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WO1999000486A1 - Compositions et procedes permettant d'induire la croissance et la differenciation de cellules souches hematopietiques - Google Patents

Compositions et procedes permettant d'induire la croissance et la differenciation de cellules souches hematopietiques Download PDF

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Publication number
WO1999000486A1
WO1999000486A1 PCT/US1998/012996 US9812996W WO9900486A1 WO 1999000486 A1 WO1999000486 A1 WO 1999000486A1 US 9812996 W US9812996 W US 9812996W WO 9900486 A1 WO9900486 A1 WO 9900486A1
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type
cells
endothelial cells
stem cells
endothelial
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PCT/US1998/012996
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Emilio Barbera-Guillem
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Cli Oncology, Inc.
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Priority to AU81606/98A priority Critical patent/AU8160698A/en
Publication of WO1999000486A1 publication Critical patent/WO1999000486A1/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
    • 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
    • C12N2501/10—Growth factors
    • C12N2501/14—Erythropoietin [EPO]
    • 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
    • C12N2501/25—Tumour necrosing factors [TNF]
    • 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
    • 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
    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production

Definitions

  • the present invention relates to a novel discovery that a certain subpopulation of liver endothelial cells can provide the microenvironment necessary for the maintenance and growth of hemopoietic cells. More particularly, the present invention relates to coculturing purified type 1 endothelial cells with primitive hemopoietic precursors
  • stem cells in an ex vivo method for supporting long-term hemopoiesis by supporting the proliferation and differentiation of the stem cells.
  • Hemopoiesis (also known as hematopoiesis) is the formation of the various differentiated blood cell lineages (hemopoietic/hematopoietic cells) from precursor cells.
  • Pluripotential or multipotential stem cells can serve as common precursor cells for granulocytes (including neutro- phils, eosinophils and basophils) , monocytes, erythrocytes, megakaryocytes (and platelets), and lymphocytes.
  • granulocytes including neutro- phils, eosinophils and basophils
  • monocytes including neutro- phils, eosinophils and basophils
  • monocytes erythrocytes
  • megakaryocytes and platelets
  • lymphocytes lymphocytes.
  • stem cells have been used therapeutically to reconstitute a depleted supply of hemopoietic cells in vivo .
  • high dose chemotherapy or radiotherapy for treating cancer results in the destruction and resultant depletion of stem cells and more differentiated hemopoietic cells in the bone marrow (aplasia) .
  • the depleted cells can be restored by the infusion of stem cells, wherein the stems cells are collected from the patient prior to treatment, or from a donor.
  • Stem cells have been used to treat marrow aplasia following chemotherapy for breast cancer, ovarian cancer, testicular cancer, brain tumors, neuroblastoma, lymphomas, multiple myeloma, and acute leuke ias. Additionally, stem cells are being used to treat aplastic anemia, sickle cell anemia, diabetes, and various autoimmune disorders.
  • stromal cells are known to include a mixed cell population comprising reticular cells, macrophages/monocytes, fibroblasts, osteoclasts, adipocytes, and endothelial cells; wherein a minority of the cells are endothelial (Clark and Keating, 1995, Ann .
  • U.S. Patent No. 5,436, 151 discloses a method for the long term culture of mammalian stem cells comprising maintaining the stem cell population (adhered to a substrata) in stromal cell culture medium in a noncontacting relationship.
  • U.S. Patent 5,460,964 disclose the same method, further comprising the addition of cytokines IL-3 and macrophage inflammatory protein-1 alpha (MIP-1 alpha) to maintain self replication and differentiation of the cultured stem cells.
  • MIP-1 alpha macrophage inflammatory protein-1 alpha
  • U.S. Patent No. 5,605,822 discloses a method for ex vivo human stem cell proliferation comprising culturing stem cells in the presence of culture medium from stromal cells, wherein the stromal cells are transformed to produce at least one growth factor.
  • the present invention provides a method for the growth and differentiation of hemopoietic cells in culture.
  • the method comprises maintaining mammalian stem cells, preferably human stem cells, in a coculture with a substantially pure population of type 1 endothelial cells without the need for adding exogenous cytokines to the coculture.
  • the type 1 endothelial cells provide both a physical matrix and soluble growth factors (cytokines) for maintaining the proliferation and differentiation of stem cells.
  • cytokines soluble growth factors
  • the majority of the stem cells in coculture are present in the cell layer adherent to the monolayer of type 1 endothelial cells.
  • this embodiment provides for hemopoietic progenitor cells generated from the active hemopoiesis ongoing in the coculture.
  • the type 1 endothelial cells provide a microenvironment that can direct stem cells to differentiate along multiple cell lineages.
  • the majority of the hemopoietic progenitor cells in co- culture may be harvested from the non-adherent fraction; i.e., suspended in the medium.
  • the stem cells are physically separated from the type 1 endothelial cells, thereby facilitating the harvesting of the stem cells. While there are several ways known to those skilled in the art for achieving such a separation, in one illustration of this embodiment a cell production system is used in which the stem cells and the type 1 endothelial cells are each adhered to separate support matrices. Thus, the supported stem cells are maintained in a non-contacting association with the supported type 1 endothelial cells. In this embodiment, the stem cells are perfused and maintained in the medium from the type 1 endothelial cells ("type 1 endothelial cell conditioned medium") during culturing or coculturing.
  • type 1 endothelial cell conditioned medium the medium from the type 1 endothelial cells
  • a population of self replicating pluripotent stem cells can be maintained.
  • This embodiment also provides for hemopoietic progenitor cells generated from the active hemopoiesis ongoing in the coculture. The majority of the hemopoietic progenitor cells may be harvested from the non-adherent fraction; i.e., from the medium.
  • FIG. 1 is a graph illustrating the ability of either unactivated type 1 endothelial cells or unactivated type 2 endothelial cells to support hemopoiesis in vi tro .
  • FIG. 2A is a photograph showing hemopoietic cell formation on type 1 endothelial cells after 5 days of coculture.
  • FIG. 2B is a photograph showing hemopoietic cell formation and active hemopoiesis on type 1 endothelial cells after 3 weeks of coculture.
  • FIG. 3A is a graph illustrating the number of erythroid progenitors present in either coculture of unactivated type 1 endothelial cells or unactivated type 2 endothelial cells.
