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WO1999023205A1 - Cellules souches hematopoietiques - Google Patents

Cellules souches hematopoietiques Download PDF

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Publication number
WO1999023205A1
WO1999023205A1 PCT/CA1998/001012 CA9801012W WO9923205A1 WO 1999023205 A1 WO1999023205 A1 WO 1999023205A1 CA 9801012 W CA9801012 W CA 9801012W WO 9923205 A1 WO9923205 A1 WO 9923205A1
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cells
lin
stem cells
human
cell
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PCT/CA1998/001012
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English (en)
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John E. Dick
Dominique Bonnet
Mickie Bhatia
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Hsc Research And Development Limited Partnership
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Priority to AU97320/98A priority Critical patent/AU9732098A/en
Priority to CA 2307624 priority patent/CA2307624A1/fr
Publication of WO1999023205A1 publication Critical patent/WO1999023205A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/20—Transition metals
    • C12N2500/24—Iron; Fe chelators; Transferrin
    • C12N2500/25—Insulin-transferrin; Insulin-transferrin-selenium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/36—Lipids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/28—Vascular endothelial cells

Definitions

  • the present invention relates to human hematopoietic stem cells and to methods of isolating and using such cells in compositions and in methods for the reconstitution of a deficient or missing cell population.
  • the present invention also provides a population of human hematopoietic stem cells that can be isolated and genetically altered for introduction in a human patient to correct various genetic disorders.
  • CD34 antigen was thought to be the distinguishing feature of stem cells because CD34 is downregulated as stem cells differentiate into more abundant mature cells (4), and CD34 has been used as a basis for isolation of stem cells.
  • CD34 does not mark stem cells exclusively, since 1% of bone marrow cells are CD34+ and include clonogenic progenitors that are not able to repopulate the hematopoietic system after transplantation. Other markers such as Thy-1 can be combined with CD34 to positively select for a cell fraction more enriched in stem cells (5, 6, 7).
  • the CD34+ cell fraction can be enriched by eliminating cells that express markers that are expressed on non-repopulating cells (e.g.lineage antigens). Nevertheless, all current clinical and experimental protocols utilizing human stem cells, including ex vivo culture, gene therapy and bone marrow transplantation, focus on CD34+ cells.
  • Figure 1A shows cell surface expression of CD34 on cord blood cells depleted for lineage markers (Lin-).
  • Lin- cells were stained with a class III monoclonal antibody for CD34 (581) conjugated to FITC (Becton
  • Diseases may include but are not restricted to congenital disorders, severe combined immunodeficiency, Wiskott-Aldrich syndrome, Fanconi's anemia, congenital red cell aplasia, lysosomal storage disease, thalassemia major, sickle cell anemia, aplastic anemia, acute lymphoblastic leukemia, acute myelogenous leukemia, megakaryoblastic leukemia, hematologic melanomas, lymphoma, multiple myeloma, myelodysplastic syndromes, carcinomas, neuroblastomas, arthritis and neurological genetic diseases (e.g. Gaucher Disease).
  • congenital disorders severe combined immunodeficiency, Wiskott-Aldrich syndrome, Fanconi's anemia, congenital red cell aplasia, lysosomal storage disease, thalassemia major, sickle cell anemia, aplastic anemia, acute lymphoblastic leukemia, acute myelogenous leukemia, mega
  • CD34-cells that do not express lineage markers exist in human hematopoietic tissues Fig. 1 A.
  • the possibility that CD34 protein was produced in the CD34-Lin- cells but not transported to the cell surface was excluded using pe ⁇ neabilized, stained cytospins of purified CD34-Lin- that were further conjugated with fluorescent monoclonal antibodies. This confirmed that no CD34-FITC signal was detected in the CD34-Lin- cells (Fig. IB).
  • mice A total of 15 of 35 mice were engrafted following transplantation with CD34-Lin- cells that had been cultured for 4 days in SF or 5% FCS at cell doses below the calculated frequency of CD34-SRC. For example, 13 of 29 mice were engrafted following transplantation of 50,000 to as few as 4,000 cultured CD34-Lin- cells. By contrast, only 1 of 7 mice were engrafted when 100,000 uncultured CD34-Lin- cells were transplanted ( Figure 2). Similarly, a higher proportion of mice (33%) transplanted with cultured CD34-CD38-Lin- cells were engrafted (Figure 7B) compared to mice transplanted with similar doses of uncultured CD34-CD38-Lin- ( Figure 7B).
