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WO1999003980A1 - Cellules de stroma derivees d'agm - Google Patents

Cellules de stroma derivees d'agm Download PDF

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Publication number
WO1999003980A1
WO1999003980A1 PCT/JP1998/003209 JP9803209W WO9903980A1 WO 1999003980 A1 WO1999003980 A1 WO 1999003980A1 JP 9803209 W JP9803209 W JP 9803209W WO 9903980 A1 WO9903980 A1 WO 9903980A1
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Prior art keywords
cells
hematopoietic stem
hematopoietic
stem cells
cell
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PCT/JP1998/003209
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English (en)
Japanese (ja)
Inventor
Tatsutoshi Nakahata
Original Assignee
Kirin Beer Kabushiki Kaisha
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Priority to AU82437/98A priority Critical patent/AU8243798A/en
Publication of WO1999003980A1 publication Critical patent/WO1999003980A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3886—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells comprising two or more cell types
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3895—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
    • 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
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0668—Mesenchymal stem cells from other natural sources
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to a cell line isolated and established from the AGM region, a method for supporting the growth or survival of hematopoietic stem cells and hematopoietic progenitor cells using this cell line,
  • the present invention relates to a graft and a composition for gene therapy using hematopoietic stem cells or hematopoietic progenitor cells proliferated by the method, a transplantation method, and a gene therapy method.
  • BACKGROUND ART Mature blood cells flowing in vivo have only a short life span (about 120 days for red blood cells and about 7 days for platelets in humans), and mature blood cells are differentiated daily from hematopoietic progenitor cells.
  • peripheral blood mature blood cells Maintains homeostasis of peripheral blood mature blood cells.
  • the number of mature blood cells supplied to peripheral blood is 200 billion Z-days for red blood cells and 700 billion days for neutrophils in humans.
  • the progenitor cells of each of these differentiation lineages proliferate while differentiating from undifferentiated hematopoietic stem cells to form a system in which peripheral blood cells do not constantly die.
  • Hematopoietic stem cells with pluripotency were found to be present in the bone marrow. Then, as a means of analyzing the properties of hematopoietic cells, a transplantation experiment system using irradiated mice, and an in vitro (in vitro) colony formation method (Bradley, TR, J. Exp. Med., 44: 287-299) , 1966), and knowledge on the differentiation of hematopoietic stem cells and hematopoietic progenitor cells has been accumulated.
  • the transplantation experiment system using irradiated mice is the most direct way to decipher the properties of hematopoietic stem cells.
  • Transplantation of bone marrow cells isolated from another mouse (Donna 1) into a mouse (recipient) that has damaged the hematopoietic system by irradiation may reconstitute the donor-derived hematopoietic system in the recipient mouse. it can.
  • Various differentiation antigens expressed on hematopoietic cells (Spangrude, GJ, Proc. Natl. Acad. Sci.
  • hematopoietic stem cells can differentiate into lymphoid cells and myeloid cells even if only one cell is transplanted, and have a long-term Has been shown to be able to build a hematopoietic system in recipients (Osawa, M., Science, 273: 242-245, 1996) Hematopoietic stem cells proliferate in transplant recipients because one cell can reconstitute the hematopoietic system in the recipient individual for a long time. It is believed that you are.
  • hematopoietic progenitor cells can differentiate only into a single lineage of mature blood cells, despite the presence of many cytoplasmic cells, hematopoietic stem cells that can build a long-term hematopoietic lineage in transplanted mice It is possible to differentiate into a cell lineage. From these results, the differentiation from hematopoietic stem cells to mature blood cells flowing in peripheral blood is interpreted as follows.
  • Hematopoietic stem cells have a pluripotency capable of differentiating into various differentiation lineages, and are capable of self-renewal while maintaining this pluripotency property (pluripotency). Hematopoietic stem cells self-renew and partially differentiate, are affected by various site forces, gradually narrow the lineage of cells that can be differentiated, differentiate into only a limited number of cell types, differentiate into hematopoietic progenitor cells Proliferates and eventually matures into blood cells.
  • mice As described above, a series of studies on hematopoietic stem cells have been performed using mice, as described above, because it is possible to confirm the long-term appearance of hematopoietic cells by the transplantation experimental system. Although it is thought that a similar hematopoietic cell differentiation mode exists in humans, it is impossible to establish an experimental system for human-to-human transplantation in humans. difficult.
  • CFU-Emix mixed colonies containing erythrocytes
  • CFU-Emix mixed colonies containing erythrocytes
  • Human hematopoietic stem cells identified using these evaluation systems express CD34 antigen and C-KIT (c-kit gene product). The presence of these hematopoietic stem cells has been confirmed in bone marrow, fetal liver, fetal bone marrow, umbilical cord blood, peripheral blood treated with anticancer drugs or cytokines.
  • hematopoietic cells The long-term maintenance of hematopoietic cells in vitro was due to the use of Dexter for fibroblasts other than bone marrow blood cells, preadipocytes, adipocytes, vascular endothelial cells, smooth muscle cells, etc. After establishing a system for co-culturing stromal cells (also called stromal cells) and bone marrow blood cells in vitro (Dexter, TM, J. Cell. Physiol., 91: 335-344). , 1977) 0 In this culture system, hematopoietic cells can be observed to grow and float in the medium for more than 6 months.