  • FIG. 3B is a graph illustrating the number of granulocyte/ macrophage progenitors present in either coculture of unactivated type 1 endothelial cells or unactivated type 2 endothelial cells.
  • FIG. 3C is a graph illustrating the number of megakatyocyte progenitors present in either coculture of unactivated type 1 endothelial cells or unactivated type 2 endothelial cells.
  • FIG. 4A is a bar graph illustrating the cytokine expression of unactivated type 1 endothelial cells.
  • FIG. 4B is a bar graph illustrating the cytokine expression of unactivated type 2 endothelial cells.
  • FIG. 4C is a bar graph illustrating the cytokine expression of type 1 endothelial cells activated by TNF- ⁇ .
  • FIG. 4D is a bar graph illustrating the cytokine expression of type 2 endothelial cells activated by TNF- ⁇ .
  • FIG. 5 is a bar graph illustrating the comparison of nitric oxide production (measured as N0 2 ) in unactivated type 1 endothelial cells, unactivated type 2 endothelial cells, and type 1 endothelial cells or type 2 endothelial cells activated by either TNF- ⁇ , or IFN- ⁇ , or by a combination of TNF- ⁇ and IFN- ⁇ .
  • coculture is used herein, for purposes of the specification and claims, to mean either the culture of stem cells in the physical presence of substantially pure type 1 endothelial cells and growth factors secreted from the type 1 endothelial cells, or the culture of stem cells in the presence of type 1 endothelial cell conditioned medium containing growth factors secreted from the type 1 endothelial cells (e.g., medium harvested from a type 1 endothelial cell culture; therefore lacking the type 1 endothelial cells themselves) .
  • hemopoietic progenitor is used herein, for purposes of the specification and claims, to mean a lineage- committed hemopoietic cell that has differentiated from a stem cell; e.g., a heterogenous mixture of cells at various stages of differentiation and maturation along the myeloid, erythroid, and megakaryocyte lineages.
  • Hemopoietic progenitors may include granulocyte-monocyte colony forming cells (GM-CFC) , myeloblasts, promonocytes, and promyelo- cytes, as cells committed to the monocyte and/or neutrophil lineages; eosinophil colony forming cells (EO-CFC) , myelo- blasts, and myelocytes, as cells committed to the eosinophil lineage; erythrocyte colony forming cells, pronormoblasts, and normoblasts, as cells committed to the erythrocyte lineage; megakaryocyte colony forming cells, megakaryo- blasts, and promegakaryocytes, as cells committed to the megakaryocyte/platelet lineage; and lymphoid stem cells, as cells committed to the lymphocyte lineages.
  • GM-CFC granulocyte-monocyte colony forming cells
  • EO-CFC eosinophil colony forming cells
  • Type 1 endothelial cell is used herein, for purposes of the specification and claims, and as more apparent by the detailed description that follows, to mean a subpopulation of endothelial cells that are found in vi vo to predominate in the periportal segment of the organ sinusoids.
  • organs may include liver, lung, brain, lymph node, bone marrow, and adrenal gland.
  • Type 1 endothelial cells may include primary cloned lines (e.g., by limiting dilution and maintenance in culture) , or immortalized
  • type 1 endothelial cells may be mammalian in origin, and in a preferred embodiment, human in origin. Additionally, if desired, the type 1 endothelial cells may be allogenic or autologous with respect to the hemopoietic stems cells cocultured therewith. In the development of the present invention it was demonstrated that type 1 endothelial cells survive for at least several weeks in culture, in addition to maintaining a stable phenotype after multiple passages (greater than 10 passages) . Thus, while not necessary to practice the present invention, type 1 endothelial cells may be immortalized for use in the present method.
  • transformations with SV40 T-antigen can include transformation with SV40 T-antigen (Moldovan et al., 1996, In Vi tro Cell Dev. Biol . Anim. 32:16-23; Yamazaki et al., 1995, In Vivo 9:421-426; Nakano et al . , 1995, Biochem. Mol . Biol . Int . 36:715-722) ; transformation with the E1A adenovirus gene (Roux et al . , 1994, J. Cell Physiol . 159:101-113); and transformation with the E6/E7 genes of human papilloma virus (Fontijn et al., 1995, Exp . Cell Res .
  • the term "substantially pure” is used herein in conjunction with type 1 endothelial cell, for purposes of the specification and claims, to mean either a heterogeneous population of cells in which type 1 endothelial cells comprise greater than or equal to approximately 70% of the cells comprising the heterogenous population (a percentage greater than any individual or collective endothelial cell populations in bone marrow stromal cells) ; or a homogeneous population of type 1 endothelial cells wherein the type 1 endothelial cells comprises greater than 90% of the cell population.
  • stem cells is used herein for purposes of the specification and claims, to mean pluripotential or multi-potential cells that can serve as common precursor cells for hemopoietic progenitors.
  • the stem cells are mammalian stem cells.
  • the mammalian stem cells are human stem cells.
  • Mammalian stem cells have been characterized by the expression of various cell surface markers, and by function. Hemopoietic stem cells have been identified, and purified (e.g., by fluorescent-activated cell sorting), on the basis of the expression or lack of expression of cell surface markers including lineage marker negative (Lin(-)); CD34(+); and stem cell antigen (Sca-1 (+) ) .
  • Other cell surface markers used for the identification of stem cells may include HLA-DR (-) and c-kit receptor (+) (see, e.g., Nishio et al . , 1996, Stem Cells 14:584-591; Messner, 1991, J3one Marrow Transplant 1 (Suppl) : 18-22) .
  • the stem cells may be allogeneic or autologous with respect to the intended recipient.
  • autologous transplants of stem cells effect complete remission in patients with multiple myeloma in up to 50% of the cases; whereas the complete response rate for allogeneic transplants ranges from 30-50% (Jagannath et al., 1994, Oncology 8:89-103).
  • the source of the stem cells may include bone marrow, cord blood, or peripheral blood.
  • Cord blood has been shown to be a source for pluripotential stem cells (see, e.g., Fishman et al., 1992, Immunol . Lett . 34:189- 193) . Compared to other sources, cord blood appears to have a higher stem cell concentration.
