WO2000034443A2 - Multiplication de cellules souches hematopoietiques induite par des megacariocytes - Google Patents

Multiplication de cellules souches hematopoietiques induite par des megacariocytes Download PDF

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WO2000034443A2
WO2000034443A2 PCT/US1999/029138 US9929138W WO0034443A2 WO 2000034443 A2 WO2000034443 A2 WO 2000034443A2 US 9929138 W US9929138 W US 9929138W WO 0034443 A2 WO0034443 A2 WO 0034443A2
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hsc
cells
culture
interleukin
tpo
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WO2000034443A3 (fr
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Stephen H. Bartelmez
Gerald J. Roth
Kindred A. Ritchie
Mayumi Yagi
Ewa Sitnicka
Carl Storey
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Bartelmez Stephen H
Roth Gerald J
Ritchie Kindred A
Mayumi Yagi
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Publication of WO2000034443A3 publication Critical patent/WO2000034443A3/fr

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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood cells
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/125—Stem cell factor [SCF], c-kit ligand [KL]
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/145—Thrombopoietin [TPO]
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/22—Colony stimulating factors (G-CSF, GM-CSF)
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/26—Flt-3 ligand (CD135L, flk-2 ligand)
    • C—CHEMISTRY; METALLURGY
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/39—Steroid hormones
    • C—CHEMISTRY; METALLURGY
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    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/11—Coculture with; Conditioned medium produced by blood or immune system cells
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    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
    • C12N2502/1394—Bone marrow stromal cells; whole marrow

Definitions

  • the present invention relates to methods for increasing a population of hematopoietic stem cells (HSC) in vitro or ex vivo by co-culturing hematopoietic stem cells with megakaryocytes and stromal cells for about one month under conditions effective to result in at least a 5-fold increase in HSC.
  • HSC hematopoietic stem cells
  • the invention also relates to HSC compositions produced by such methods.
  • the hematopoietic stem cell is a pluripotent progenitor cell that has been characterized as a cell that is transplantable, can self-replicate or generate daughter cells that are destined to commit to mature cells of different specific lineages.
  • transplantation studies have shown that a single HSC can repopulate the marrow of a lethally irradiated mouse, demonstrating that self-renewal of HSC occurs in vivo, as indicated by transplantation studies wherein a single HSC repopulated the marrow of an immunodef ⁇ cient mouse (Smith, LG et al, Proc Natl Acad Sci USA 88, 2788-92, 1991: Osawa M et al, Science 273, 242- 245, 1996).
  • hematopoietic stem cells can be infected with recombinant retroviruses, and can serve as cellular targets for gene therapy (Keller and Snodgrass, 1990).
  • Attempts to attain practical expression of exogenous genes in HSC using retroviral vectors have shown limited success (Tisdale JF et al., Blood 92, 1131-41, 1998), however efforts are underway using both viral-mediated and non-viral methods to introduce and express trabsgenes in HSC (See, e.g., Kume A et al., Int J Hematol 69(4):227-33, 1999)
  • patients suffering from various cell-based diseases including, but not limited to, myeloproliferative diseases, blood cell proliferative diseases and autoimmune diseases often have an imbalance in the number of cells of particular lineages.
  • patients undergoing chemotherapy or irradiation often have defective hematopoiesis.
  • High-dose chemotherapy and/or radiation therapy together with bone marrow transplantation or transplantation of a cell population enriched for hematopoietic stem cells are standard treatment regimens for some malignancies, including, acute lymphocytic leukemia, chronic myelogenous leukemia, neuroblastoma, lymphoma, breast cancer, colon cancer, lung cancer and myelodysplastic syndrome, as well as for other non-malignant hematopoietic diseases. e.g. thrombocytopenia.
  • HSC have been demonstrated to be capable of repopulating non-hematopoietic tissues, including but not limited to liver (Petersen BE et al. Science 284: 1 168-70, 1999) and neuronal tissue (Bjornson CRR et al, Science 283:534-7, 1999). Clinical trials are underway using such regimens for the treatment of various cancers, including ovarian cancer, thymomas, germ cell tumors, multiple myeloma, melanoma, testicular cancer, lung cancer, and brain tumors.
  • Cell preparations enriched for hematopoietic stem cells generally contain a low percentage of cells capable of long-term hematopoietic reconstitution. Hence, there remains need to develop techniques for purification and expansion of hematopoietic stem cells.
  • Tpo Thrombopoietin
  • HSCs hematopoietic stem cells
  • Tpo increases the probability that both daughter cells of murine LTR-HSC would retain their HPP after the first cell division of single LTR-HSC (Sitnicka et al, Blood 92, Suppl 1, 59a, 1998).
  • HSC decline in long term bone marrow culture (LTBMC) in the absence of Tpo, and the continuous presence of Tpo results in the generation of both long- and short-term repopulating HSC as detected by an in vivo competitive repopulation assay (Yagi et al. , PNAS 96: 8126-8131, 1999).
  • LTBMC long term bone marrow culture
  • Tpo directly increases the probability that HSC daughter cells will self- replicate and become LTR-HSC (like the mother cell) during the 2-8 cell stage, the expansion is not sustained at later stages, but reaches a level of about 3-4 fold, then begins to reverse in culture. Accordingly, remains a need for ex vivo expanded hematopoietic stem cells that are pluripotent, capable of developing into a wide variety of cellular lineages and capable of transplantation. Research efforts are under way to develop a procedure for ex vivo expansion of hematopoietic stem cells, that retain such pluripotent status and both short- and long-term repopulating capability.
  • the invention provides a method of increasing the number of hematopoietic stem cells in vitro or ex vivo by obtaining a population of cells containing HSC from a mammalian subject; initiating an HSC culture by adding the HSC-containing population of cells to a culture containing stromal cells and megakaryocytes; and culturing the cells in vitro under conditions effective to result in at least a five-fold increase in cells having the phenotype and function of HSC about one month after initiating the culture.
  • the population of cells containing HSC are human HSC, derived from mobilized human peripheral blood, human bone marrow, human fetal liver or human cord blood.
  • the stromal cell component of the culture includes marrow fibroblasts and/or marrow-derived endothelial cells expanded within the culture; and/or cloned marrow fibroblasts and/or cloned marrow-derived endothelial cell lines generated exogenously and added to the culture.
  • the megakaryocyte component of the culture includes megakaryocytes expanded within the culture, and/or megakaryocytes derived from a biological source, cultured exogenously in the presence of one or more of thrombopoietin (Tpo), interleukin-3 (IL-3), interleukin-6 (IL-6), interleukin-1 1 (IL-1 1 ), and stem cell factor (SCF), then added to the culture.
  • Tpo thrombopoietin
  • IL-3 interleukin-3
  • IL-6 interleukin-6
  • IL-1 1 interleukin-1 1
  • SCF stem cell factor
  • conditions effective to result in at least a five-fold increase in cells having the phenotype and function of HSC after about one month of initiating the culture include, culturing the cells in medium containing horse serum, hydrocortisone and one or more cytokines, as detailed herein.
  • the method results in an expanded HSC composition comprising long term repopulating-HSC (LTR-HSC).
  • the population of cells containing HSC are murine HSC.
  • the method results in an expanded HSC composition, wherein the number of HSC is increased at least 100-fold within a one month of initiating the culture.
  • the invention also provides a cultured HSC composition having a number of hematopoietic stem cells that represents at least a five-fold increase in the number of HSC over the number of initiating HSC following about one month of culture, wherein the composition comprises HSC, stromal cells, megakaryocytes, cultured under conditions effective to result in at least a five-fold increase in cells having the phenotype and function of HSC.