  • stromal cells also called stromal cells
  • bone marrow blood cells In this culture system, hematopoietic cells can be observed to grow and float in the medium for more than 6 months.
  • hematopoietic stem cells and hematopoietic progenitor cells are maintained viable for a long period of time by providing a favorable culture environment for hematopoietic stem cells and hematopoietic progenitor cells.
  • This Dexter culture system has a very large number of cell types, and it is not clear which cell types are involved in the maintenance of hematopoietic stem cells. Is also considered to exist. Therefore, it was considered possible to isolate cells that support the proliferation and maintenance of hematopoietic stem cells from bone marrow.
  • stromal cells which are bone marrow stromal cells that are not hematopoietic cells, have been established from bone marrow cells, and the growth support of hematopoietic stem cells and hematopoietic progenitor cells has been analyzed.
  • hematopoietic stem cells and hematopoietic progenitor cells.
  • International patent applications disclose stromal cell lines that maintain and proliferate hematopoietic progenitor cells. No activity to proliferate is found. Also, US Pat. No.
  • 5,599,703 (Davis et al.) Discloses a hematopoietic stem cell expansion system using porcine brain microvasculature vascular endothelial cells. In both cases, it is necessary to add one kind of site force-in, and there is no description about the proliferation of hematopoietic stem cells when only vascular endothelial cells are used without adding the site force-in. In such a culture system with the addition of site force, it is hard to imagine that the performance of the cells themselves has been accurately evaluated.
  • hematopoiesis mainly occurs in the yolk sac, mainly erythrocytes.
  • hematopoiesis in the yolk sac is transient, and it is thought that hematopoietic cells in this area disappear in this area without migrating to other organs.
  • Subsequent major hematopoietic organs migrate to the liver and bone marrow, and the hematopoietic cells from the liver are thought to become hematopoietic stem cells that have proliferated and differentiated to maintain adult blood cell homeostasis. (Zon, LI, Blood, 86: 2876-2891, 1995).
  • Hematopoietic stem cells responsible for hematopoiesis in the liver are thought to develop in the AGM (Aorta-Gonad-Mesonephros) region prior to hematopoiesis in the liver (Tavian, M. Blood, 87: 66-72 1996; Sanchez, MJ., Immunity, 5: 513-525, 1996).
  • the AGM region refers to the region where the aorta (Aorta), the gonad primordium (Gonad), and the mesonephros primordium (Mesonephros) are present in the early stage of mammalian embryo development.
  • mice In humans, it is often found around 20 to 50 days from embryonic age, and in mice it is often found around 9 to 12 days before embryonic age. In other words, it is thought that adult hematopoietic stem cells, after developing in AGM, migrate to the liver and bone marrow, self-renew and differentiate, and supply peripheral blood cells circulating throughout the body.
  • bone marrow transplantation is one of the treatments for aplastic anemia and congenital immunity deficiency, which are diseases caused by pluripotent stem cell disorders.
  • aplastic anemia and congenital immunity deficiency which are diseases caused by pluripotent stem cell disorders.
  • systemic X-ray therapy and high-dose chemotherapy for leukemia are effective treatments, but bone marrow cells are destroyed by these treatments, and bone marrow transplantation is performed in combination with these treatments. ing.
  • problems with bone marrow transplantation such as the extremely low probability of match between donor and patient histocompatibility antigens and the need for strong immunosuppression.
  • bone marrow used for transplantation is collected by puncturing the bone marrow with a iliac comb under general anesthesia, there is also a pain and danger for the donor.
  • umbilical cord blood has attracted attention as a source of stem cells because it has a lower frequency of graft-versus-host disease (GVHD) and a lower need for immunosuppression than bone marrow cells.
  • GVHD graft-versus-host disease
  • the present invention has been made from the above viewpoint, and is a hematopoietic stem cell or hematopoietic progenitor cell that can be used for blood cell transplantation instead of bone marrow transplantation or umbilical cord blood transplantation, or a hematopoietic stem cell that can be used for gene therapy
  • Another object of the present invention is to provide a means for obtaining hematopoietic progenitor cells, and specifically, a stromal cell line capable of supporting the proliferation or survival of hematopoietic stem cells and hematopoietic progenitor cells, It is an object of the present invention to provide a method for supporting the proliferation or survival of hematopoietic stem cells and hematopoietic pro
  • the present inventors have conducted intensive studies in order to solve the above problems, and as a result, the AGM region is suitable as a source of supporting cells for efficiently growing hematopoietic stem cells and hematopoietic progenitor cells. They found that the established Stoma cell line was able to proliferate hematopoietic stem cells and hematopoietic progenitor cells. Further, as a result of examining the use of the obtained stoma cell line, hematopoietic stem cells or hematopoietic progenitor cells cultured together with the stromal cell line of the present invention can be used as a material for blood cell transplantation or gene therapy.
  • the present invention is a cell line that is isolated and established from the mammalian GM AGM region and that can support the growth or survival of hematopoietic stem cells and hematopoietic progenitor cells. Further, the present invention provides the above-mentioned cell line capable of proliferating hematopoietic stem cells or hematopoietic progenitor cells while at least partially maintaining pluripotency, and hematopoietic stem cells such that at least a part thereof involves cell cycle rotation. Is provided.
  • the present invention also relates to the above-mentioned cell line in which an oncogene or an apobutosis-related gene is introduced, and the self-proliferation or survival is regulated by the gene; and the above-mentioned cell in which a gene encoding a cell stimulating factor is introduced. Offer stocks.