  • Human stem cells from peripheral blood may be collected by cytapheresis during the recovery from short-term aplasia induced by drug treatment (such as with chemotherapeutic agents or sargramostim) , wherein the drug-induced stem cell mobilization comprises an expansion of the circulating pool of stem cells (Fermand and Brouet, 1995, Annu . Rev. Med. 46:299-307; see also, Nishio et al., 1996, supra) .
  • Stem cells may also comprise stem cells transfected with an expression vector. It has been reported that transfected stem cells can replicate (J3iove.nture View, 1996, Vol. 1, No. 2) and generate persistent populations of differentiated cells expressing the transgene long-term in various tissues ⁇ Vaccine Weekly, 1996, February 26) .
  • preferentially expressed by type 1 endothelial cells is used herein, for purposes of the specification and claims, to mean a molecule expressed on the surface of type 1 endothelial cells, wherein the level of expression (measured directly or indirectly, and including by presence or by activity) of such molecule is at least 3 to 4 times that expressed by other subpopulations of endothelial cells contained in the sinusoidal area of an organ.
  • preferential expression may include detection of expression of the molecule on or by type 1 endothelial cells, and absence of detection of the same molecule on or by type 2 endothelial cells; or a log greater expression of the same molecule on or by type 1 endothelial cells as compared to expression on or by type 2 endothelial cells.
  • a method for preserving and inducing proliferation of stem cells by maintaining the stem cells in a coculture with a substantially pure population of type 1 endothelial cells (a) without the need for adding exogenous cytokines to the coculture; and (b) in direct contact with the type 1 endothelial cells.
  • the type 1 endothelial cells provide both a physical matrix and soluble growth factors (cytokines) for supporting the maintenance and proliferation of stem cells.
  • a monolayer of substantially pure type 1 endothelial cells is preformed and adhered onto the surface of a vessel, such as a reactor, followed by the addition of stem cells to the culture resulting in a coculture promoting the direct contact between the stem cells and the preformed monolayer of type 1 endothelial cells.
  • the coculture is then incubated in conditions (with culture medium and incubator temperatures) suitable for promoting the maintenance and proliferation of stem cells ("physiological acceptable conditions") .
  • physiological acceptable conditions serum supplemented or serum-free substitute supplemented standard tissue culture medium may be used, with incubation at temperatures around 37°C.
  • maintenance of the cell coculture will require the introduction of nutrients and the removal of metabolic waste products (typically achieved by periodically removing culture medium and adding fresh medium) , and by maintaining the appropriate atmospheric conditions.
  • the incubation conditions can be varied.
  • the proliferating stem cells can be gently removed (by enzymatic or physical means) from the type 1 endothelial cells, allowing for the harvesting of stem cells and thereby providing additional space for continued stem cell proliferation.
  • physical means for separating cells of different types may be achieved by their differential tolerance to shear stress (see, e.g. U.S. Patent No. 5,605,822).
  • enzymatic conditions such as trypsin concentration and time of incubation in trypsin
  • this embodiment provides for hemopoietic progenitors generated from the active hemopoiesis ongoing in the coculture.
  • the type 1 endothelial cells provide a microenvironment that can direct stem cells to differentiate along multiple cell lineages.
  • type 1 endothelial cells tend to select for differentiation at early stages of hemopoiesis resulting in the accumulation of hemopoietic progenitors, and thereby tends to restrict the accumulation of mature hemopoietic cells. This tendency has several attendant advantages. For example, even with the infusion of mobilized stem cells, engraftment time is 8 days on average. Engraftment time is the time necessary for the stem cells to find, and implant themselves in, the bone marrow.
  • the stem cells need to grow and differentiate for some time before an individual's immune system, destroyed by chemotherapy or radiotherapy, can be reconstituted. Because it takes a while for the peripheral blood cell populations to be reconstituted, this time period leaves the individual at risk for infection, as well as susceptible to other morbidity associated with relatively depleted peripheral blood cell populations.
  • hemopoietic progenitors generated and cultivated from the method of the present invention may be suitable for transfusion into recipients (autologous or allogeneic) .
  • Such a transfusion may reduce the morbidity associated with relatively depleted peripheral blood cell populations by accelerating the hematological and immunological reconsti- tution of the relatively depleted populations.
  • hemopoietic progenitors can multiply and differentiate into the mature, highly specialized cells constituting the peripheral blood cell populations including red blood cells (for carrying oxygen throughout the body) ; platelets (for blood clotting) ; and lymphocytes, granulocytes (basophils, eosinophils, neutrophils) and macrophages/monocytes (for disease-fighting and immune system functions) . It is also believed that the hemopoietic progenitors may tend to be more "naive" (like stem cells) to the immune system, than the mature hemato- poietic cells.
  • hemopoietic progenitors can be frozen and stored for long periods of time; and, if autologous, can obviate the need for cross-matching and immune sensitiza- tion, and risk of blood-borne infections, as compared to use of allogeneic cells. As with any transfusion or infusion, a careful monitoring of the recipient and donor should be considered to watch for side effects.
  • the majority of the hemopoietic progenitors in coculture may be harvested from the non-adherent fraction; i.e., as present in the medium from the reactor.
  • the hemopoietic cells can be harvested by removing the medium from the coculture, and separating the medium from the cells present, such as by physical means. Such physical means may include, but is not limited to, filtration, chromatography, or centrifugation. It may be desirable to analyze or further purify the harvested hemopoietic progenitors before transfusing a therapeutically effective amount of the harvested cells.
  • active hemopoiesis By maintaining a supply of proliferating stem cells, and by periodically removing the non-adherent hemopoietic progenitors, active hemopoiesis can be maintained in culture thereby generating a supply of hemopoietic progenitors .
  • the stem cells are cocultured in physical separation from the substantially pure type 1 endothelial cells, thereby facilitating the harvesting of the stem cells. Also, by maintaining a supply of proliferating stem cells, and by periodically removing the non-adherent hemopoietic progenitors, active hemopoiesis can be maintained in culture thereby generating a supply of hemopoietic progenitors.
  • the type 1 endothelial cell conditioned medium provides necessary growth factors for maintenance and proliferation of stem cells in culture.
  • a cell production system in which the stem cells and the type 1 endothelial cells are each adhered to separate support surfaces; whether it is at least two separate support matrices, or one support matrix having on one side the type 1 endothelial cells and having on the other side the stem cells.
  • proteins or compositions known to those skilled in the art to facilitate hemopoietic cell adhesion and attachment to a support matrix.