  • the HSC composition comprises human HSC.
  • the stromal cell component of the HSC composition includes marrow fibroblasts and/or marrow-derived endothelial cells expanded within the culture, and/or cloned marrow fibroblasts and/or cloned marrow-derived endothelial cell lines generated exogenously and added to the culture.
  • the megakaryocyte component of the HSC composition includes megakaryocytes expanded within the culture, and/or megakaryocytes derived from a biological source, cultured exogenously (as described above) and added to the culture.
  • the culture conditions for preparing the HSC composition include culturing the HSC in medium containing horse serum, hydrocortisone and one or more cytokines.
  • the HSC composition comprises LTR-HSC.
  • the invention further provides an HSC composition comprising murine HSC.
  • the HSC composition comprises HSC which represent at least a 100- fold increase over the number of initiating HSC.
  • Figure 1 shows the effect of SCF+IL-6 ⁇ Tpo on the repopulating ability of LTR-HSC induced to divide 2-3 times in vitro, then transplanted into immunosuppressed mice;
  • Figure 3 shows the kinetics of lineage negative, c-kit+, Sca-1+ cells in ⁇ Tpo-LTMC from initiation of the culture up to four months in culture;
  • Figure 2 shows the cumulative production of HPPs in ⁇ Tpo-LTMC from initiation of the culture up to four months in culture;
  • Figure 4 shows the proportion of CD41+/61+ megakaryocytes in the NA fraction of ⁇ Tpo- LTMC
  • Figure 5A shows multilineage donor repopulation by 550,000 freshly harvested normal bone marrow cells
  • Figure 5B shows multilineage donor repopulation by 550,000 NA cells from 2-month Tpo-
  • FIG. 6 shows cumulative production of RU in ⁇ Tpo-LTMC.
  • RU were determined by limiting cell dilution transplant assays. Total number of RU was the frequency of RU x the average total cell number per flask (10 replicates); and
  • Figure 7 illustrates the frequency of human colony-forming cells at 10 and 12.5 weeks after initiation of long-term bone marrow culture in the presence and absence of Tpo.
  • a cell population enriched for hematopoietic stem cells refers to a cell population obtained using the positive and negative selection techniques described herein, wherein the hematopoietic stem cells are LTR- or STR-HSCs.
  • HSC expansion and an “increased number of HSC” refer to an increase in the number of LTR- HSC and STR-HSC.
  • long term repopulating hematopoietic stem cells or “LTR-HSC” refers to hematopoietic stem cells that are transplantable, and contribute to all lineages of hematopoietic cells for an undefined period of time, when transplanted into totally immunosuppressed recipients and do not undergo clonal extinction, as exemplified herein by murine LTR-HSC.
  • candidate hematopoietic stem cells may be evaluated in an in vivo sheep model or an in vivo NOD-SCID mouse model for human HSC and normal immunosupressed mice for murine HSC, respectively, as further described herein.
  • LTR-HSC have been isolated and characterized in mice using fluorescence-activated cell sorter (FACS) selection of density gradient-enriched, lineage-depleted bone marrow cells which are negative for expression of the CD34 antigen, positive for expression of the CD1 17 (c-kit) antigen, and exhibit low-level binding of the DNA binding dye, Hoechst 33342 (Ho-33342) and the mitochondrial binding dye, Rhodamine 123 (Rh-123), (Wolf, et al, 1993).
  • FACS fluorescence-activated cell sorter
  • STR- HSC short term repopulating hematopoietic stem cells
  • the STR-HSC population may be selected by FACS sorting and are phenotypically defined as light density gradient-enriched bone marrow cells which lack the expression of lineage markers (lin-), are positive for c-kit (CD 117), Seal and CD34, exhibit low- level binding of the DNA binding dye, Hoechst 33342 (Ho-33342) and high-level binding of the mitochondrial binding dye, Rhodamine 123 (Rh-123).
  • clonal extinction refers to the terminal differentiation of a single hematopoietic stem cell and all the progeny produced by clonal expansion of that cell, such that no more daughter cells are produced from the initial clone.
  • pluripototent hematopoietic stem cells refers to hematopoietic stem cells, capable of differentiating into all the possible cell lineages.
  • high proliferative potential colony forming cells or "HPP- CFCs”, as used herein relative to hematopoietic stem cells refers to murine or human cells that proliferate in response various cytokines and other culture conditions.
  • murine HPP-CFC are produced by culture of murine HSC in the presence of rat rSCF, mouse rIL-3 and human rIL-6.
  • the cells proliferate in semi-solid media, such as agar or methyl cellulose or as single cells in liquid culture, and form macroclones which have a diameter greater than 1 mm, generally having greater than 100,000 cells per clone with dense multicentric centers.
  • This population includes all murine HSCs, however, not all HPP-CFC are HSCs, and the HPP-CFC assay is not a specific assay for LTR-HSC.
  • low proliferative potential (LPP) clones contain from 2 to 100,000 cells per clone.
  • lineage-committed hematopoietic stem cells are hematopoietic stem cells that have differentiated sufficiently to be committed to one or more particular cell lineages, but not all cell lineages.
  • the term "lin -” or “lineage-depleted”, refers to a cell population which lacks expression of cell surface antigens specific to T-cells, B-cells, neutrophils, monocytes and erythroid cells, and does not express antigens recognized by the "YW 25.12.7” antibody. (See, e.g.,
  • purified relative to hematopoietic stem cells refers to HSCs that have been enriched (isolated or purified) relative to some or all of the other types of cells with which they are normally found in a particular tissue in nature, e.g., bone marrow or peripheral blood.
  • a "purified" population of HSCs has been subjected to density gradient fractionation, lineage depletion and positive selection for c-kit and Sea- 1 expression in addition to low level staining with both Hoechst 33342 and Rhodamine 123.
  • megakaryocytes refers to cells having an immunophenotype characterized as CD41+ and CD61+, with the potential to mature into platelet-producing cells, and having a specific morphology when stained by Romanovsky dyes.
  • CD41+ and CD61+ cells that range from immature megakaryoblasts to mature megakaryocytes.
  • the term "mid-differentiation megakaryocytes" refers to CD41 +/CD61 + cells that express transforming growth factor beta receptors 1 and 2.
  • tumor and cancer refer to a cell that exhibits a loss of growth control and forms unusually large clones of cells. Tumor or cancer cells generally have lost contact inhibition and may be invasive and/or have the ability to metastasize.
  • treatment of an individual or a cell is any type of intervention used in an attempt to alter the natural course of the individual or cell. Treatment includes, but is not limited to, administration of e.g., a cellular or pharmaceutical composition, and may be performed either prophylactically, or subsequent to the initiation of a pathologic event or contact with an etiologic agent.
  • improved therapeutic outcome relative to a cancer patient refers to a slowing or diminution of the growth of cancer cells or a solid tumor, or a reduction in the total number of cancer cells or total tumor burden.
  • cytokines including stem cell factor (SCF, or c-kit ligand), thrombopoietin (Tpo, c-mpl ligand), and the ligand for the Flt3/Flk2 receptor (FL) have been shown to act directly on HSC (Ogawa M et al, Stem Cells L5 Suppl 1, 7-1 1, 1997; Ku H et al, Blood 87, 4544-51, 1996; Ramsfjell V et al, Blood 88, 4481-92, 1996; Sitnicka E et al, Blood 87, 4998-5005, 1996; Young JC et al, Blood 88, 1619-31, 1996; Yoshida M et al, Br J Haematol 9%, 254-64, 1997; Matsunaga T et al, Blood ⁇ , 452-61 , 1998).