  • the present invention provides a transplant containing hematopoietic stem cells or hematopoietic progenitor cells cultured and proliferated or maintaining survival by culturing with the cell line, and cultured and proliferated or maintained on survival with the cell line, and A gene therapy composition comprising a hematopoietic stem cell or hematopoietic progenitor cell into which a foreign gene has been introduced.
  • the present invention also provides an isolated and established mammalian GM AGM region, and a hematopoietic stem cell. Culturing a cell group or a fraction thereof containing at least hematopoietic stem cells or hematopoietic progenitor cells, together with a cell line capable of supporting the proliferation or survival of the cells and hematopoietic progenitor cells. Methods for supporting growth or survival are provided. Examples of the cell group include a cell group derived from umbilical cord blood, fetal liver, bone marrow, fetal bone marrow, or peripheral blood.
  • the present invention provides a method of isolating and establishing a cell line that is isolated from the AGM region of a mammalian fetus and that is cultured with a cell line that can support the proliferation or survival of hematopoietic stem cells and hematopoietic progenitor cells, and that has grown or has maintained its survival.
  • the present invention provides a method for transplanting hematopoietic stem cells or hematopoietic progenitor cells, which comprises transplanting hematopoietic stem cells or hematopoietic progenitor cells.
  • the present invention provides a hematopoietic cell isolated and established from an AGM region of a mammalian fetus, which is cultured with a cell line capable of supporting the proliferation or survival of hematopoietic stem cells and hematopoietic progenitor cells, and which is proliferated or whose survival is maintained. It is intended to provide a gene therapy method characterized by introducing a foreign gene into stem cells or hematopoietic progenitor cells, and transplanting the transduced cells.
  • the AGM (Aorta-Gonad-Mesonephros) region refers to the fetal aorta, gonads, and mesonephros.
  • the AGM region is an anatomically identified region where fetal adult hematopoietic stem cells appear.
  • a stroma cell is a cell group that exists in a hematopoietic tissue other than hematopoietic cells that supports the survival, differentiation, and proliferation of the hematopoietic cell group, and that constitutes the cell.
  • These cells include cell types such as fibroblasts, preadipocytes, adipocytes, and vascular endothelial cells.
  • the hematopoietic stem cells are cells having pluripotency to differentiate into all of the lineage of blood cells, and stem cells c current human is a cell capable of self-renewal while maintaining their pluripotency
  • stem cells are identified as cells that can form colonies (CFU-Emix) containing erythrocytes in an in vitro Atsushi system.
  • Hematopoietic progenitor cells refer to cells that can differentiate into a single hematopoietic lineage or multiple but not all lineages, and these cells can differentiate into single or multiple lineage cells .
  • the AGM region-derived stromal cell line refers to a single stromal cell isolated and established from the AGM region.
  • the AGM region-derived stoma cell line may be simply referred to as “stroma cell line” or “cell line of the present invention”.
  • stroma cell line or “cell line of the present invention”.
  • the stromal cell line of the present invention is a cell line that is isolated and established from the AGM region of a mammalian fetus and can support the growth or survival of hematopoietic stem cells and hematopoietic progenitor cells.
  • the method for isolating and establishing the cell line of the present invention is exemplified below.
  • mice Male and female mice are bred under SPF (specific pathogen-free) environment, females are placed in the same cage with males overnight, and the following morning, female mice confirmed to have a vaginal plug are transferred to new cages. Breed. The day when the vaginal plug is confirmed is defined as 0.5 day of gestation, and the fetus is removed from the mouse on day 8 to 13 of gestation, preferably at day 10.5. Methods for isolating the AGM region from the fetus are described in Godin et al. (Godin, I., Proc. Natl. Acad. Sci. USA, 92: 773-777, 1997), Medvinsky et al. (Medvinsky, AL, Blood, 87: 557).
  • SPF specific pathogen-free
  • the fetus is placed in a culture dish filled with phosphate buffered saline so that the fetus is immersed, and the AGM region is excised under a stereoscopic microscope so as not to include other regions, and transferred to a new culture dish.
  • a medium for example, a MEM medium containing 10% FCS (male fetal serum) at 37 ° C, 5% CO 2 and 100% humidity. Incubate. When the cells in the AGM region have adhered to the culture dish, the culture is continued by further adding a medium, and stromal cells appear around the tissue piece of the AGM region. After further culturing for about one week, the adherent cells are detached by trypsin treatment, washed in the medium, and seeded on a culture dish. On the next day, remove the cells that have not adhered to the culture dish together with the medium, and add fresh medium.
  • a medium for example, a MEM medium containing 10% FCS (male fetal serum) at 37 ° C, 5% CO 2 and 100% humidity.
  • FCS male fetal serum
  • ⁇ - rays of about 900 rad are applied to remove endogenous hematopoietic cells.
  • the culture system is trypsinized to suspend the cells, and placed in a 24-well culture dish at 10 to 20,000 cell wells, preferably 50 to 100 cells / well. Sowing.
  • the cells do not proliferate, so the number of cells seeded in one well is increased, and After acclimating the cells so that they can be tolerated, it is preferable to carry out cloning by the limiting dilution method.
  • the cells were seeded in a 96-well culture dish by limiting dilution so that the cells became 0.05-1 cell Nowell, preferably 0.3 cells Z cells. Expand the cells growing from the well where only one cell has been seeded.