  • extracellular matrix collagen, vitrogen, entactin, nidogen, glycosaminoglycans, proteoglycans, laminin, fibronectin, synthetic peptides, and combinations thereof.
  • a commercially available cell culture system that is suitable for, and illustrates, the use of two separate support matrices is the TranswellTM system (Corning Costar Corp., Cambridge, MA).
  • TranswellTM system Corning Costar Corp., Cambridge, MA
  • two cell culture chambers are provided, wherein a first chamber fits inside a larger second chamber.
  • the type 1 endothelial cells may be added to the inner surface forming the bottom of the second chamber.
  • the stem cells may be added to the inner surface forming the bottom of the first chamber wherein that bottom comprises a microporous membrane.
  • the microporous membrane is prepared such that it facilitates adhesion and attachment of the stem cells, and provides pores of sufficient size (e.g., 0.4 to 0.5 micro m) to allow for transport of culture medium therethrough but prevent the passage of cells therethrough.
  • the membrane allows for physical separation (i.e., in a non-contacting association) between the type 1 endothelial cells and the stem cells, while allowing for the stem cells to be perfused in and maintained by the medium from the type 1 endothelial cells in coculture.
  • a similarly functioning cell culture device and its operation is also described in U.S. Patent No. 5,605,822 (herein incorporated by reference).
  • U.S. Patent No. 5,605,822 also illustrates the mode of this embodiment wherein one support matrix is utilized in coculture to maintain the type 1 endothelial cells and the stem cells in a non-contacting association.
  • the liquid growth medium in the coculture may be in simultaneous contact with both the type 1 endothelial cells and the stem cells, wherein the medium is exchanged at fixed intervals thereby enhancing the maintenance of the coculture.
  • type 1 endothelial cell conditioned medium may be controllably circulated into, and may be exchanged for existing medium in, the chamber containing the stem cells thereby perfusing the stem cells, and wherein the medium is exchanged at fixed intervals thereby enhancing the maintenance of the coculture.
  • bioreactor systems that may be useful in practice of the method of the present invention include systems marketed by Aastrom Biosciences Inc. (Ann Arbor, Michigan) .
  • This example illustrates isolation of type 1 endothelial cells found in the unique capillary zones in organ sinusoids, and identification of this cell sub- population as an endothelial cell subpopulation.
  • the sinusoidal walls of certain organs are composed of at least two kinds of endothelial cells.
  • type 1 endothelial cells are located proximal to the peri- portal side of the liver sinusoids.
  • type 2 endothelial cells are located proximal to the perihepatic side of the sinusoids.
  • Type 1 endothelial cells were isolated and purified as essentially taught by Vidal- Vanachlocha et al . (1993, supra, herein incorporated by reference) .
  • a portion of sinusoidal tissue from the organ in which is enzymatically treated e.g., pronase E (0.02%), type 1 collagenase (0.05%), and DNase (0.03%)
  • the cell suspension is filtered through a nylon screen, washed and centrifuged several times, and treated (17.5% metrizamide) to remove cell debris and erythrocytes.
  • an enriched population of type 1 endothelial cells was achieved by counterflow elutriation at 10°C and 2,400 rpm with a flow rate of approximately 37 ml/minute.
  • the collected cell fraction contained essentially type 1 endothelial cells (>90% of endothelial cells) together with Kupffer cells in a proportion of about 2:3. Further purification of the type 1 endothelial cells was achieved on discontinuous arabinogalactin density gradient centri- fugation (containing densities of 1.02, 1.03, 1.04, and 1.06) at 20,000 rpm for 30 minutes at 25°C. Cells sedimenting in the 1.04 to 1.06 interphase were exclusively type 1 endothelial cells as characterized by more than 90% high-wheat germ agglutinin (WGA) binding. Using this procedure, for example, a yield of at least 4 x 10 6 type 1 endothelial cells can be obtained from 8 grams of organ tissue. Yields of type 1 endothelial cells may be improved by using the portal vein as tissue from which the cells are isolated, consistent with the anatomy of the location of type 1 endothelial cells.
  • type 1 endothelial cells isolation and purification of type 1 endothelial cells may be facilitated at any stage by using one or more targeting molecules having affinity for cell surface receptors preferentially expressed by type 1 endothelial cells (as compared to expression by other cells in the area) as an immobilized affinity molecule in a process of affinity chromatography.
  • the process of affinity chromatography is a standard technique known to those skilled in the art.
  • fluorescent activated cell sorting may be used to prepare a population of substantially pure type 1 endothelial cells.
  • the process of fluorescent activated cell sorting is a standard technique known to those skilled in the art.
  • subpopulations of type 1 endothelial cells and of type 2 endothelial cells were isolated and purified using a method outlined above, and evaluated for expression of endothelial cell markers by nucleic acid amplification using commercially available primers specific for the endothelial cell markers.
  • the endothelial cell markers included stem cell factor (SCF, see e.g., Koenig et al., 1994, Blood 83:2836-43), vascular endothelial cell growth factor receptor flt-1 (see, e.g., Mochida et al., 1996, Biochem. Bi ophys . Res .
  • VW von Willebrand factor
  • LIF leukocyte inhibitory factor
  • CD34 CD34
  • TGF- ⁇ transforming growth factor beta
  • EPO erythro- poietin
  • IL-3 interleukin-3
  • EPO erythropoietin
  • M-CSF macrophage colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • VEGF vascular endothelial cell growth factor
  • Table 1 illustrates that certain cell markers (e.g., cytokines) expressed by endothelial cells are expressed by the subpopulation of type 1 cells thereby confirming that this subpopulation is of endothelial cell type .
  • cell markers e.g., cytokines
  • This example illustrates characterization of unactivated type 1 endothelial cells and unactivated type 2 endothelial cells in analyzing if these endothelial cells subpopulations can provide some or all of the various factors necessary for the maintenance and growth of circulating hemopoietic stem cells during extramedullary hemopoiesis.
  • circulating hemopoietic stem cells must first arrest in the organ. Such arrest appears to be due a local environment provided by type 1 endothelial cells, e.g.
  • endothelial cell adhesion molecules or other cell surface receptors which (a) are preferentially expressed in higher amounts by type 1 endothelial cells (as compared to other cell populations in the sinusoids of the organ) , and (b) initially mediate the colonization of the seeding hemopoietic stem cell to the endothelium comprising the hematopoietic environment.
