytes.
• MPc were culture under standard conditions for 5 (FIG. 20A) or 8 days (FIG. 21 B).
• G-CSF was added 300ng/ml was added at day 5 or 8 MPc (day zero on graphs) to the medium and the cell growth monitored over time and compared to control cultures, which did not receive G-CSF (w/o).
• the data shows that G-CSF can be used to increase cell numbers over long period of times when added at later stages to the cultures. It also shows that our MP are responsive to G-CSF and are likely to be direct progenitors of granulocytes/neutrophilesm and suggests G-CSF in combination with MPc transplant to increase neutrophils numbers in patients.
• FIG. 21 is a schematic to show responsiveness of human MP cells to G-CSF in vivo.
• the scheme shows a transplantation experiment of day 8 MPc into NOD/SCID mice to evaluate their potential to engraft, developmental potential and response to G-CSF in vivo.

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