  • CD33+, CD14+, CD15+ and CD13+ cells indicated the differentiation potential of CD38-CD38-Lin- cells to the myeloid lineages (Fig. 8G-H).
  • the engraftment pattern of mice transplanted with expanded CD34-CD38-Lin- cells is similar to that observed with unstimulated purified CD34-Lin- cells.
  • the presence of human T-cells is a unique feature of CD34-Lin- engraftment, since T-cells have not been detected in mice transplanted with purified CD34+CD38-Lin- cells either before or after ex vivo culture. We had previously found that the CD34+SRC are lost if CD34+CD38-
  • the stem cells of the present invention can be identified and isolated from bone marrow, peripheral blood and cord blood.
  • the most clinically advantageous source is peripheral blood due to the fact that the procedure for obtaining such is easy and non- invasive. Collection of peripheral blood also has no health effect on the donor. While peripheral blood is the most convenient and least invasive source for use in isolating the stem cells of the present invention, it is understood by those skilled in the art that the bone marrow and cord blood are more ideal as a starting point due to the larger percentage of stem cells present in such.
  • Other techniques which may be used to isolate the stem cells of the present invention include immunoseparation where antibodies against specific receptor molecules are used together with immunoaffinity columns to bind cells having the specific target receptor. The targeted cells are then removed from the antibody complex by the use of shear fluid force.
  • the cells of the present invention can be used to screen compounds which may affect their proliferation and/or differentiation into other cell types.
  • an isolated population of CD34-Lin- cells may be suitably cultured in vitro to which selected growth factors, cytokines, chemicals, peptides and other agents are added individually or in specific combination.
  • selected growth factors, cytokines, chemicals, peptides and other agents are added individually or in specific combination.
  • One skilled in the art would readily comprehend the conditions and procedures for preparing such a cell culture and the amounts of agent to add for such testing.
  • the CD34-Lin- cells may be phenotypically characterized and counted in order to determine the effect of the added agent(s). In this manner, one may establish a simple method for producing a specific cell type for a clinical application.
  • the cells of the present invention can be used for understanding the origin of hematopoietic diseases such as leukemia and for clinical procedures such as stem cell transplantation and gene therapy for the treatment of various diseases.
  • the stem cell of the present invention is also important for the treatment or prophylaxis against disease or infection, for the reconstitution of deficient or missing cell populations, as for example in cancer patients after myeloablative therapy, and for the treatment of congenital or acquired genetic abnormalities and defects by the introduction of desired genetic infoimation into the patient.
  • the CD34-Lin- or CD34- CD38-Lin- stem cells can be utilized for stem cell transplantation in order to reconstitute missing or deficient cell populations.
  • Bone marrow transplants are typically done in order to restore hematopoiesis in cancer patients receiving high doses of chemotherapy and/or radiation therapy as well as in leukemia patients and aplastic anemia patients.
  • Cord blood has recently been used in order to reconstitute hematopoiesis as an alternative to bone marrow transplants.
  • bone marrow transplants there are several disadvantageous with bone marrow transplants as they are highly invasive and require major surgery. One also must find a suitable phenotypically matched donor.
  • Peripheral blood transplantation can also be used to isolate and provide back an enriched culture of CD34-Lin- or CD34-CD38-Lin- cells.
  • Diseases may include but are not restricted to congenital disorders, severe combined immunodeficiency, Wiskott-Aldrich syndrome, Fanconi's anemia, congenital red cell aplasia, lysosomal storage disease, thalassemia major, sickle cell anemia, aplastic anemia, acute lymphoblastic leukemia, acute myelogenous leukemia, megakaryoblastic leukemia, hematologic melanomas, lymphoma, multiple myeloma, myelodysplastic syndromes, carcinomas, neuroblastomas, arthritis and neurological genetic diseases (e.g. Gaucher Disease).