  • VW von Willebrand factor
  • GlcNAC N-acetylglucosamine
  • GalNAC N-acetyl galactosamine
  • ICM-1 intracellular cell adhesion molecule-1
  • VCAM-1 vascular cell adhesion molecule-1
  • PCAM-1 platelet cell adhesion molecule-1
  • MHC-II major histocompatibility antigen-II
  • phagocytic ability VW was measured by immunofluorescence using a commercially available rabbit anti-human factor VIII antigen antibody.
  • GlcNAC was measured by binding specificity with the lectin wheat germ agglutinin (WGA) .
  • WGA wheat germ agglutinin
  • Viable type 1 endothelial cells and type 2 endothelial cells were separately assayed for GlcNAc expression by flow cytometry.
  • each subpopulation was incubated in vi tro at 37°C for 5 minutes in the presence of fluorescein isoythiocyanate- labelled wheat germ agglutinin lectin (FITC-WGA, lO ⁇ g/ml) .
  • FITC-WGA fluorescein isoythiocyanate- labelled wheat germ agglutinin lectin
  • FITC-conjugated OA fluorescence-labeled ovalbumin
  • Endothelial cells contain membrane-associated polypeptides that bind Amadori-modified glycated albumin (GA) , the predominant form of in which nonenzymatically glycated albumin exists in vivo, but do not bind to unmodified albumin.
  • G Amadori-modified glycated albumin
  • Endothelial cell adhesion molecules and the integrin family to which they bind, are important for the colonization of hematopoietic cells, homing of lymphocytes, and proliferation of lymphocytes.
  • Viable type 1 endothelial cells and type 2 endothelial cells were separately assayed for expression of ICAM-1, VCAM-1, and PCAM-1 by flow cytometry. First, each subpopulation was incubated in vi tro at 4°C for 40 minutes in the presence of saturating doses of either fluorescein isoythiocyanate labelled mouse monoclonal antibody against ICAM-1 (FITC-anti-ICAM-1) , FITC-labelled anti-VCAM-1, or FITC-labelled anti-PCAM-1.
  • the monoclonal antibodies used are commercially available.
  • the cell subpopulations were then analyzed using the flow cytometric methods, data collection, and analysis as outlined above.
  • ICAM expression "very high” represents a log of fluorescence intensity (e.g., 10 ) greater than "low” expression (between 10 and 10 ) .
  • VCAM the expression by type 1 endothelial cells was similar to that by type 2 endothelial cells ("high to very high” is 10 to 10 log fluorescence intensity) .
  • (-) correlates with absence of detectable expression.
  • MHC-II Major histocompatibility antigen-II
  • (+) means presence of detectable activity
  • (-) means absence of detectable activity.
  • Table 2 illustrates that certain cell surface molecules preferentially expressed on unactivated type 1 endothelial cells may be responsible for the arrest and/or survival of any hemopoietic stem cells which may subsequently attempt to seed the organ.
  • Type 1 endothelial cells may contribute to initially mediating the colonization of the seeding hemopoietic stem cell to the endothelium comprising the hematopoietic environment.
  • growth factors secreted by type 1 endothelial cells interact directly with hemopoietic stem cells for the colonized hemopoietic stem cells to subsequently develop into hematopoietic foci.
  • Subpopulations of unactivated type 1 endothelial cells and of unactivated type 2 endothelial cells were characterized and compared for cytokine expression.
  • cytokines including stem cell factor (SCF), interleukin-l ⁇ (IL-l ⁇ ) , interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-10 (IL-10), erythropoietin (EPO), thrombopoietin (TPO) , macrophage colony stimulating factor (M-CSF) , granulocyte colony stimulating factor (G-CSF) , granulocyte-macrophage colony stimulating factor (GM-CSF) , transforming growth factor beta (TGF- ⁇ ), and interferon gamma (IFN- ⁇ ).
  • SCF stem cell factor
  • IL-l ⁇ interleukin-3
  • IL-4 interleukin-4
  • IL-6 interleukin-6
  • IL-10 interleukin-10
  • EPO erythropoietin
  • TPO thrombopoietin
  • M-CSF macrophage colon
  • cytokine expression was evaluated by commercially available enzyme linked immunosorbent assays (ELISA) and performed using standard methods known to those skilled in the art (see, e.g., Endo et al . , 1996, Res . Commun . Mol . Pathol . Pharmacol . 94:23-38).
  • ELISA enzyme linked immunosorbent assays
  • Table 3 illustrates that certain cytokines expressed on unactivated type 1 endothelial cells play a role in the survival of hemopoietic stem cells after arrest in a hematopoietic microenvironment. More particularly, both IL-l ⁇ and GM-CSF secreted by type 1 endothelial cells may play a role in maintenance of arrested hemopoietic stem cells in the hematopoietic microenvironment.
  • GM-CSF can stimulate a single bone marrow stem cell to proliferate and differentiate into mature neutrophils, eosinophils, granulocytes, or macrophages (see for example, Fan et al., 1991, In Vivo, 5:571-7).
  • IL-3 supports terminal differentiation of hemopoietic progenitors to mature cells (Valent et al., 1990, Bl ut 61:338-45). From these results it is noted that type 1 endothelial cells do .not express detectable levels of IL-3, which may be one reason why in vi tro type 1 endothelial cells tend to select for differentiation at early stages of hemopoiesis and tends to restrict the accumulation of mature hemopoietic cells.
  • EXAMPLE 3 The mechanisms for extramedullary hematopoiesis are not well defined.
  • the liver is an important hematopoietic organ during fetal life; however, liver hematopoiesis ceases after birth. From an in vivo experimental model of extramedullary hematopoiesis, it appears that the hematopoietic microenvironment components are probably expressed in the liver sinusoids (Barbera- Guillem et al., 1989, Hepatology 9:29-36).
  • a hematopoietic microenvironment is known to those skilled in the art as a complex system composed mainly of stromal cells comprising fibroblasts, endothelial cells, adipocytes, osteoclasts, and monocytes.
  • the stromal cells secrete cytokines, produce extracellular matrix and mediate direct cellular contact, and perform other functions which contribute to in vivo and in vi tro hematopoiesis (Wolf et al . , 1968, J. Exp. Med. 127:205; Trentin et al . , 1971, Am. J. Pa thol . 65:3; Dexter et al., 1977, J. Cell Physiol . 91:335-44).