  • SCF stem cell factor
  • Tpo c-mpl ligand
  • FL Flt3/Flk2 receptor
  • Tpo as a single growth factor has been demonstrated to support survival and modest proliferation of highly purified HSC in vitro (Ramsfjell V et al, Blood 88, 4481-92, 1996; Sitnicka E et al, Blood 87, 4998-5005, 1997). Tpo is also known to be the major cytokine to facilitate the proliferation and differentiation of megakaryocytes(Kaushansky, K. Blood 86, 419-31, 1995) and administration of Tpo has been shown to speed hematopoietic recovery in myelosuppressed animals (Grossmann A et al, Exp Hematol 24, 1238-46, 1996).
  • Tpo or its receptor, c-mpl results not only in thrombocytopenia and megakaryocytopenia, but also in a decreased number of HSC and their progeny (Kimura S et al, Proc Natl Acad Sci USA 95:1195-200, 1998; Murone M et al, Stem Cells 16: 1-6, 1998), indicating that Tpo modulates the biological responses of HSC both in vitro and in hematopoietic tissues in vivo (Kaushansky K, Blood 92, 1-3, 1998).
  • LTMC Long term bone marrow cultures
  • Human hematopoietic stem cells for use in the present invention may be derived from human bone marrow, human newborn cord blood, fetal liver or adult human peripheral blood, after appropriate mobilization.
  • hematopoietic stem cells can be dramatically increased by treatment of a subject with certain compounds including cytokines.
  • Such "mobilized" peripheral blood hematopoietic stem cells have become an important alternative to bone marrow-derived hematopoietic stem cells transplantation procedures primarily because engraftment is more rapid. (See, e.g., Tanaka, J, et al, Int J Hematol 69(2):10-4, 1999).
  • Such mobilization may be accomplished using for example, one or more of granulocyte colony-stimulating factor (G-CSF), stem cell factor (SCF), thrombopoietin (Tpo), and a chemotherapeutic agent (i.e., cyclophosphamide).
  • G-CSF granulocyte colony-stimulating factor
  • SCF stem cell factor
  • Tpo thrombopoietin
  • a chemotherapeutic agent i.e., cyclophosphamide
  • hematopoietic stem cell enrichment/isolation generally include obtaining bone marrow, newborn cord blood, fetal liver or adult human peripheral blood which contains hematopoietic stem cells. Once obtained, a hematopoietic stem cell population may be enriched by performing various separation techniques such as density gradient separation, immunoaffinity purification using positive and/or negative selection by panning, FACS or magnetic bead separation. Following such enrichment steps, the cell population is further characterized phenotypically and functionally.
  • Tpo has been shown to support megakaryocyte differentiation and some expansion of CD34+, Thy-1+, lin- and Rhol23'° cells.
  • both the above phenotype as well as the CD34+, CD38-, KDR+ phenotype have been shown to contain most HSC and comprises only about 1 out of 100 CD34+ human cells, it appears that only approximately 1/100 CD34+ cells is an HSC. Therefore, assay for CD34+ cells may not accurately assay HSC present in a cell sample.
  • HPP-CFC high proliferative potential colony-forming cells
  • two pre- enrichment steps based on density gradient centrifugation e.g., using Nycodenz 1.080 g/ml, Nygaard, Oslo, Norway
  • negative selection using Dynal beads coupled to myeloid and lymphoid specific monoclonal antibodies
  • positive selection based on FACS sorting of cells based on staining with Rhodamine 123 (Rh), Hoescht 3342(Ho) and antibodies to c-kit.
  • Such candidate HSC are characterized in a variety of in vitro and in vivo assays generally known in the art, as further described below.
  • assays include, but are not limited to, an HPP-CFC assay, a single-cell HPP daughter cell assay, a single-cell IL-3 response assay, a single-cell assay for time to the first cell division, a cobblestone area-forming cell assay and an in vivo limiting dilution transplant assay to quantitate STR- and LTR-HSC.
  • Hematopoietic stem cells have been historically defined as transplantable cells, capable of self-renewal which possess the ability to generate daughter cells of any hematopoietic lineage.
  • Lineage-committed progenitor cells are defined as more differentiated cells derived from hematopoietic stem cells.
  • hematopoietic stem cell The phenotypic markers which characterize the hematopoietic stem cell have been the subject of extensive debate and numerous publications. As yet, there is no consensus as to which markers are definitive for murine or human hematopoietic stem cells, however, the markers for LTR-HSCs and STR-HSCs, as used herein, are provided above.
  • Functional readouts that have been used to detect and characterize hematopoietic stem cells include the ability to form colonies under particular conditions in cell culture (in vitro), such as in the long term culture initiating cell (LTCIC) assay (Pettengell R et al, Blood 84(11):3653-9, 1994), long term bone marrow culture (LTBMC; Dexter T M et al, Prog Clin Biol Res 148, 13-33, 1984) and the high proliferative potential-colony-forming cell (HPP-CFC) assay.
  • LTCIC long term culture initiating cell
  • LTBMC long term bone marrow culture
  • HPP-CFC high proliferative potential-colony-forming cell
  • Further functional characterization includes in vivo assay for long-term repopulating hematopoietic stem cells (LTR-HSC) and short-term repopulating hematopoietic stem cells (STR-HSC), as described above.
  • LTR-HSC long-term repopulating hematopoietic stem cells
  • STR-HSC short-term repopulating hematopoietic stem cells
  • LTBMC (Dexter T M et al, 1984) develop a complex adherent stromal layer containing a large variety of cell types, and can generate nonadherent (NA) hematopoietic cells for periods of several months. Hematopoietic stem cells are also often characterized functionally by activity in the high proliferative potential colony-forming cell (HPP-CFC) assay, as defined above.
  • HPP-CFC high proliferative potential colony-forming cell
  • HPP-CFC are generally characterized by: ( 1 ) a relative resistance to treatment in vivo with the cytotoxic drug 5-fluorouracil; (2) a high correlation with cells capable of repopulating the bone marrow of lethally irradiated mice; (3) their ability to generate cells of the macrophage, granulocyte, megakaryocyte and erythroid lineages, and (4) their multifactor responsiveness. (See, e.g., McNiece, I.K., fntJCell Cloning 8(3): 146-60, 1990).
  • Preferred cytokines for the culture of hematopoietic stem cells include one or more of interleukin-3 (IL-3), interleukin-6 (IL-6), interleukin- 1 1 (IL-11), interleukin-12 (IL-12), stem cell factor tyrosine kinase-3 (flt-3), an early acting hematopoietic factor, described, for example in WO 91/05795, and thrombopoietin (Tpo).
  • IL-3 interleukin-3
  • IL-6 interleukin-6
  • IL-12 interleukin-12
  • flt-3 stem cell factor tyrosine kinase-3
  • Tpo thrombopoietin
  • LTR-HSCs Long-term reconstitution of mice with murine LTR-HSCs following complete immunosuppression has been shown to require the transplantation of unfractionated bone marrow cells together with less differentiated long term repopulating cells, in order to provide initial, albeit unsustained engraftment, such that the completely immunosuppressed host may survive until the long term repopulating cells differentiate sufficiently to repopulate the host.
  • LTR-HSCs may take several months to effectively repopulate the hematopoietic system of the host following complete immunosuppression.
  • Methods have been developed to distinguish the cells of the donor and recipient in murine hematopoietic reconstitution studies, by using donor hematopoietic stem cells, congenic at the CD45 locus, defined as CD45.1 and recipient hematopoietic stem cells defined as CD45.2, such that monoclonal antibodies may be used to distinguish donor and recipient cells, i.e., by FACS analysis and or sorting.