  • the cell strain of the present invention can be obtained.
  • Such cell selection can be performed by co-culturing the candidate strain with hematopoietic stem cells or hematopoietic progenitor cells, and then evaluating the degree of proliferation of hematopoietic stem cells or hematopoietic progenitor cells.
  • the degree of proliferation of hematopoietic stem cells or hematopoietic progenitor cells is determined by culturing the cells at the start of coculture and after coculture in the presence of cytokines, and comparing the number of blood cell colonies.
  • the AGM region used for establishing a stoma cell line is limited to mouse origin. Instead, it may be another mammal, for example, an AGM region such as pig, sheep, and human.
  • an AGM region such as pig, sheep, and human.
  • a The appearance of hematopoietic stem cells in the GM region was confirmed in both human (Tavian, M., Blood, 87: 67-72, 1996) and mouse (Sanchez, MJ., Immunity, 5: 513-525, 1996). Therefore, it is considered that a common mechanism exists in the developmental process of mammals.
  • human umbilical cord blood-derived hematopoietic stem cells can be expanded using a mouse AGM region-derived stroma cell line. It is strongly supported that stromal cells equivalent to the stomal cell line obtained in the Examples can be isolated from the mammalian GM region of the present invention, and that they can be used equally.
  • a single stromal cell can be isolated from the AGM region and established.
  • This stromal cell line can support the proliferation and survival of hematopoietic stem cells and hematopoietic progenitor cells. That is, a stromal cell line and a hematopoietic stem cell, a hematopoietic progenitor cell, or a hematopoietic cell group containing at least one of them are co-cultured.
  • hematopoietic stem cells rotate through the cell cycle, partly renew themselves as stem cells, partly proliferate while differentiating into hematopoietic progenitor cells, and hematopoietic stem cells are maintained in this culture system.
  • hematopoietic progenitor cells proliferate while differentiating into cells of a single or multiple lineages.
  • the proliferation or survival of the stromal cell line is regulated. It becomes possible. If the growth of stromal cell lines could be regulated, it would be possible to efficiently increase the number of stromal cell lines and extend their lifespan. In general, oncogenes are thought to have an advantageous effect on proliferation.
  • the stromal cell line may have an adverse effect in the body, and avoid bringing it into the body. Is preferred. If the survival of stromal cell lines could be regulated, it would be possible to kill and eliminate only stromal cell lines after hematopoietic cell culture. Therefore, a gene that induces cell death should be in a form that allows its expression to be regulated by an external stimulus, and introduced into a stoma cell line to artificially regulate its growth and viability. You can also create a system that makes it possible.
  • the oncogenes include the human papilloma virus gene (Halbert, C.L., J. Virol., 65: 473, 1991; Ryan, M.J., Kidney Int 45:48, 1994)) and the SV40 gene (Chou,
  • Fas Itoh, N., Cell, 66: 233-243, 1991
  • TNFreceptor type II Litoson, H., Cell 61 (2): 351-359, 1990
  • Death receptor 3 Kitoson, J., Nature, 384: 372-375, 1996)
  • Death receptor 4 Pan, G. ICE (IL-1 ⁇ converting enzyme) (Cerretti, DP, Sc), a proteolytic enzyme belonging to the signal transduction system of the receptor. ience 256: 97-100, 1992), and Caspase Family (Patel, T, FASEB.
  • AAV Adetin, RM Hum Gene Ther 5: 793, 1994
  • a method using a virus-derived animal cell vector such as a simple herpes virus vector, a calcium phosphate coprecipitation method, The DEAE-dextran method, the electoral poration method, the ribosome method, the lipofection method, the microinjection method and the like can be used.
  • a marker gene such as a drug resistance gene is used in addition to the apobutosis-related gene, the selection of a stromal cell line into which the target gene has been introduced is facilitated.
  • the method of the present invention for supporting the growth or survival of hematopoietic stem cells or hematopoietic progenitor cells utilizes the properties of the above-mentioned stromal cell line, and, together with the stromal cell line, at least hematopoietic stem cells or hematopoietic progenitor cells. Culturing the cell group containing the cells or the fraction thereof.
  • the cell group may be one in which one of hematopoietic stem cells or hematopoietic progenitor cells is isolated, or both of them. It may also contain at least one of hematopoietic stem cells or hematopoietic progenitor cells, and may further contain other hematopoietic cells.
  • the fraction refers to a fraction containing hematopoietic stem cells or hematopoietic progenitor cells, which is fractionated from a cell group containing hematopoietic stem cells or hematopoietic progenitor cells.
  • a cell group or a fraction thereof may be simply referred to as hematopoietic stem cells and hematopoietic progenitor cells.
  • stem cells can be obtained by administering fetal liver, bone marrow, fetal bone marrow, peripheral blood, cytokine ⁇ y ⁇ Z or an anticancer agent of a mammal such as a human or a mouse.
  • the mobilized peripheral blood, umbilical cord blood and the like can be mentioned, and any tissue may be used as long as it contains hematopoietic stem cells.
  • a culture method using a so-called culture dish or flask is possible, but the medium composition, pH, etc. are controlled mechanically to achieve high density.
  • the culture system can also be improved by a bioreactor capable of culturing in E. coli (Schwartz, Proc. Natl. Acad. Sci. USA, 88: 6760, 1991; Roller, MR, Bio / Technology, 11: 358). Roller, MR, Blood, 82: 378, 1993; Palsson, B. 0., Bio / Technology, 11: 368, 1993).