  • congenital disorders severe combined immunodeficiency, Wiskott-Aldrich syndrome, Fanconi's anemia, congenital red cell aplasia, lysosomal storage disease, thalassemia major, sickle cell anemia, aplastic anemia, acute lymphoblastic leukemia, acute myelogenous leukemia, mega
  • compositions of the present invention may comprises substantially pure populations of CD34- Lin- human hematopoietic stem cells or CD34-CD38-Lin- human hematopoietic stem cells.
  • the compositions may also comprise enriched cultures of CD34- Lin- or CD34-CD38-Lin- cells.
  • the compositions of the present invention may additionally comprise cells selected from the group consisting of CD34+ cells, Thy-1 cells, CD4+ cells, CD56+ cells, CD33+ cells, CD9+ cells, CD11+ cells, CD41+ cells, CD45 cells and mixtures thereof.
  • the type of composition made depends on the end use. For example, for the treatment of leukemia it is desired to provide a composition comprising substantially homogenous populations of CD34-Lin- or CD34-
  • the composition may comprise a mixture of CD34-Lin- or CD34-CD38-Lin- cells together with CD34+ cells.
  • compositions substantially enriched populations of cells characterized phenotypically as CD34-Lin- or CD34-CD38-Lin- but which also may be further phenotypically characterized by the presence or absence of other antigenic markers.
  • the CD34-Lin- or CD34-CD38-Lin- cells may be used as a method of gene therapy.
  • the CD34-Lin- or CD34-CD38-Lin- cells may be isolated and enriched in in vitro culture where a desired genetic sequence can be inserted into the cells prior to their reintroduction into a patient.
  • the genetic element introduced can simply be one to correct a defect in the cells themselves or to target a specific recombinant gene sequence to a specific area of the patient.
  • diseases which may be treated with genetically altered stem cells of the present invention include but are not restricted to hemophilia A, thalassemia, sickle-cell anemia, SCID and Gaucher's disease.
  • CD34-Lin- or CD34-CD38-Lin- cells are isolated from a source and cultured according to the method of the present invention.
  • the cell culture is maintained under suitable conditions and the cells are subjected to techniques for the introduction and stable incorporation of a desired genetic sequence into the cells.
  • introduction techniques may include transfection (calcium-mediated or microsome-mediated transfection), cell fusion, electroporation, microinjection or infection using recombinant vaccinia or retrovirus vectors.
  • the cells which acquire the selected genetic sequence are then screened for, and reintroduced into a patient.
  • the identifed cells may be further cultured to allow the cells to enrich and/or further differentiate to another cell type prior to being reintroduced into a patient.
  • the cells of the present invention may be trans fected with a selected
  • DNA sequence encoding for a therapeutic agent such as an antibiotic, anticancer agents, peptides, cytotoxic compounds and antisense RNA.
  • the cells may be transfected with an antigenic or immunogenic product which creates an immune response in a patient and reintroduction. In this manner, such cells would produce a vaccine like effect.
  • the cells of the present invention can be used for fetal genetic testing.
  • the stem cells may be isolated from samples of peripheral blood taken from a pregnant woman which contain some fetal cells. Isolated fetal cells may be cultured and the stem cells isolated and tested for genetic abnormalities.
  • the stem cells of the present invention may be isolated and cultured in vitro and treated with specific factors and/or cytokines in order to produce a specific cell lineage such as immune cells, granulocytic, megakaryocytic, etc. which carry out a specific function.
  • a specific cell lineage such as immune cells, granulocytic, megakaryocytic, etc. which carry out a specific function.
  • Such specialized cells may be generated in large numbers and transplanted back into patients in order to treat them of a disease such as for example an autoimmune disease or one of the diseases listed supra.