  • the hematopoietic microenvironment in organs other than bone marrow remain undefined.
  • the unknowns include which cell populations are necessary to provide the in vivo hematopoietic microenvironment in organs other than bone marrow; how the cells support and regulate such a hematopoietic microenvironment; and whether such cells can support in vi tro long-term hemopoiesis.
  • This example illustrates that it is the type 1 endothelial cells, and not type 2 endothelial cells or other cell subpopulations, in the sinusoids of organs that represent the main element of the hematopoietic microenvironment.
  • type 1 endothelial cells maintain and induce proliferation of ste cells in a process of supporting in vi tro long-term hemopoiesis which generates hemopoietic progenitors.
  • Unactivated type 1 endothelial cells and unactivated type 2 endothelial cells were evaluated for their ability to support hematopoiesis in vi tro by providing the microenvironment necessary for the maintenance and growth of hemopoietic cells.
  • Type 1 endothelial cells and type 2 endothelial cells were isolated from murine liver using the methods according to Example 1. To obtain pure endothelial cell subpopulation cultures, type 1 endothelial cells and type 2 endothelial cells were each cloned by limiting dilution.
  • Bone marrow cells were harvested two days post-injection. This population of bone marrow cells includes primitive multipotent precursors which are uncommitted cells expressing very early hemopoietic stem cell markers such as Sea, and the c-kit receptor, while having no detectable expression of Lin.
  • This population of bone marrow cells was resuspended in cell culture medium (Iscove's modified Dulbecco's medium) supplemented with 20% fetal calf serum and cultured in tissue culture dishes for 1 hour to remove adherent cells.
  • the non-adherent bone marrow cells were removed and resuspended in the cell culture medium at 5x10 cells/ml. These non-adherent cells represent a population of hemopoietic stem cells from 5-FU treated donors.
  • hemopoietic stem cells (Lin(-), Sca(+) cells) were isolated from normal mice (i.e., not treated with 5-FU) by immunomagnetic separation according to previously described methods (Spangrude et al., 1988, Science 241:58; 1990, Exp. Hematol . 18:920-23). Briefly, the bone marrow cells were incubated with a cocktail of antibodies having binding specificities for cell surface receptors including B220, GR-1, MAC-1, Lyt-2 (CD8), Ly-1 (CD5) , L3T4 (CD4), and TER119.
  • B220 GR-1, MAC-1, Lyt-2 (CD8), Ly-1 (CD5) , L3T4 (CD4), and TER119.
  • type 1 endothelial cells Having an isolated and purified subpopulation of type 1 endothelial cells, an isolated and purified subpopulation of type 2 endothelial cells, and two isolated and purified subpopulations of hemopoietic stem cells (one subpopulation from 5-FU treated mice, and one subpopulation from untreated mice) , analyzed was the ability of either type 1 endothelial cells or type 2 endothelial cells to support in vi tro hemopoiesis. That is, type 1 endothelial cells and type 2 endothelial cells were each tested for ability in vi tro to maintain and to induce proliferation, in the absence of exogenous cytokines, of hematopoietic stem cells isolated from 5-FU treated mice.
  • Collagen-precoated control wells to which the stem cells were added, included those containing tissue culture medium alone, or tissue culture medium with a combination of cytokines (stem cell factor (SCF) at 50ng/ml; IL-3 at lng/ml; IL-6 at lOng/ml; and erythropoietin (EPO) at 5U/ml) .
  • SCF stem cell factor
  • EPO erythropoietin
  • the number and viability of non-adherent cells present in the respective collected coculture medium were counted with a hemocytometer . After 21 days, the average count of non-adherent cells present in the type 1 endothelial cell coculture medium was 44,200 + 13,200; whereas the average count of non-adherent cells present in the type 2 endothelial cell coculture medium was 3,900 + 750. Thus, a statistically significant (p ⁇ 0.01) increase of hemopoietic cells was observed in the type 1 endothelial cell cocultures as compared to the type 2 endothelial cell cocultures. FIG.
  • unactivated type 1 endothelial cells can maintain stem cells for more than 5 weeks, induce the stem cells to proliferate and differentiate into hemopoietic progenitors, and support hemopoiesis for more than 5 weeks (long-term) ; whereas unactivated type 2 endothelial cells ( ⁇ ) do not support such cell maintenance nor hemopoiesis.
  • type 1 endothelial cells and type 2 endothelial cells were also analyzed in coculture with the stem cell subpopulation purified by flow cytometry.
  • Non-adherent cells (10 3 /week + std. error)
  • Lin-, Sca+ cells isolated by flow cytometry
  • type 1 endothelial cell-stem cell coculture without adding exogenous cytokines
  • HC hematopoietic foci
  • FIG. 2B illustrated are cells after 3 weeks of coculture, wherein the cobblestone areas are areas indicative of active hemopoiesis.
  • Cytospin preparations of the non-adherent cells were stained with Giemsa solution for morphologic analysis. Aliquots of the non-adherent cells were incubated with conjugated monoclonal antibodies against murine cell surface receptors including B220, Gr-1, MAC-1, Lyt-2, Ly-1, L3T4, c-mpl, and terll9 for pheno-typical analysis by flow cytometry (with data collection and analysis of fluorescent intensities) .
  • the main phenotype of the non-adherent cells in the type 1 endothelial cell-stem cell cocultures consisted of hemopoietic progenitors wherein 47% were of the monocyte/macrophage lineage (MAC-1+) , 33% were of the granulocyte lineage (Gr-1+) , 16% were of the megakaryocyte lineage (c-mpl+), and present were low percentages ( ⁇ 5%) of erythroblasts (terll9+) and B cells (B220+) .
  • MAC-1+ monocyte/macrophage lineage
  • Gr-1+ granulocyte lineage
  • c-mpl+ megakaryocyte lineage
  • B cells B220+
  • type 1 endothelial cells In another illustration of ability of type 1 endothelial cells to support the maintenance and growth of hemopoietic stem cells (without addition of exogenous cytokines) , analyzed were the number of stem cells adhering to the type 1 endothelial cell monolayer or the type 2 endothelial cell monolayer after long-term coculture in vi tro . After 6 to 8 weeks, the cocultures were terminated and the adherent layer was removed gently with scraping, and the cells of the adherent layer were assayed for colony forming cells (CFC) using the fibrin clot assay described in Example 5 (see also, Bruno et al., 1991, Blood, 77:2339-46).