  • the medium used for the culture is not particularly limited as long as the growth and survival of hematopoietic stem cells or hematopoietic progenitor cells are not impaired.
  • SF-02 medium Sudo Junyaku
  • Opti-MEM medium GIBC0 BRL
  • MEM medium GIBC0 BRL
  • IMDM medium GIBC0 BRL
  • PRMI1640 medium GIBC0 BRL
  • the culture temperature is usually from 25 to 39 ° C, preferably from 33 to 39 ° C.
  • Materials added to the culture medium include fetal calf serum, human serum, calf serum, insulin, transferrin, lactoferrin, ethanolamine, sodium selenite, monothioglycerol, 2-mercaptoethanol, ⁇ Serum albumin, sodium pyruvate, polyethylene glycol, various vitamins, various amino acids, various growth factors, preferably EGF (epidermal growth factor), PDGF (platelet-derived growth factor), bFGF (basic fibroblast growth factor) , O 2 is typically 4-6%, and preferably 5%.
  • the closed cell line of the present invention can be used to support the growth and survival of hematopoietic stem cells and hematopoietic progenitor cells, but is more effective by adding a cell stimulating factor to the co-culture system. Proliferation and survival can be supported.
  • a cell stimulating factor is not particularly limited as long as it does not prevent the Stoma cell line from supporting the growth and survival of hematopoietic stem cells or hematopoietic progenitor cells.
  • SCF stem cell factor
  • IL-3 interleukin-3
  • GM-CSF granulocyte / macrophage colony-stimulating factor
  • IL -6 interleukin-6
  • TP0 thrombopoetin
  • G-CSF granulocyte colony-stimulating factor
  • TGF- ⁇ transforming growth factor-3
  • MIP-1a Davatelis, G., J. Exp. Med.
  • hematopoietic hormones such as EP0 (erythropoietin), Wnt (Thimoth, AW, Blood, 89: 3624 -3635, 1997) Differentiation and growth regulators such as gene products, or developmental regulation such as Notch / Delta (Moore KA, Proc. Natl. Acad. Sci. USA, 94: 4011-4016, 1997) gene products Factors and the like.
  • a gene that encodes a cell stimulating factor as described above is introduced into a stromal cell line in such a manner that the gene can be expressed in the cell, and the resulting gene-introduced cell is used.
  • the culture system can be improved.
  • the culture supernatant obtained by culturing the Stoma cell line shows activity that contributes to the growth and survival of hematopoietic stem cells or hematopoietic progenitor cells
  • only the culture supernatant is used for hematopoiesis.
  • Stem cells or hematopoietic progenitor cells can be subjected to culture.
  • stromal cells and hematopoietic stem cells or hematopoietic progenitor cells are separated by a porous membrane that allows the penetration of factors involved in the growth and survival of these hematopoietic cells. It is also possible to culture.
  • cell stimulating factors as described above to the culture system in order to favor the survival and proliferation of hematopoietic cells (Verfaillie, CM, Blood, 84: 1442-1449, 1994).
  • the above-mentioned cell stimulating factor, a gene encoding a cell stimulating factor, an oncogene, an apoptosis-related gene, and addition to a cell, a method of introducing a gene into a cell, and a method of culturing a cell are known to those skilled in the art. This is performed by a known method.
  • hematopoietic stem cells or hematopoietic progenitor cells grown and cultured with the cell line of the present invention can be used as a blood substitute for conventional bone marrow transplantation or cord blood transplantation. It can be used as a transplant for cell transplantation.
  • Hematopoietic stem cell transplantation can improve conventional blood cell transplantation treatments because the transplant is semi-permanently engrafted.
  • Conventionally a large amount of bone marrow was collected for bone marrow transplantation, but according to the present invention, a small amount of bone marrow can be collected.
  • the stem cell expansion technology according to the present invention can be used to expand autologous or non-autologous stem cells.
  • Transplantation of hematopoietic stem cells by the method of the present invention can be performed by systemic X-ray therapy or leukemia for leukemia.
  • chemotherapy it can be used for various diseases in addition to combining with these treatments.
  • a treatment that causes bone marrow suppression as a side effect such as chemotherapy or radiation therapy for solid cancer patients
  • bone marrow is collected before the procedure, and hematopoietic stem cells and hematopoietic progenitor cells are expanded in vitro
  • hematopoietic disorders due to side effects can be recovered early, and more powerful chemotherapy can be performed, and the therapeutic effect of chemotherapy can be improved.
  • a patient or another person's hematopoietic stem cells and hematopoietic progenitor cells are differentiated into various blood cells and transferred into the patient's body, thereby reducing the dysfunction due to hypoplasia of various blood cells.
  • Patients presenting can be improved.
  • it can improve hematopoietic failure caused by bone marrow hypoplasia presenting anemia such as aplastic anemia.
  • Other diseases for which hematopoietic stem cell transplantation by the method of the present invention is effective include chronic granulomatosis, double immunodeficiency syndrome, agammaglobulinemia, Wiskott-Aldrich syndrome, acquired immunodeficiency syndrome (AIDS), and the like.
  • Immunodeficiency syndrome thalassemia, hemolytic anemia due to enzyme deficiency, congenital anemia such as sickle cell disease, lysosomal storage diseases such as Gaucher disease and mucopolysaccharidosis, adrenal leukemia, various cancers and tumors, etc. .