  • the recipient is infused with sufficient CD45.2 positive bone marrow cells to keep the mouse alive until differentiation of CD45.1 donor cells occur to an extent sufficient to repopulate the hematopoietic system of the recipient.
  • Such methods may be used to differentiate LTR-HSC from STR-HSC and donor cells from recipient cells.
  • HSC human fetal liver
  • LTMC assay conditions include commercially available media, e.g. , Fishers medium; horse serum (Hyclone, Logan, UT) from a lot selected based on optimal HSC generation in murine Tpo-LTMC assays; purified recombinant human Tpo (rhuTpo, Genentech, South San Francisco, CA); hydrocortisone; a human stromal cell component which includes, but is not limited to, cells of mesenchymal origin, including fibroblasts, adipocytes, endothelial cells; and megakaryocytes.
  • media e.g. , Fishers medium
  • horse serum Hyclone, Logan, UT
  • purified recombinant human Tpo rhuTpo, Genentech, South San Francisco, CA
  • hydrocortisone a human stromal cell component which includes, but is not limited to, cells of mesenchymal origin, including fibroblasts, adipocytes, endotheli
  • the human stromal cell and megakaryocyte component of such cultures may be produced within the culture or provided from an exogenous source.
  • Marrow epithelial cells and marrow fibroblasts are available as cloned cell lines or may be obtained from biological sources, separately cultured and added to the HSC culture.
  • Exemplary cloned marrow fibroblast cell lines include HS- 27a, HS-5, HS-19, HS-21, HS-23 (Dr. Beverly Torok-Storb).
  • Human HSC produced in such assays are initially characterized by immunophenotype, e.g., as lineage negative and CD34+ or lineage negative and CD 34+/38-/KDR+ cells 1 and by telomere length for both HuTpo-LTBMC and control-LTBMC (where cells with high proliferative capacity have longer telomeres).
  • HSC expansion occurs in cultures that contain both non-adherent megakaryocytes at different stages of differentiation and adherent clusters of f ⁇ broblasts/immature megakaryocytes and associated endothelial cells.
  • In vivo assays for human HSC may be carried out by using approximately 10,000-20,000 purified lineage negative CD34+ cells derived from culture in an in utero fetal sheep assay (Zanjani ED et al, Stem Cells 13(2): 101-11, 1995).
  • the cells may be used immediately or frozen in liquid nitrogen and stored for long periods of time, using standard conditions, such that they can later be thawed and used, e.g., for administration to a patient.
  • the cells will usually be stored in 10% DMSO, 50% fetal calf serum (FCS), and 40% cell culture medium.
  • FCS fetal calf serum
  • the present invention is directed to compositions comprising an expanded population of
  • HSC HSC
  • the culture must include: (1) HSC; (2) megakaryocytes at different degrees of maturation; (3) one or more stromal cell components, e.g., marrow fibroblasts and/or marrow endothelial cells; (4) culture medium which comprises horse serum; hydrocortisone; and one or more of the following cytokines, Tpo, IL-6, TGF-beta, IL- 1 1 , IL- 12, flt-3 , SCF, and IL-3.
  • Effective concentrations for such cytokines include: Tpo (10-50 ng/ml), IL-6 (5-20 ng/ml), TGF-beta (0.1-1.0 femtogram/ml), IL-1 1 (10-50 ng/ml), IL-12 (10-50 ng/ml), flt-3 (10-50 ng/ml), SCF (10-50 ng/ml), and IL-3 (10-100 ng/ml).
  • Tpo (20 ng/ml), IL-6 (20 ng/ml), TGF-beta (1.0 femtogram/ml, IL-1 1 (20 ng/ml), IL-12 (20 ng/ml), flt-3 (20 ng/ml), SCF (20 ng/ml), and IL-3 (100 ng/ml).
  • Megakaryocytes at different degrees of maturation include, but are not limited to, CD41+ and CD61+ cells that range from immature megakaryoblasts to mature megakaryocytes.
  • the term mid-differentiation megakaryocyte refers to a CD41+/CD61+ cell that expresses transforming growth factor beta receptors 1 and 2.
  • hematopoietic cell types are also present and generated under Tpo-LTMC conditions, and include granulocyte series cells, red blood series cells (RBC), B-lymphocyte series cells, and T-lymphocyte series cells.
  • RBC red blood series cells
  • B-lymphocyte series cells include granulocyte series cells, red blood series cells (RBC), B-lymphocyte series cells, and T-lymphocyte series cells.
  • the stromal cell component includes, but is not limited to, cells of mesenchymal origin, including fibroblasts, adipocytes, endothelial cells.
  • a preferred HSC composition (murine or human) will develop after about one month in culture and contains: (1) 30-50% megakaryocytes at different stages of development that are both non-adherent and adherent to marrow fibroblasts ; (2) 10-20% marrow fibroblasts; (3) 5-10% marrow endothelial cells; (4) 20-40% mature hematopoietic cells (myeloid, lymphoid and erythyroid series cells); and (5) 0.01-0.0001% hematopoietic stem cells.
  • Each of the above cell types may be generated from a normal human bone marrow aspirate, human fetal liver as described for mouse bone marrow, or in some cases the various components are generated as follows: (1) 30-50% human megakaryocytes generated in separate culture from CD34+ bone marrow, peripheral blood, cord blood or fetal liver cells in the presence of one or more of Tpo, IL-6, TGF-beta, IL-11, IL-12, flt-3, SCF, and IL-3; (2) 10-20% cloned human bone marrow fibroblast cell lines generated in separate cultures; (3) 5-10% marrow endothelial cells; and (4) about 0.01% hematopoietic cells from a source such as (a) CD34+ cells from human bone marrow, peripheral blood, cord blood or fetal liver, (b) lineage negative, 34+38- human bone marrow, peripheral blood, cord blood or fetal liver cells, or (c) partially purified or unfractionated human bone
  • HSC composition characterized by: (1) an increased number of HSC and other primitive hematopoietic cells relative to the number of HSC and other primitive hematopoietic cells present at initiation of the culture (assayed in vitro or by in vivo reconstitution assay; (2) an increased number of HSC relative to the nadir of HSC which usually occurs between two to four weeks; and (3) an increase in population of megakaryocytes relative to the number of megakaryocytes present at initiation of the culture.
  • compositions and methods described herein reflect an increased number of HSC which is at least 5-fold and preferably 100-fold greater than the number of HSC used to initiate the culture.
  • initiate the culture or initiating HSC is meant the HSC added to the culture from a source of HSC, which self-replicate under the conditions described herein.
  • An increased number of megakaryocytes means an increased number of megakaryocytes which is at least 5-fold and preferably at least 40-fold greater than the number of megakaryocytes present at the initiation of the culture.
  • the invention is also directed to a method for obtaining such expanded HSC compositions, where the method includes the steps of: (1) obtaining a population of HSC for initiating a HSC culture, as further described above; (2) adding the HSC to a culture containing either mouse or human stromal cells and megakaryocytes; and (3) culturing the HSC under conditions effective to result in an increase in the population of megakaryocytes relative to the number of megakaryocytes present at initiation of the culture.
  • a population of HSC for initiating such a HSC culture is LTR-HSC (characterized by phenotype as described above), generally derived from bone marrow, mobilized peripheral blood, fetal liver or fetal cord blood, with bone marrow and mobilized peripheral blood preferred.
  • Hematopoietic stem cells for initiating such a HSC culture can be extracted from a subject, purified and cultured in vitro in the presence of one or more cytokines.
  • Preferred cytokines include Tpo, IL-6, TGF-beta, IL- 1 1 JL- 12, flt3 , SCF, and IL-3.