  • Example 1 Analysis of CD34-Lin- cells found in human hematopoietic tissue Mononuclear cells were isolated from various human hematopoietic cell sources and stained with monoclonal antibodies for CD2, CD3, CD4, CD7, CD13, CD14, CD15, CD16, CD19, CD20, and glycophorin conjugated to FITC, CD38 conjugated to PE and CD34 conjugated to Cy-5 . Cells gated Rl did not express lineage associated markers (Lin-) and were further analyzed for the expression of CD34 and CD38.
  • CD34- cells that do not express lineage markers exist in human hematopoietic tissues
  • human cord blood cells were first depleted of mononuclear cells that express 15 different lineage-specific antigens from human cord blood. This Lin- population was 99% pure (data not shown). The Lin- cells were then stained with the most widely used CD34 class III monoclonal antibody conjugated to FITC. Flow cytometric analysis showed two distinct populations of CD34+ and CD34- cells (Fig 1A panel 1). The CD34- cells (gated Rl, Fig. 1A, I) were collected by flow sorting and reanalysis demonstrated their high purity (99%; Fig. 1 A, II).
  • cytospins of purified CD34- Lin- cells were permeabilized, stained with CD34 monoclonal antibodies conjugated to FITC and counter stained with DAPI (Fig. IB).
  • No CD34-FITC signal was detected in the CD34-Lin- cells.
  • the specificity of the procedure was shown by the detection of cell surface and intracellular expression of CD34 on a population of purified CD34+Lin- cells under similar conditions. Background fluorescence was indicated by staining cells with IgG conjugated to FITC as isotype control (Fig. IB).
  • CD34+Lin- cells Heterogeneity within human CD34+Lin- cells is well documented and further subdivision for the most primitive cells is typically based on the cell surface markers CD38, c-kit, Thy-1 and HLA-DR. The expression of these markers on both the CD34+Lin- and CD34-Lin- cells was compared (Fig. IC).
  • CD34-Lin- cells displayed a bi-modal distribution of CD38, clearly dividing the population into two fractions in contrast to the high proportion of CD34+Lin- cells that express CD38 (Fig. IC).
  • Cell surface expression of c-kit was similar between that two populations, while the CD34-Lin- cells are almost exclusively Thy-1- and HLA-DR- (Fig. IC).
  • CD34-Lin- population derived from cord blood is a distinct population which differs not only in CD34 expression from primitive CD34+Lin- cells but also in phenotypic heterogeneity based on additional markers associated with stem cells.
  • Example 3 Cell Engraftment Purified cell populations at the indicated dose were transplanted by tail vein injection into sublethally irradiated mice (375 cGy using a 137Cs g- irradiator) according to a standard protocol as previously described (16, 17). Mice were sacrificed 8 to 12 weeks post transplant and the bone marrow from the femurs, tibiae and iliac crests of each mouse were flushed into IMDM containing 10% FCS.
  • Mouse bone marrow was analyzed using FACS analysis and by southern analysis using genomic DNA extracted by standard protocols in which the level of human cell engraftment was determined by comparing the characteristic 2.7 kb band with those of huma mouse DNA mixtures as controls (limit of detection 0.05% human DNA) (16, 17). The results are shown in Figures 2 and 7.
  • Example 4 Determination of Hematopoietic Progenitor Activity of CD34-Lin.
  • CD34+CD38-Lin- and CD34-CD38+Lin- Highly purified cells were plated in clonogenic methlycellulose assays and seeded on MS-5 stroma in order to quantitate the CFC and LTC-IC content, respectively.
  • Clonogenic capacity of CD34-Lin- cells was extremely low in comparison to CD34+CD38-Lin- cells (250 CFC vs. 8.9 CFC per 800 cells) (Table I).
  • CD34-CD38-Lin- cells As many as 10,000 cells needed to be seeded on MS-5 stroma to detect a single LTC-IC within the CD34-Lin- cell fraction, while further purification demonstrated that detection of LTC-IC in the CD34-CD38-Lin- fraction required seeding of at least 2000 cells. By contrast, as few as 10 CD34+CD38-Lin- cells contain an LTC-IC.
  • the CD34-CD38+Lin- cells were devoid of LTC-IC activity (limit of detection at 10,000 cells) but contained a much higher capacity to form CFC specifically committed to the erythroid lineage (Table I).
  • Example 5 Multilineage Differentiation of Human CD34-Lin- cells in NOD/SCID mice after ex vivo Culture
  • a representative mouse was transplanted with 50,000 expanded CD34- Lin- cord blood cells after 2 days of ex-vivo culture in the presence of SF medium supplemented with 5% FCS.
  • Mouse bone marrow was extracted 10 weeks after transplant and analyzed by multiparameter flow cytometry (11, 12). The results are shown in Figure 8.