  • transplantation of hematopoietic stem cells using the graft of the present invention may be performed in the same manner as conventional bone marrow transplantation or cord blood transplantation, except for the cells used.
  • the origin of hematopoietic stem cells that may be used for hematopoietic stem cell transplantation as described above is not limited to bone marrow, but can be obtained by the administration of fetal liver, fetal bone marrow, peripheral blood, cytokines and anticancer drugs as described above. Mobilized peripheral blood, umbilical cord blood, and the like can be used.
  • the transplant of the present invention may be a composition containing a buffer solution or the like in addition to the hematopoietic stem cells and hematopoietic progenitor cells grown by the method of the present invention.
  • Hematopoietic stem cells or hematopoietic progenitor cells cultured and grown together with the cell line of the present invention can be used for ex vivo gene therapy.
  • Gene therapy includes an in vivo method in which DNA is directly administered to patients and an ex vivo method in which DNA is introduced into target cells and the transfected cells are transplanted into patients.
  • bone marrow cells are used as target cells in the ex vivo method, a large amount of bone marrow must be collected, but according to the present invention, a small amount of bone marrow can be collected.
  • the ability of hematopoietic stem cells to self-renew and supply hematopoietic cells in the long term is considered to be a suitable target for gene therapy.
  • the cell cycle could not be rotated, making gene transfer difficult.
  • co-culturing the cell line of the present invention with hematopoietic stem cells or hematopoietic progenitor cells the cell cycle of these cells can be rotated, and gene transfer can be performed easily. .
  • Gene therapy using the present invention is performed by introducing a foreign gene (therapeutic gene) into hematopoietic stem cells or hematopoietic progenitor cells cultured and grown together with stromal cells, and using the resulting transfected cells.
  • Gene therapy using the gene therapy composition of the present invention is the same as conventional gene therapy except that hematopoietic stem cells or hematopoietic progenitor cells co-cultured and expanded with stromal cells are used as target cells. You can go to The foreign gene to be introduced is appropriately selected depending on the disease.
  • Diseases targeted for gene therapy targeting blood cells include chronic granulomatosis, double immunodeficiency syndrome, agammaglobulinemia, Wiskott-Aldrich syndrome, and acquired immunodeficiency syndrome
  • AIDS thalassemia
  • hemolytic anemia due to enzyme deficiency
  • congenital anemia such as sickle cell disease
  • lysosomal storage disease such as Gaucher disease and mucopolysaccharidosis
  • adrenal white matter degeneration various cancers or Tumors and the like.
  • the origin of hematopoietic stem cells used as target cells is not limited to bone marrow, and fetal liver, fetal bone marrow, peripheral blood, peripheral blood obtained by mobilizing stem cells by administration of cytokine and Z or an anticancer agent, umbilical cord blood, and the like can be used.
  • retrovirus vector such as Moroni murine leukemia virus, adenovirus vector, adeno-associated virus (MV) Vector
  • MV adeno-associated virus
  • a method using a virus-derived animal cell vector such as a simple virus vector, calcium phosphate co-precipitation method, DEAE-dextran method, electrification method, ribosome method, lipofection method, A microinjection method or the like
  • retrovirus vector or an adeno-associated virus vector is preferable, since it can be integrated into the chromosome DNA of the target cell and gene expression can be expected permanently.
  • an adeno-associated virus (AAV) vector can be constructed as follows. First, ITRs (inverted t) at both ends of the wild-type adeno-associated virus DNA Then, transfection of 293 cells with vector plasmid containing the therapeutic gene inserted between them (erminal repeat) and helper plasmid to supplement viral proteins is performed. Subsequent infection with the helper virus adenovirus produces virus particles containing the MV vector. Alternatively, instead of adenovirus, a plasmid expressing an adenovirus gene responsible for helper function may be transfected. Next, the obtained virus particles are used to infect hematopoietic stem cells or hematopoietic progenitor cells.
  • a suitable promoter and enhancer upstream of the target gene in the vector DNA it is preferable to insert a suitable promoter and enhancer upstream of the target gene in the vector DNA, and to regulate the expression of the gene by these.
  • a marker gene such as a drug resistance gene in addition to the therapeutic gene facilitates selection of cells into which the therapeutic gene has been introduced.
  • the therapeutic gene may be a sense gene or an antisense gene.
  • hematopoietic stem cells are mostly in the G0 phase in the cell cycle and cannot be infected with retrovirus. Therefore, IL-1, IL-3, IL-6 and SCF are added to hematopoietic stem cells. It must be allowed to act and enter the cell cycle before infection with the virus (Nolta, J., Exp. Hematol, 20: 1065-1071 1992). Since the cell line of the present invention can rotate the cell cycle of hematopoietic stem cells, efficient virus infection is possible. In addition, it has been reported that the co-existence of bone marrow feeder cells during viral infection increases the efficiency of infection (Moore, KA, AB Blood, 79: 1393-1399, 1992). A possible method would be to infect the virus with the coexisting stromal cell line and hematopoietic stem cells, and then kill the stromal cell line by expressing apobutosis-related genes.
  • the composition for gene therapy of the present invention may be a composition containing a buffer, a novel active substance, and the like, in addition to the hematopoietic stem cells and hematopoietic progenitor cells grown by the method of the present invention.