  • Such HSC may be derived from any mammal, with preference for HSC that are autologous or allogeneic human HSC.
  • the number of HSC for initiating such an HSC culture is generally from about 1,000 to 1,000,000 HSC, dependent upon the size of the culture.
  • an HSC culture in a T-25 flask may initiated by addition of about 10 X 10 6 cells.
  • such cultures may be scaled up in proportion to the surface area of the culture vessel such that the number of HSC and other cells in the source used to initiate the culture can be accommodated.
  • the method for obtaining such an expanded HSC composition is effective to result in: (1) an increase in population of HSC relative to the number of HSC present at initiation of the culture (in vitro) or reconstitution assay (in vivo) and (2) an increase in population of megakaryocytes relative to the number of megakaryocytes present at initiation of the culture.
  • HSC are characterized by one or more of: (1) phenotype, by FACS analysis for cells which are negative for expression of the CD34 antigen, positive for expression of the CD1 17 (c-kit) antigen, and exhibit low-level binding of the DNA binding dye, Hoechst 33342 (Ho-33342) and the mitochondrial binding dye, Rhodamine 123 (Rh- 123); long term culture, e.g., in a long term bone marrow assay; and in vivo reconstitution experiments.
  • Ho-33342 Hoechst 33342
  • Rh- 123 Rhodamine 123
  • LTR-HSC can be achieved ex vivo (in vitro) and such self-replication (i.e., expansion) of LTR-HSC is facilitated by culture conditions which: (1) are effective to expand the population of megakaryocytes at different degrees of maturation; (2) include mouse or human marrow fibroblasts and/or other stromal cells; (3) include hydrocortisone; and (4) include one or more of the following cytokines: Tpo, IL-6, TGF-beta, IL-1 1, IL-12, flt-3, SCF and IL-3.
  • Tpo increased the probability that both daughter cells of murine LTR-HSC would retain their HPP after the first cell division of single LTR-HSC (Sitnicka E et al, Blood 92, Suppl 1, 59a, 1998). See, also Example 1, wherein the effect of Tpo alone on HSC (in the absence of stroma), is further described.
  • the results suggest that Tpo may be directly involved in cell fate decisions during at least the first cell division in vitro, as indicated the observation of an increased number of daughter cells with a HPP when Tpo was included in the culture medium in addition to other cytokines.
  • HSC When HSC are cultured in the presence of stromal cells and Tpo, LTR-HSC survive for several months but do not expand, as indicated by HPP and transplantation assays.
  • HSC When HSC are co-cultured with megakaryocytes in the presence of Tpo, a rapid loss of
  • HSC occurs as indicated by HPP assay and in vivo transplant studies.
  • the present invention is directed to culture conditions which results in prolonged expansion of HSC as demonstrated by in vitro culture assays and/or in vivo reconstitution studies.
  • the invention is based on the discovery that culture of HSC in the presence of megakaryocytes at different degrees of maturation; a stromal cell component and culture medium which comprises horse serum; hydrocortisone; and one or more of the following cytokines; Tpo, IL-6, TGF-beta, IL-11, IL-12, flt-3, SCF and IL-3 is effective to result in at least a five-fold increase in the number of HSC in ex vivo culture over a period of from about one to four months.
  • Transplantation of hematopoietic stem cells derived from peripheral blood and/or bone marrow is increasingly used in combination with chemotherapy and/or radiation therapy for the treatment of a variety of disorders including numerous forms of cancer.
  • the percentage of cells in such transplants that are capable of long-term hematopoietic reconstitution is very low and therefore there is a need to develop techniques for purification and expansion of hematopoietic stem cells.
  • Complications of transplantation therapy with a cell population enriched for hematopoietic stem cells include removal of cells in the transplant that pose a risk to the transplant recipient, including T-cells that are responsible for graft versus host disease (GVHD) in allogeneic grafts and tumor cells in autologous transplants that may cause recurrence of disease.
  • GVHD graft versus host disease
  • the invention provides hematopoietic stem cells for initiating an HSC culture.
  • Such initiating HSC may be contained in a biological sample, enriched or purified, and are cultured ex vivo under the conditions described herein, resulting in an HSC composition which has at least 5-fold more HSC than added to the initial culture.
  • Such a hematopoietic stem cell composition finds utility in a variety of applications, including, but not limited to, (1) expanding or multiplying the population of hematopoietic stem cells ex vivo for subsequent in vivo administration to a subject for purposes of (a) hematopoietic stem cell replacement therapy, (b) gene therapy, (c) treatment of autoimmune disease; (d) reducing the immune response to allogeneic transplants and (e) treatment of HIV- infection in a subject; (2) increasing the population megakaryocytes ex vivo for subsequent in vivo administration to a subject for (a) cell replacement therapy; and (b) enhancing engraftment of HSC.
  • Autologous hematopoietic stem cell transplantation has been used to treat many solid tumors, including but not limited to, breast cancer and ovarian cancer.
  • a chemotherapy regimen to reduce the amount of tumor present, generally followed by: (1) the collection of the patient's hematopoietic stem cells from either bone marrow or mobilized peripheral blood, (2) culture of hematopoietic stem cells in the presence of cytokines or cryopreservation in liquid nitrogen, (3) high-dose chemotherapy administration intravenously (in most cases), and (4) reinfusion of the patient's hematopoietic stem cells (IV), approximately 48 hours after the chemotherapy administration is complete, and (5) further treatment of the patient with growth factors to promote the differentiation of the hematopoietic stem cells and repopulation of the patients hematopoietic system. In general, during this time the patient is immunocompromised and protective isolation is required.
  • Allogeneic hematopoietic stem cell transplantation has been used to treat patients with leukemia, aplastic anemia, lymphomas (Hodgkin's disease and non-Hodgkin's lymphoma), and immune deficiency diseases.
  • An allogeneic hematopoietic stem cell transplantation protocol is similar to that used for autologous transplantation with the exception that in allogeneic transplantation, the donor and recipient must be matched based on the similarity of HLA cell surface antigens in order to minimize the immune response of both donor and recipient cells against the other.
  • GVHD is a frequent complication of allogeneic transplantation. About half of the patients undergoing an allogeneic bone marrow transplant develop some GVHD, which is generally mild, but can be life threatening in some cases. In GVHD, the donor's cells attack the recipient's organs and tissue. Patients with GVHD have an increased susceptibility to infection and the skin, liver, and gastrointestinal tract may be attacked in GVHD.
  • GVHD graft versus host disease
  • such treatment often includes, T-cell depletion (i.e., by elutriation which removes T-cells based on density gradient centrifugation) alone, or in combination with hematopoietic stem cell enrichment by selection using monoclonal antibodies with hematopoietic stem cell markers, and drug therapy for prevention of GVHD, e.g., by administration of cyclosporine (an immunosuppressive drug), alone or together with mehtotrexate.
  • T-cell depletion i.e., by elutriation which removes T-cells based on density gradient centrifugation
  • drug therapy for prevention of GVHD e.g., by administration of cyclosporine (an immunosuppressive drug), alone or together with mehtotrexate.
  • the culture of hematopoietic stem cells under conditions described herein results in a hematopoietic stem cell composition that wherein the number of HSC is increased at least 5-fold the number of hematopoietic stem cells used to initiate the ex vivo culture.
  • HSC within such an ex vivo expanded HSC composition lack immunological memory of self and non-self antigens, such that transplantation of the hematopoietic stem cells into an allogeneic host is unlikely to result in GVHD.
  • the methods of the invention may be used to generate increased numbers of hematopoietic stem cells together with increased numbers of megakaryocytes, based on the duration of culture, the composition of the cytokine component, the lot of horse serum, the source of the stem cells, and the composition of the stromal cell and megakaryocyte components.