  • CD34-Lin- cells were incubated in 50 ml of SF condition consisting of IMDM supplemented with 1% BSA (Stem Cell Technologies), 5 mg/ml of human insulin (Humulin R from Eli Lilly and Co.), 100 mg/ml of human transferrin (Gibco, BRL), 10 mg/ml of low density lipoproteins (Sigma Chemical Co.), 10-4 M Beta-mercaptoethanol and growth factors (GF).
  • GF cocktail was used at final concentrations of 300 ng/ml of SCF (Amgen) and Flt- 3 (Immunex), 50 ng/ml of G-CSF (Amgen), 10 ng/ml of IL-3 (Amgen) and IL- 6 (Amgen).
  • condition media obtained from a fresh umbilical vein endothelial cell culture in a low percentage of serum ( 10%) and passaged four times, was added in some wells.
  • Cells were cultured in flat bottomed suspension wells of 96-well plates (Nunc), incubated for 2 and 4 days at 37oC and 5% C02 and 50 ml of fresh GF cocktail was added to each well every other day.
  • Example 6 Effect of ex vivo culture on the number of clonogenic progenitors present in the CD34-Lin-.
  • CD34-CD38-Lin- and CD34-CD38+Lin- cell fractions An aliquot of 800 to 2, 500 CD34-Lin-, CD34-CD38-Lin- or CD34-
  • Example 7 The effect of liquid culture on the development and potential differentiation of CD34-CD38-Lin- cells
  • CD34-CD38-Lin- cells seeded in SF media began to express CD34 which could be enhanced with the addition of serum (Fig. 6).
  • HUNEC human umbilical vein endothelial cell
  • CD34-Lin- and more highly purified CD34-CD38-Lin- cell fractions were injected into ⁇ OD/SCID mice and the level of human engraftment evaluated after 8 to 10 weeks.
  • Purified cell fractions from 44 CB and 3 BM samples cultured for 2 and 4 days in SF, SF supplemented with 5% FCS or 25% HUVEC-CM were transplanted at various doses into 144 recipient ⁇ OD/SCID mice and the level of human cell engraftment was determined (Figure 7B).
  • mice A total of 15 of 35 mice were engrafted following transplantation with CD34-Lin- cells that had been cultured for 4 days in SF or 5% FCS at cell doses below the calculated frequency of CD34-SRC. For example, 13 of 29 mice were engrafted following transplantation of 50,000 to as few as 4,000 cultured CD34-Lin- cells. By contrast, only 1 of 7 mice were engrafted when 100,000 uncultured CD34-Lin- cells were transplanted ( Figure 2). Similarly, a higher proportion of mice (33%) transplanted with cultured CD34-CD38-Lin- cells were engrafted (Figure 7B) compared to mice transplanted with similar doses of uncultured CD34-CD38-Lin- ( Figure 7B).
  • the bone marrow of engrafted mice was analyzed by multiparameter flow cytometry to determine whether cultured CD34-Lin- repopulating cells possessed the same in vivo proliferative and differentiative capacity as uncultured cells.
  • a representative analysis of the bone marrow of a NOD/SCID mouse transplanted with an initial population of 40,000 CD34-Lin- cells after 4 days of culture is shown in Figure 8.
  • the bone marrow of this mouse contained 7% human cells as detected by expression of CD45, a human specific pan- leukocyte marker (Fig. 8). Both B and T-lymphoid cells were present in the murine bone marrow as shown by staining for CD 19, CD20 and CD4, CD3 antigens (Fig. 8D-F).
  • CD33+, CD14+, CD15+ and CD13+ cells indicated the differentiation potential of CD38-CD38-Lin- cells to the myeloid lineages (Fig. 8G-H).
  • the engraftment pattern of mice transplanted with expanded CD34-CD38-Lin- cells is similar to that observed with unstimulated purified CD34-Lin- cells.
  • the presence of human T-cells is a unique feature of CD34-Lin- engraftment, since T-cells have not been detected in mice transplanted with purified CD34+CD38-Lin- cells either before or after ex vivo culture.
  • CD34+CD38-Lin- or CD34+Lin- cells were cultured for 4 days under the dame serum containing conditions.
  • CD34+CD38-Lin- and CD34-Lin- cells were cultured for 4 days under the dame serum containing conditions.
  • 3 out of 6 mice were engrafted following transplantation with cultured CD34- Lin- cells ( Figure 9).
  • 5,000 and 10,000 CD34+CD38-Lin- cells, containing 10-20 CD34+SRC, cultured under the same conditions were unable to engraft NOD/SCID mice.
  • NOD/SCID mice were transplanted with CD34-Lin- or CD34+CD38-Lin- cells purified from the same cord blood sample and cultured in the presence of 5% FCS.
  • a southern blot was performed using standard techniques and was hybridized with a human chromosome 17-specific ⁇ -satellite probe.
  • CD34-Lin- ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇
  • Phenotype of purified CFC/800 cells Frequency of population LTC-IC
  • Phenotype of purified Day Medium Number l ⁇ ?l population