  • CFC colony forming cells
  • the number of CFC reflected the number of hemopoietic stem cells (from the 5-FU induced subpopulation) present in the adherent cell fraction of the respective cocultures.
  • adherent cells from bone marrow cultures (BMC) were cultured on collagen precoated wells.
  • the adherent cell fraction from the respective cocultures were harvested after 6 weeks of coculture, and then cultured in a fibrin clot system in the presence of SCF and IL-3. After 7 days of culture, the number of CFC (defined as a colony containing >40 cells) were counted.
  • the results, shown in Table 5, are the mean + standard error, with the statistical significance estimated by the Student's t-test (P values >0.05 were considered statistically non significant).
  • type 1 endothelial cells In another illustration of ability of type 1 endothelial cells to support the proliferation of hemopoietic stem cells into hemopoietic progenitors, analyzed were the ability of type 1 endothelial cells, as compared to type 2 endothelial cells, to support the survival and proliferation of specific hemopoietic progenitors.
  • the non-adherent cells from type 1 endothelial cell-stem cell coculture and from type 2 endothelial cell- stem cell cocultures were assayed in a fibrin clot assay to detect granulocyte/macrophage (CFU-GM) , erythroid (burst forming unit, BFU-E) , megakaryocytic (CFU-Mk) and colony forming cells (CFC) using methodology described previously (Kuriya et al., 1989, Exp. Hematol . 15:896-901).
  • CFU-GM granulocyte/macrophage
  • BFU-E erythroid
  • CFU-Mk megakaryocytic
  • CFC colony forming cells
  • the non-adherent cells harvested from the respective cocultures were placed in 20% bovine fibrinogen and 10% human plasma thrombin (lxlO 4 cells/clot) in the presence of different cytokines (SCF, IL-3, IL-l ⁇ , and erythropoietin (Epo) ) . Aliquots of 0.4 ml were placed in the center of 35 mm culture dishes. Once the fibrin clots had formed, the clots were bathed with 1 ml of serum-free medium (X-Vivo; BioWhittaker, Inc.). The dishes were then incubated at 37°C in a humidified atmosphere flushed with 5% C0 2 .
  • serum-free medium X-Vivo; BioWhittaker, Inc.
  • CFC CFU-GM
  • CFU-Mk CFU-Mk
  • BFU-E BFU-E
  • BFU-E progenitors were observed in the non-adherent cells from the beginning of the type 1 endothelial cell-stem cell coculture, and reaches peak production during the fourth week of coculture. This erythropoiesis continued until this study was terminated. In contrast, the type 2 endothelial cell-stem cell coculture did not support the survival of BFU-E.
  • CFU-GM progenitors were also observed in the non-adherent cells from the beginning of the type 1 endothelial cell-stem cell coculture, and reaches peak production during the third week of coculture.
  • CFU-Mk progenitors were also observed in the non-adherent cells throughout the experimental period of the type 1 endothelial cell-stem cell coculture, and reaches peak production during the second week of coculture.
  • EXAMPLE 6 This example illustrates that the ability of type 1 endothelial cells to sustain stem cell growth and maintenance, and to support the proliferation of hemopoietic stem cells into hemopoietic progenitors, in an in vi tro coculture is due to the presence of factors secreted by the type 1 endothelial cells in coculture.
  • the medium of either type 1 endothelial cell cultures or type 2 endothelial cell cultures was investigated for the possible presence of soluble factors which could support the maintenance and proliferation of stem cells and promote in vi tro hemopoiesis.
  • type 1 endothelial cells and type 2 endothelial cells were grown in separate cultures to 90% confluence, wherein the culture supernatant was removed, 10 mis of serum-free culture medium was added, and the cells were cultured overnight. Then, the medium from each culture was removed ("conditioned medium"), centrifuged at 2,000 x g for 15 minutes, and passed through a 0.2 ⁇ m filter before use to ensure that no cells were present.
  • conditioned medium the medium from each culture was removed
  • the capacity of either type 1 endothelial cell conditioned medium or type 2 endothelial cell conditioned medium to promote hemopoiesis from normal bone marrow cells was assayed using the fibrin clot system described in Examples 5 and 6 herein.
  • the number of hemopoietic CFC was evaluated according to their morphology (e.g., compact colonies with >40 cells/colony) and by their cell content.
  • normal bone marrow cells were cultured with medium alone.
  • the results, shown in Table 6, are the mean + standard error, with the statistical significance estimated by the Student's t-test (P values >0.05 were considered statistically non significant) .
  • type 1 endothelial cell conditioned medium contains soluble factors which can maintain stem cells in an in vi tro long-term coculture; and induce those stem cells to proliferate and differentiate into hemopoietic cells capable of forming colonies (CFC) . Further, the soluble factors are produced by the type 1 endothelial cells.
  • type 1 endothelial cells can be activated by cytokines (growth factors) , thereby causing the activated type 1 endothelial cells to secrete growth factors that may interact with the hemopoietic stem cells in a hematopoietic microenvironment.
  • Cytokines causing the activation of type 1 endothelial cells have been identified herein to include tumor necrosis factor alpha (TNF- ⁇ ) , IL- l ⁇ , and IFN- ⁇ (acting separately, and also synergistically when in combination) .
  • subpopulations of unactivated type 1 endothelial cells and of unactivated type 2 endothelial cells were activated by TNF- ⁇ , and then characterized and compared for cytokine expression.
  • Each subpopulation was isolated and purified using the methods according to Example 1. Cultures of each subpopulation were treated for four hours with 25 ng/ml TNF ⁇ at 37°C. The TNF ⁇ was then removed by washing the TNF ⁇ - activated cells.
  • respective cytokine expression was quantitated in unactivated type 1 endothelial cells and unactivated type 2 endothelial cells.
  • the activated endothelial cells were then evaluated by commercially available ELISAs for quantitating the expression of cytokines including IL-l ⁇ , IL-2, IL-4, IL-6, IL-10, GM-CSF, IFN- ⁇ , and TNF- ⁇ .