  • the present method may be used to expand a population of the recipient's own hematopoietic stem cells in vitro (ex vivo) preserving the multilineage potential of such cells without lineage commitment, e.g., for use in autologous hematopoietic stem cell transplantation.
  • An exemplary therapeutic regimen involves ex vivo expansion of HSC derived from a cancer patient, wherein HSC are purified from an HSC-containing population of cells taken from the pateint in a manner effective to eliminate cancer-containing cells and the cells are cultured under the conditions described herein such that the number of viable cancer-free HSC is expanded, followed by reinfusion of the expanded HSC composition into the patient.
  • the therapeutic regimen further includes additional intervention such as radiation therapy and/or chemotherapy. The treatment may occur prior to, during or subsequent to re-infusion of ex vivo expanded HSC.
  • hematopoietic stem cells differentiate they are exposed to the various antigens present on the cells and tissue of the host and immunological tolerance is established during T cell development within the thymus. In general, T cells that would be reactive to host proteins do not survive. However, in some cases, the immune system may recognize self antigens as foreign resulting in an immune reaction against one or more endogenous antigens, leading to an autoimmune condition or disease.
  • Exemplary autoimmune conditions include organ specific forms wherein the immune response is directed against, e.g., the cells of the adrenal glands, causing Addison's disease, against the thyroid causing auto- immune thyroiditis (Hashimoto's disease) or against the beta cells of the islets of Langerhans in the pancreas, resulting in insulin-dependent diabetes mellitus; and non- specific forms wherein the immune response is directed against an antigen that is ubiquitous, e.g., an immune reaction against DNA, resulting in the disease systemic lupus erythematosus.
  • Further examples include, Sj ⁇ gren's syndrome, caused by the production of auto-antibodies against salivary ducts, rheumatoid arthritis.
  • Auto- immunity may be the result of attack by antibodies, T-cells or both.
  • the invention provides methods and compositions for the treatment of autoimmune disease.
  • hematopoietic stem cells are obtained from a patient, followed by treatment of the patient with chemotherapy, radiation therapy or other means to deplete the patient of residual T-cells.
  • the patients' hematopoietic stem cells or hematopoietic stem cells from an allogeneic donor are cultured ex vivo under conditions effective to result in an increase the number of viable hematopoietic stem cells in vitro (as detailed herein), which are then re-infused into the patient.
  • the expanded population of hematopoietic stem cells develops in the presence of the antigenic repertoire of the host, the newly developed T-cells should not recognize host antigens as foreign and GVHD should not occur.
  • Such an in vitro expanded hematopoietic stem cell composition lacks immunological memory of self antigens, such that transplantation of the HSC composition finds utility in transplantation regimens for treatment of a patient with an autoimmune disease, in order to minimize or eliminate the autoimmune condition.
  • ex vivo hematopoietic stem cell treatment and re- infusion is generally used in combination with additional therapeutic intervention to minimize the autoimmune response of the patent's cells which are present prior to and during hematopoietic stem cell isolation and in vitro HSC expansion.
  • additional treatment components include compositions and procedures known in the art for the treatment of autoimmune disease.
  • Gene therapy is a fast evolving area of medical and clinical research. Gene therapy encompasses gene correction therapy, and transfer of therapeutic genes and is being applied for treatment of cancer, infectious diseases, multigenic diseases, and acquired diseases.
  • Exemplary disease targets include, but are not limited to cancer such as prostate cancer, breast cancer, lung cancer, colorectal cancer, melanoma and leukemia; infectious diseases, such as HIV, monogenic diseases such as CF, hemophilia, phenylketonuria, ADA, familial hypercholesterolemia, and multigenic diseases, such as restenosis, ischemia, and diabetes.
  • infectious diseases such as HIV, monogenic diseases such as CF, hemophilia, phenylketonuria, ADA, familial hypercholesterolemia, and multigenic diseases, such as restenosis, ischemia, and diabetes.
  • HSC hematopoietic stem cells
  • An exemplary therapeutic gene therapy regimen may include the steps of obtaining a source of HSC from a subject, HSC enrichment or purification, in vitro or ex vivo HSC expansion, transduction of HSC with a vector containing a gene of interest, and reintroduction into a subject.
  • the transfer of genetic material into cells can be achieved by physical and chemical methods or by the use of recombinant viruses.
  • chemical and physical methods such as calcium phosphate, electroporation and pressure mediated transfer of genetic material into cells are often used.
  • viral vectors for example, retroviral vectors, adenovirus vectors, adenovirus-associated vectors (AAV), herpes virus vectors, pox virus vectors; non-viral vectors, for example naked DNA delivered via liposomes, receptor-mediated delivery, calcium phosphate transfection, electroporation, particle bombardment (gene gun), or pressure-mediated gene delivery.
  • HSC compositions described herein may be evaluated, e.g., by conventional FACS assays for the phenotype of cells produced by in vitro culture or at various time points after in vivo administration of HSC.
  • Phenotypic analysis is generally carried out using monoclonal antibodies specific to the cell type being analyzed.
  • the use of monoclonal antibodies in such phenotypic analyses is routinely employed by those of skill in the art for cellular analyses.
  • Hematopoietic stem cells are characterized phenotypically as detailed above. Such phenotypic analyses are generally carried out in conjunction with biological (functional) assays for a given cell type of interest, for example; (1) hematopoietic stem cells, LTCIC, cobblestone forming assays, and assays for HPP-CFC; (2) granulocytes or neutrophils, clonal agar or methylcellulose assays wherein the medium contains G-CSF or GM-CSF; (3) megakaryocytes, clonal agar or methyl cellulose assays wherein the medium contains Tpo, IL-3, IL-6 and IL-1 1; and (4) erythroid cells, clonal agar or methyl cellulose assays wherein the medium contains EPO and SCF or EPO, SCF and IL-3.
  • an in vitro or ex vivo expanded hematopoietic stem cell composition may serve as a source of hematopoietic stem cells or an expanded population of megakaryocytes for various cellular and gene therapy applications.
  • Such an in vitro or ex vivo expanded hematopoietic stem cell composition finds utility in both autologous and allogeneic hematopoietic engraftment when readministered to a patient, where the cells are freed of neoplastic cells and graft-versus-host disease can be avoided.
  • such an in vitro or ex vivo expanded hematopoietic stem cell composition may be used for gene therapy to treat any of a number of diseases.
  • HSC containing a transgene of interest directed toward a particular disease target is prepared in vitro and reinfused into a subject such that the cell type(s) targeted by the disease are repopulated by differentiation of cells in the HSC composition following reinfusion into the subject.
  • treatment of the recipient's own bone marrow or hematopoietic stem cells e.g., bone marrow extracted from the patient before commencing chemotherapy or radiation therapy
  • the conditions described herein h result in an expanded population of HSC will avoid the current need for immune suppression by minimizing the potential for GVHD following transplantation.
  • the methods of the invention can be used to expand HSC and megakaryocytes derived form bone marrow or purified hematopoietic stem cell compositions, prior to their storage in cell banks in order to retain the cells in a form devoid of tissue histocompatibility antigens.
  • the expanded HSC composition described herein finds utility in therapeutic regimens directed to repopulation of various tissues, including but not limited to liver (Petersen BE et al, Science 284: 1168-70, 1999) and neuronal tissue (Bjornson CRR et al, Science 283:534-7, 1999).