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Abstract

La présente invention concerne des cellules souches hématopoïétiques humaines appelées CD34-Lin- et CD34-CD38-Lin- et des procédés d'isolement et d'utilisation de ces cellules dans des compositions et dans des méthodes de reconstitution d'une population cellulaire déficiente ou absente. La présente invention concerne également une population de cellules souches hématopoïétiques humaines pouvant être isolées et modifiées génétiquement et destinées à être introduites chez un patient humain, afin de corriger divers dysfonctionnements génétiques, mises en culture et encore différenciées in vitro pour obtenir une nouvelle population de cellules.
PCT/CA1998/001012 1997-10-31 1998-10-30 Cellules souches hematopoietiques WO1999023205A1 (fr)

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Application Number Priority Date Filing Date Title
AU97320/98A AU9732098A (en) 1997-10-31 1998-10-30 Hematopoietic stem cells
CA 2307624 CA2307624A1 (fr) 1997-10-31 1998-10-30 Cellules souches hematopoietiques

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CA002219869A CA2219869A1 (fr) 1997-10-31 1997-10-31 Cellule de souche humaine cd-34 hematopoietique
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WO2002039109A3 (fr) * 2000-11-07 2003-05-01 British Columbia Cancer Agency Procede de detection de cellules hematopoietiques humaines capables de reconstitution a court terme
US6632620B1 (en) 2000-06-22 2003-10-14 Andrew N. Makarovskiy Compositions for identification and isolation of stem cells
WO2005056026A1 (fr) * 2003-12-04 2005-06-23 Regents Of The University Of Minnesota Compositions et methodes de traitement des maladies lysosomales
US7799324B2 (en) 2001-12-07 2010-09-21 Geron Corporation Using undifferentiated embryonic stem cells to control the immune system
EP2292734A1 (fr) 2001-12-07 2011-03-09 Geron Corporation Cellules hématopoïétiques à partir de cellules souches embryonnaires humaines
US7927587B2 (en) 1999-08-05 2011-04-19 Regents Of The University Of Minnesota MAPC administration for the treatment of lysosomal storage disorders
US9005964B2 (en) 2006-11-24 2015-04-14 Regents Of The University Of Minnesota Endodermal progenitor cells
CN110082515A (zh) * 2012-12-21 2019-08-02 干细胞生物科技公司 利用干细胞数据评估行为效果的方法
US10617721B2 (en) 2013-10-24 2020-04-14 Ospedale San Raffaele S.R.L. Methods for genetic modification of stem cells
CN112585261A (zh) * 2018-06-07 2021-03-30 布里格姆妇女医院 产生造血干细胞的方法

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Cited By (14)

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Publication number Priority date Publication date Assignee Title
US7927587B2 (en) 1999-08-05 2011-04-19 Regents Of The University Of Minnesota MAPC administration for the treatment of lysosomal storage disorders
US6632620B1 (en) 2000-06-22 2003-10-14 Andrew N. Makarovskiy Compositions for identification and isolation of stem cells
US7223549B2 (en) 2000-06-22 2007-05-29 Andrew N. Marakovskiy Compositions for identification and isolation of stem cells
WO2002039109A3 (fr) * 2000-11-07 2003-05-01 British Columbia Cancer Agency Procede de detection de cellules hematopoietiques humaines capables de reconstitution a court terme
EP2292734A1 (fr) 2001-12-07 2011-03-09 Geron Corporation Cellules hématopoïétiques à partir de cellules souches embryonnaires humaines
US7799324B2 (en) 2001-12-07 2010-09-21 Geron Corporation Using undifferentiated embryonic stem cells to control the immune system
JP2007513188A (ja) * 2003-12-04 2007-05-24 リージェンツ オブ ザ ユニバーシティ オブ ミネソタ リソソーム蓄積症の処置のための組成物および方法
WO2005056026A1 (fr) * 2003-12-04 2005-06-23 Regents Of The University Of Minnesota Compositions et methodes de traitement des maladies lysosomales
US8580249B2 (en) 2003-12-04 2013-11-12 Regents Of The University Of Minnesota Cell therapy for the treatment of lysosomal storage disorders
US9005964B2 (en) 2006-11-24 2015-04-14 Regents Of The University Of Minnesota Endodermal progenitor cells
CN110082515A (zh) * 2012-12-21 2019-08-02 干细胞生物科技公司 利用干细胞数据评估行为效果的方法
US10617721B2 (en) 2013-10-24 2020-04-14 Ospedale San Raffaele S.R.L. Methods for genetic modification of stem cells
CN112585261A (zh) * 2018-06-07 2021-03-30 布里格姆妇女医院 产生造血干细胞的方法
US12286643B2 (en) 2018-06-07 2025-04-29 The Brigham And Women's Hospital, Inc. Methods for generating hematopoietic stem cells

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