  • BRIEF DESCRIPTION OF THE DRAWINGSFIG. 1 shows the results of colony assembly 4 weeks after the start of co-culture of mouse AGM-derived stoma cell line and CD34-positive human cord blood-derived hematopoietic stem cells. is there.
  • FIG. 2 is a diagram showing the results of colony attachment three weeks after the start of co-culture of a mouse AGM-derived stoma cell line and CD34-positive human cord blood-derived hematopoietic stem cells.
  • FIG. 3 is a graph showing the results of colony attachment 6 weeks after the start of co-culture of a mouse AGM-derived stroma cell line and CD34-positive human cord blood-derived hematopoietic stem cells.
  • FIG. 4 is a graph showing the results of colony atsieties 10 days after the start of co-culture of mouse AGM-derived stromal cell line and mouse bone marrow-derived C-KIT + Sca-1 + Lin_ hematopoietic stem cells. .
  • FIG. 5 is a diagram showing the ratio of donor cells to myeloid cells and lymphoid cells in mouse peripheral blood transplanted with mouse bone marrow-derived hematopoietic stem cells.
  • ⁇ indicates hematopoietic stem cells co-cultured with AGM-S3
  • FIG. 6 is a diagram showing the ratio of donor cells to myeloid and lymphoid cells in mouse peripheral blood transplanted with mouse fetal hematopoietic stem cells.
  • ⁇ indicates hematopoietic stem cells co-cultured with AGM-S3, indicates hematopoietic stem cells not co-cultured with AGM-S3.
  • BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described more specifically with reference to examples.
  • Example 1 Hematopoietic stem cells can support proliferation or survival of hematopoietic progenitor cells
  • mice Males and females of C3H / HeNSLc mice (purchased from Japan SLC Co., Ltd.) were bred under SPF (special pathogen-free) environment. One or two females were placed in the same cage with one male overnight, and the following morning, female mice confirmed to be vaginal were transferred to a new cage for rearing. The day when the vagina was confirmed was defined as 0.5 gestation. Mice at 10.5 days of gestation were killed by cervical dislocation, and the fetuses were removed. Separation of AGM is described in Godin et al. (Godin, I., Proc. Natl. Aacd. Sci. USA, 92: 773-777, 1995) and Medvinsky et al.
  • the adherent cells were detached by trypsin treatment (in PBS containing 0.05% trypsin, 0.53 mM EDTA (Gibco BRL), 37. C, 3 to 5 minutes). After washing twice with the medium, the cells were seeded on 6-well culture dishes (Nunc # 15 2795). The next day, cells that did not adhere to the culture dish were removed together with the medium, and fresh medium was added. Two weeks after transfer to a 6-well culture dish, the cells were irradiated with 900 Rad gamma rays to remove endogenous hematopoietic cells.
  • the cells were directly cloned from this culture system by the limiting dilution method, but no cell growth was observed, and the cells could not be cloned. Therefore, the number of cells seeded in one well was increased, and cells were adapted to withstand proliferation from a small number of cells, and then cloned by the limiting dilution method.
  • AGM was excised and cultured, and the culture system, which had been exposed for 2 weeks after ⁇ -irradiation, was treated with trypsin (in PBS containing 0.05% trypsin and 0.53m EDTA). (37 ° C., 3-5 minutes), and the cells were suspended and seeded on a 24-well culture dish at 50 to 100 cells / well. After culturing for 3 weeks, seed cells in a 96-well culture dish (Nunc # 167008) by limiting dilution to a 0.3-cell Z-well, and seed only one cell. The cells that had proliferated from were expanded and cultured. As a result, fibroblast-like cells and cobblestone-like cells were obtained, and the cloning was successful.
  • Human cord blood-derived CD34-positive cell fraction was co-cultured with fibroblast-like cells for two weeks, Examination of the presence or absence of colony forming cells in the culture system revealed no colony forming cells in the co-culture system with fibroblast-like cells. Therefore, the same examination was performed on 7 clones showing a pavement-like morphology, and three clones having the activity of supporting the growth of human hematopoietic stem cells were obtained. These were named AGM-sl, AGM-s2 and AGM-s3. Using these three clones, the ability to support hematopoietic cells was examined.
  • AGM-derived stromal cell line established as described above was examined using human CD34-positive stem cells.
  • Human umbilical cord blood was collected at the time of normal delivery according to the guidelines of the Institute of Medical Science, the University of Tokyo. According to the method of Sui et al. (Sui, X., Pro Natl, Acad. Sci. U.S.A., 92: 2859-2863, 1995), CD34-positive stem cells were fractionated as follows. Human cord blood was added to silica at a volume of 1/10 (IBL) and left at 37 ° C for 30 minutes with thorough mixing every 10 minutes. Thereafter, the cord blood added with silica was subjected to Ficoll / Hypaque (Pharmacia Biotech) specific gravity centrifugation to separate mononuclear cells.
  • the mononuclear cells are reacted with magnetic beads (Dynabeads M-450 CD34) coated with anti-CD34 antibody, and the cells expressing CD34 are bound to the beads.
  • the CD34-positive cell fraction was eluted using another CD34 antibody (DETACHaBEAD CD34; Dynal, Oslo).
  • This cell population was used as a human umbilical cord blood hematopoietic stem cell population.
  • This cell population was confirmed to contain 85 to 95% of CD34-positive cells by FACS (fluorescence activated cell sorting) analysis using a CD34 antibody (using a Senor Resolator manufactured by Ortho Diagnostics Systems).