  • LTMC Long term murine bone marrow cultures
  • B6.SJL-Pt/?rc ⁇ Pep3 b /BoyJ (Ly5.1) (CD45.1 ) (B6.SJL) mice (Jackson Labs, Bar Harbor, ME) with medium consisting of Fischer's medium (Gibco BRL Life Technologies, Gaithersburg, MD) supplemented with 20% heat-inactivated defined horse serum (HyClone Laboratories, Logan, UT), 100 units/ml penicillin- 10 ⁇ g/ml streptomycin, 2 mM L- glutamine, and 1 ⁇ M hydrocortisone succinate (Sigma, St.
  • recombinant mouse thrombopoietin (R & D Systems, Minneapolis, MN) was added to a final concentration of 10 ng per ml. Cultures were incubated in a humidified incubator at 37°C in 5% CO2 in air. LTBMC were fed twice weekly beginning after week one by removing 4 (for the subsequent four weeks) or 6 ml (thereafter) medium and NA cells, and replacing with an equivalent volume of medium (and Tpo in the appropriate cultures).
  • long term murine bone marrow cultures may support the replication of human HSC derived from (1) bone marrow, (2) mobilized peripheral blood, (3) fetal liver, or (4) fetal cord blood using procedures routinely employed by those of skill in the art.
  • LTBMC cells Immunophenotyping of LTBMC cells.
  • NA cells from Tpo-containing and control LTBMC were centrifuged and resuspended in 1% (w/v) bovine serum albumin in Dulbecco's phosphate- buffered saline.
  • Fluorochrome-conjugated monoclonal antibodies to various mouse CD antigens, or biotinylated anti-mouse CD34 and FITC- or PE-conjugated strepavidin (Pharmingen, San Diego, CA) were incubated with the cells on ice (1 ⁇ g antibody/ l-2xl0 5 cells). Cells were washed and analyzed by flow cytometry (FACScan, Becton-Dickinson, Mountain View, CA) in the presence of propidium iodide to exclude dead cells.
  • Clonogenic cell assays were performed in soft agar cultures (murine) or methylcellulose (human) in the presence of recombinant cytokines (R & D Systems or PeproTec, Rose Hill, NJ) (Sitnicka E et al, Blood 87, 4998-5005, 1996). Two thousand to 5,000 cells were added per ml of culture and plated in 35mm dishes. Cultures were incubated for 12 days, and colonies were counted using an inverted microscope. In some experiments, cells were plucked from colonies and their morphology assessed after staining with Giemsa.
  • Cytokines were used at the following concentrations: for CFC, 5ng/ml mouse GM-CSF and 10% (v/v) L929 supernatant (mouse M-CSF); for HPP-CFC, 50ng/ml rat SCF, 20ng/ml human IL-6, and lOng/ml mouse IL-3.
  • Colony formation assays for murine versus human CFC differ in that human HPP-CFC are carried out in methylcellulose medium (Stem Cell Tech., Cat. No. H4435), in the presence of SCF (50 ng/ml), IL-3 (50 ng/ml), IL-6 (20 ng/ml), erythropoietin (EPO, 1 unit/ml) and GM-CSF (5 ng/ml).
  • SCF 50 ng/ml
  • IL-3 50 ng/ml
  • IL-6 20 ng/ml
  • EPO erythropoietin
  • GM-CSF 5 ng/ml
  • NA cells from LTBMC established from B6.SJL mice (CD45.1) were harvested, washed, and used unfractionated for transplant.
  • 2-10 recipient C57B16 mice (CD45.2) were irradiated (950 rad, l- ⁇ Cesium source ) anc j transplanted by injection via the tail vein with the indicated number of test cells mixed with 4 xlO ⁇ fresh unfractionated CD45.2 marrow cells.
  • fresh unfractionated bone marrow cells from CD45.1 animals were transplanted in parallel with the cells from LTBMC. Animals were maintained in microisolator cages in an SPF facility.
  • Peripheral blood samples were obtained by retro-orbital bleeding 3, 6, 12, and 24 weeks post transplant. Expression of the donor CD45.1 allele and lineage specific antigens was assessed by two-color flow cytometry analysis of peripheral blood leukocytes using directly labeled monoclonal antibodies as described above for cultured cells. The frequency of long-term repopulating units was estimated using the maximum likelihood model that requires limiting dilution cell transplants of the test cells (Taswell C, J Immunol 126, 1614-9, 1981).
  • Single LTR-HSC were sorted into 96-well roundbottom plates and clones visually (microscopically) identified at their first cell division (between 2-7 days of culture), then re-plating the two daughter cells into a mini agar culture containing SCF, IL-3 and IL-6 to assay for HPP ability.
  • the presence of purified, recombinant Tpo ( 10 ng/ml), in the culture increased the probability of generating daughter cells with a HPP when combined with SCF+IL-6, or SCF+IL- 6+IL-3 or SCF+flt-3, as indicated by the results presented in Table 1.
  • Tpo increases the number of HPP generated from single LTR-HSC at the 8-cell stage.
  • Tpo to a modified LTMC resulted in a sustained production of LTR-HSC for several months.
  • the modified cultures were initiated and carried forward as follows: 10 X10 6 unfractionated bone marrow cells were seeded into T-25 flasks in Fisher's medium, 20% horse serum (selected lots), 10 "6 M hydrocortisone and 10 ng/ ml purified, recombinant Tpo.
  • Cultures were incubated at 37°C, fed with medium during weeks 2-4, when cultures were fed twice a week by removing half of the medium and non-adherent (NA) cells and replacing with fresh medium; and during week 5 onward, when cultures were fed by removing two thirds of the medium and NA cells and replacing with fresh medium, without addition of marrow at any point in the culture process.
  • the clonogenic potential of the NA cells from Tpo-LTBMC was analyzed by colony formation in soft agar in the presence of various cytokines. As shown in Table 2, both the frequency and absolute number of CFC in the NA cells from Tpo-LTBMC was greater than that observed for control LTBMC. Both the number and frequency of CFC in control LTBMC decreased with time in culture. In contrast, the frequency of CFC in the NA cells from Tpo- LTBMC remained approximately constant up to four months in culture. The observed differences in CFC frequency were more striking for the primitive HPP-CFC than for the more mature myeloid CFC (Table 2A and Figure 2).
  • Purified recombinant murine Tpo (rmuTpo) was titered and the total number of NA cells/flask and the number of HPP/flask determined after 2 weeks. As indicated by the results presented in Table 2B, the increase in HPP was correlated with the concentration of Tpo.
  • the proportion of immature or lineage negative c-kit+/Sca-l+ cells was determined after 1, 2.5 and 4 months in LTMC in the presence and absence of Tpo. As shown in Figure 3, the percentage of lineage negative c-kit+/Sca-l+ cells increased in both plus and minus Tpo-LTMC after the first month, however only in the Tpo-LTMC was production sustained. In the absence of Tpo, essentially all cell production had ceased by 2.5 months.
  • hematopoietic cell markers on the NA cells from LTBMC in the presence and absence of Tpo was assessed by flow cytometry. Many of the NA cells from Tpo- LTBMC (45 to 98%) were found to express the megakaryocytic markers CD41 (platelet GPIIb) and CD61 (GPIIIa) ( Figure 4). In addition, a high number of NA cells from Tpo-LTBMC expressed cell surface antigens characteristic of primitive HSC. At 4 weeks of culture, approximately 0.5% of the NA cells from both control and Tpo-LTBMC expressed high levels of c-kit and Sea- 1 (Table 3).
  • Peripheral blood was assayed at various times post-transplant for the presence of CD45.1 + donor cells expressing lineage-specific markers to assess the contribution of the fresh BM or Tpo- LTBMC cells to hematopoiesis.