  • cytokines including IL-l ⁇ , IL-2, IL-4, IL-6, IL-10, GM-CSF, IFN- ⁇ , and TNF- ⁇ .
  • FIG. 4 illustrates the comparison of cytokine expression (measured as pg/ml) between unactivated type 1 endothelial cells (FIG. 4A) , unactivated type 2 endothelial cells (FIG. 4B) , type 1 endothelial cells activated by TNF- ⁇ (FIG. 4C) , and type 2 endothelial cells activated by TNF- ⁇ (FIG. 4D) . Similar cytokine expression was observed when type 1 endothelial cells were activated with either IL-l ⁇ or IFN- ⁇ .
  • IL-6 (TNF ⁇ -induced IL-6) is a signal for cell proliferation that organ cells use in regenerative processes (see, e.g., liver regeneration, Michalopoulos and DeFrances, 1997, Sci ence 276:60-66) .
  • Nitric oxide (NO) is a molecule formed in cells from L-arginine residues by the enzyme nitric oxide synthase (NO synthase) . Prolonged exposure to nitrous oxide has been shown to impair a hematopoietic microenvironment as well as to impair the maintenance of hemopoietic stem cells (Suzuki et al., 1990, Anesth . Analg. 71:389-93; Konno, 1991, Masui 40:702-12). Induction of NO in mammalian vessel endothelial cells has also been shown (Shuler et al . , 1995, J. Leukoc . Biol . 57:116-21; Inoue et al . , 1995, Arteriorscler. Thromb. Vase . Biol . 15:1255-61).
  • each subpopulation was activated by either TNF- ⁇ , IFN- ⁇ , or a combination of TNF- ⁇ and IFN- ⁇ , and then characterized and compared for NO expression.
  • Each subpopulation was isolated and purified using the methods according to Example 1. Cytokine induced-activation was performed using the methods herein. The supernatants from type 1 endothelial cell cultures and from type 2 endothelial cell cultures were assayed for NO production according to previously described methods for determining NO (Kolb et al., 1994, J. Biol . Chem.
  • FIG. 5 illustrates the comparison of NO production (measured as N0 2 in micromoles- ⁇ M) in unactivated type 1 endothelial cells (control D) , unactivated type 2 endothelial cells (control ⁇ ) , type 1 endothelial cells activated by TNF- ⁇ (TNF- ⁇ D) , type 2 endothelial cells activated by TNF- ⁇ (TNF- ⁇ ⁇ ) , type 1 endothelial cells activated by IFN- ⁇ (IFN- ⁇ D) , type 2 endothelial cells activated by IFN- ⁇ (IFN- ⁇ ⁇ ) , type 1 endo-thelial cells activated by a combination of TNF- ⁇ and IFN- ⁇ (TNF- ⁇ + IFN- ⁇ D) , and type 2 endothelial cells activated by TNF- ⁇ and IFN- ⁇ (TNF- ⁇ + IFN- ⁇ ⁇ ) .
  • N0 2 As shown in Fig. 5, very little N0 2 is constitutively produced in either unactivated type 1 endothelial cells or unactivated type 2 endothelial cells. Also, little induction (as measured by N0 2 ) is observed in type 1 endothelial cells or type 2 endothelial cells when activated by either TNF- ⁇ or by IFN- ⁇ . In contrast, activation of type 2 endothelial cells by a combination of TNF- ⁇ and IFN- ⁇ resulted in significant amounts (e.g., > 50 ⁇ M) of N0 2 produced by such activated type 2 endothelial cells .
  • Non-adherent ceillllss ((1100 : /week + std. dev. ) in a coculture containing TNF ⁇ -activated endothelial cells
  • type 1 endothelial cells may be an additional factor important in providing a hematopoietic microenvironment in which hemopoietic stems cells can be maintained, and be induced to proliferate and differentiate into hemopoietic progenitors.

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Abstract

On décrit des procédés et des compositions permettant de réaliser l'hématopoïèse de longue durée et produire ainsi des cellules souches hématopoïétiques et des cellules parents hématopoïétiques. Les procédés consistent à cocultiver des cellules souches hématopoïétiques de mammifères avec des cellules endothéliales de type 1 sensiblement pures ou avec un milieu conditionné de cellules endothéliales du type 1. Sans qu'il soit nécessaire d'ajouter des cytokines exogènes à la coculture, les cellules endothéliales du type 1 maintiennent et induisent la prolifération des cellules souches et incitent les cellules souches à se différencier en cellules parents hématopoïétiques.
PCT/US1998/012996 1997-06-26 1998-06-23 Compositions et procedes permettant d'induire la croissance et la differenciation de cellules souches hematopietiques WO1999000486A1 (fr)

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EP1171165A4 (fr) * 1999-03-30 2002-07-31 Ran Kornowski Injection intramyocardique de moelle osseuse autologue
US7097832B1 (en) 1999-03-30 2006-08-29 Myocardial Therapeutics, Inc. Intramyocardial injection of autologous bone marrow

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WO1995019793A1 (fr) * 1994-01-21 1995-07-27 The United States Of America, Represented By The Secretary Of The Navy Procede permettant l'expansion de cellules hematopoïetiques et procedes de greffe associes

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WO1995019793A1 (fr) * 1994-01-21 1995-07-27 The United States Of America, Represented By The Secretary Of The Navy Procede permettant l'expansion de cellules hematopoïetiques et procedes de greffe associes

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RAFII S., ET AL.: "HUMAN BONE MARROW MICROVASCULAR ENDOTHELIAL CELLS SUPPORT LONG-TERM PROLIFERATION AND DIFFERENTIATION OF MYELOID AND MEGAKARYOCYTIC PROGENITORS.", BLOOD, AMERICAN SOCIETY OF HEMATOLOGY, US, vol. 86., no. 09., 1 November 1995 (1995-11-01), US, pages 3353 - 3363., XP002913230, ISSN: 0006-4971 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1171165A4 (fr) * 1999-03-30 2002-07-31 Ran Kornowski Injection intramyocardique de moelle osseuse autologue
AU767402B2 (en) * 1999-03-30 2003-11-06 Myocardial Therapeutics, Inc. Intramyocardial injection of autologous bone marrow
US7097832B1 (en) 1999-03-30 2006-08-29 Myocardial Therapeutics, Inc. Intramyocardial injection of autologous bone marrow

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