  • chimeras were generated in which CD45.1 + donor cells comprised from about 30 to 80% of mature cells in the peripheral blood up to 4 months after transplant, demonstrating the presence of long-term repopulating HSC in Tpo-LTBMC.
  • transplant of over 10 NA cells from two-month-old control LTBMC resulted in only transient repopulation, with few CD45.1+ cells detectable after 1.5 months.
  • Tpo-LTMC chimeras were similar to those produced in mice injected with fresh BM chimeras (36% myeloid, 64% lymphoid; Table 4, Figures 5A and B). As set forth above, such cultures contain HSC, megakaryocytes, stromal cells and Tpo.
  • the non-adherent cell fraction of 2-month Tpo- LTMC contains transplantable cells that have a long term repopulating ability similar that of normal marrow; (2) the non-adherent cells from control LTMC have significantly less repopulating ability; (3) the donor repopulation by NA cells from control LTMC is transient in that donor derived cells are essentially absent by three months post-transplant (indicating that they are STR- HSC); (4) such NA cell fractions contain megakaryocytes at different stages of development; (5) the NA cells are not generated in cultures containing Tpo alone, but in cultures which have a stromal component, including, e.g., about 10-20%) marrow fibroblasts, about 5-10% marrow endothelial cells, about 20-40% mature hematopoietic cells (myeloid, lymphoid and erythyroid series cells) and about 0.01-0.0001% HSC.
  • a limiting cell dilution transplant analysis was used to quantitate the number of HSC in +/- Tpo-LTMC at 1-4 months in culture.
  • the minimum number of transplantable cells in a sample was calculated based on the cell dose at which mice become negative for donor cells (Taswell, C. J Immunol 126, 1614-9, 1991; Szilvassy et al, Proc Natl Acad Sci USA. 87(22):8736-40, 1990).
  • Negative donor repopulation is defined as ⁇ 0.5% CD45.1+ donor cells (which is based on the sensitivity of FACS detection by empirically measuring standards made by diluting CD45.1 cells into CD45.2 cells).
  • the HPP assay may be used as an indicator of HSC frequency.
  • mice transplanted with Tpo-LTMC cells were carried out to 1 1 months after transplant of 2-3 month Tpo-LTMC cells and 10 6 femoral marrow cells were retransplanted to secondary lethally irradiated mice without competitor/support marrow, all mice survived and CD 45 J donor cell chimerisms of 60-80% developed in the secondary host.
  • Cells were suspended at a concentration of 0.5-2xl0 7 per ml in medium consisting of MEM-alpha containing L-glutamine, deoxyribonucleosides and ribonucleosides (Gibco #12571) supplemented with 12.5% fetal bovine serum, 12.5% horse serum, 0.1 mM 2-mercaptoethanol, l ⁇ M hydrocortisone, 0.2mM inositol, 20mM folic acid, and extra glutamine to a final concentration of 2mM.
  • Recombinant human Tpo (R&D Systems) was added to half the flasks to a final concentration of 50 ng/ml (Experiment A), 37.5 ng/ml (Experiment B), or 10 ng/ml medium (Experiments C and D). Cultures were maintained in a humidified incubator at 37°C in 5% C0 2 in air. Cultures were fed weekly by removal and replacement of half the medium.
  • Colony forming assay were carried out according to the method described in Sitnicka et al, 1996, and colony formation evaluated at 4, 8, 10 and 12.5 weeks after initiation.
  • results indicate that the initiation and maintenance of long-term cultures in the presence of Tpo results in a consistent increase in the frequency of colony-forming cells for 8-12.5 weeks.
  • Figure 7 illustrates the results of colony forming assays preformed at 10 and 12.5 weeks after initiation of culture in the presence and absence of Tpo (Experiment A).

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Abstract

Cette invention concerne des compositions à base de cellules souches hématopoïétiques (HSC) comprenant des HSC, des mégacaryocytes et des cellules du stroma. L'invention concerne également des conditions de culture permettant de multiplier par au moins cinq le nombre de cellules présentant le phénotype et la fonction de HSC après un mois de culture ainsi que des méthodes permettant d'atteindre ce résultat.
PCT/US1999/029138 1998-12-07 1999-12-07 Multiplication de cellules souches hematopoietiques induite par des megacariocytes WO2000034443A2 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1517988B1 (fr) * 2002-06-20 2013-05-08 Augustinus Bader Utilisation de l'érythropoïétine pour la régénération de tissus in vivo
US20140205582A1 (en) * 2011-07-06 2014-07-24 Cellerant Therapeutics, Inc. Megakaryocyte progenitor cells for production of platelets

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996000779A1 (fr) * 1994-06-28 1996-01-11 Cornell Research Foundation, Inc. Procede de developpement ex vivo de cellules parentes hematopoietiques

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996000779A1 (fr) * 1994-06-28 1996-01-11 Cornell Research Foundation, Inc. Procede de developpement ex vivo de cellules parentes hematopoietiques

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
SITNICKA EWA ET AL: "The effect of thrombopoietin on the proliferation and differentiation of murine hematopoietic stem cells." BLOOD 1996, vol. 87, no. 12, 1996, pages 4998-5005, XP000907258 ISSN: 0006-4971 *
WAEGELL W O ET AL: "GROWTH ACCELERATION AND STEM CELL EXPANSION IN DEXTER-TYPE CULTURES BY NEUTRALIZATION OF TGF-BETA" EXPERIMENTAL HEMATOLOGY,US,NEW YORK, NY, vol. 22, no. 11, 1 January 1994 (1994-01-01), pages 1051-1057, XP000572237 ISSN: 0301-472X *
YAGI MAYUMI ET AL: "Sustained ex vivo expansion of hematopoietic stem cells mediated by thrombopoietin." PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA JULY 6, 1999, vol. 96, no. 14, 6 July 1999 (1999-07-06), pages 8126-8131, XP002138207 ISSN: 0027-8424 *
YAGI MAYUMI ET AL: "Sustained, substantial ex vivo production of hematopoietic stem cells mediated by thrombopoietin." 40TH ANNUAL MEETING OF THE AMERICAN SOCIETY OF HEMATOLOGY;MIAMI BEACH, FLORIDA, USA; DECEMBER 4-8, 1998, vol. 92, no. 10 SUPPL. 1 PART 1-2, 15 November 1998 (1998-11-15), page 504A XP002138206 Blood Nov. 15, 1998 ISSN: 0006-4971 *
YOUNG J C ET AL: "Thrombopoietin stimulates megakaryocytopoiesis, myelopoiesis, and expansion of CD34+ progenitor cells from single CD34+Thy-1+Lin- primitive progenitor cells" BLOOD,US,W.B. SAUNDERS, PHILADELPHIA, VA, vol. 88, no. 5, 1 September 1996 (1996-09-01), pages 1619-1631, XP002111602 ISSN: 0006-4971 *
ZEIGLER F C ET AL: "IN VITRO MEGAKARYOCYTOPOIETIC AND THROMBOPOIETIC ACTIVITY OF C-MPL LIGAND (TPO) ON PURIFIED MURINE HEMATOPOIETIC STEM CELLS" BLOOD,US,W.B. SAUNDERS, PHILADELPHIA, VA, vol. 84, no. 12, 15 December 1994 (1994-12-15), pages 4045-4052, XP000644741 ISSN: 0006-4971 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1517988B1 (fr) * 2002-06-20 2013-05-08 Augustinus Bader Utilisation de l'érythropoïétine pour la régénération de tissus in vivo
US20140205582A1 (en) * 2011-07-06 2014-07-24 Cellerant Therapeutics, Inc. Megakaryocyte progenitor cells for production of platelets

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AU3115000A (en) 2000-06-26

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