  • AGM-derived stoma cell lines (AGM-sl, AGM-s2, AGM-s3) established in 1) above, or MS-5, a mouse bone marrow-derived stoma cell line (Itoh, ⁇ ⁇ , Exp. Hematol., 17: 145-153, 1989) was grown in MEM medium containing 10% FCS until it covered the entire bottom of a 24-well culture dish.
  • MS-5 is Kazuhiro Mori, Department of Biology, Faculty of Science, Niigata University The one provided by the professor and maintained in a MEM medium containing 10% FCS was used.
  • AGM- scan Toroma cells were grown sl, AGM- s2, AGM- s3 each 5 x 1 0 3 / Ueru or is the mouse bone marrow-derived stromal cell line MS- 5 to 1 X 10 4
  • the seeds were inoculated into 24 / well / dwell culture dishes and cultured in a MEM medium containing 10% FCS for 2 days, and grown until the cells covered the entire bottom of the culture dish.
  • a MEM medium containing 10% FCS for 2 days, and grown until the cells covered the entire bottom of the culture dish.
  • 1500 or 500 CD34-positive human cord blood-derived hematopoietic stem cells purified in (1) were overlaid and co-cultured in 1 ml of MEM medium containing 10% FCS.
  • the cells cultured in the above co-culture system were appropriately diluted, applied to a 1 ml methylcellulose culture system, and analyzed in triplicate.
  • the methylcellulose culture system consists of 0.9% methylcellulose (Shin-Etsu Chemical), 30% fetal serum (HyClone), and 1% crystallized deionized serum albumin fraction in ⁇ -medium (Flow Laboratories).
  • the colonies that emerged after two weeks of culture were observed under a microscope, and the properties of the emerging cells were analyzed. Specifically, the emerged CFU-G (granulocyte colony-forming unit) ⁇ CFU-M macrophage co ⁇ ony-forming unit) s CFU-GM (granulocyte-macrophage colony-forming unit) N BFU-E (erythroid burst forming unit), CFU-GEMM (granulocyte-erythrocyte megakaryocyte-macrophage colony- for The number of ming units was counted, and the value obtained by dividing the number of colonies that appeared when human CD34-positive cells at the start of co-culture with stromal cells in a methylcellulose culture system was defined as the growth rate.
  • Fig. 1 Fig. 3 Fig. 3 CFU-Gi granulocyte colony-forming unit
  • CF U-M is macrophage colony-forming unit
  • CFU-GM is granulocyte-macrophage colony-forming unit
  • BFU- E is an erythroid burst forming unit
  • CFU-GEM is an abbreviation for granulocyte-erythrocyte- megakaryocyte- macrophage colony-forming unit. Characteristic of these results is that CFU-GEMM, which can differentiate into neutrophils, erythrocytes, macrophages, and megakaryocytes, is remarkably amplified in the co-culture system with AGM-sl and AGM_s3 cells That is.
  • CFU-GEMM is a very undifferentiated cell of the hematopoietic system, and the above results were obtained by co-culturing with a stromal cell line derived from mouse AGM without differentiation of the undifferentiated human hematopoietic cell line. It shows that it is amplified. So far, stromal cell lines established from bone marrow cannot amplify human CFU-GE marauders in this way (Nishi, N., Exp. Hematol. 24: 1312-1321, 1996). Also in this example, as shown in FIGS. 2 and 3, almost no CFU-GEMM was observed after co-culture in MS-5, a stromal cell established from mouse bone marrow.
  • this activity to support human hematopoietic stem cells is considered to be characteristic of the AGM-derived stromal cell line. Furthermore, no increase in CFU-GEMM was observed in AGM-s2 cells. In other words, it is thought that, among the AGM-derived stromal cell lines having the same morphology, cells capable of supporting human hematopoiesis are limited. As mentioned above, the activity of maintaining human hematopoietic stem cells was not observed in the fibroblast-like cells as well, and specific cell types among the cells in the AGM region have the ability to maintain human hematopoietic stem cells. It was revealed.
  • CFU-GM, CFU-G, and CFU-M human hematopoietic progenitor cells could also be expanded.
  • the mouse AGM-derived stroma cell line can support the proliferation of human hematopoietic stem cells and hematopoietic progenitor cells.
  • the proliferation mechanism of hematopoietic stem cells is conserved across species, and the proliferation of human hematopoietic stem cells and hematopoietic progenitor cells can be carried out using AGM-derived stromal cells derived from heterologous organisms other than human. It has been shown that this is possible.
  • Example 2 A GM-derived stromal cell line against mouse bone marrow hematopoietic stem cells
  • mice AGM-derived stromal cell line obtained in Example 1 was confirmed to have a growth supporting activity on mouse bone marrow-derived hematopoietic stem cells.
  • Bone marrow cells in femurs of C57BL / 6 mice (8-week-old, male) (3 SLC, Inc.) were removed and suspended in a MEM medium containing 10% FCS.
  • a standard method Takashi Takatsu, Basic Technology for Immunological Research, Yodosha 1995
  • the mouse bone marrow mononuclear cell fraction was concentrated by specific gravity centrifugation, followed by staining buffer (5% FCS, 0.05% sodium azide).
  • a hematopoietic stem cell fraction was obtained by the following method (Osawa, M., J. Immunol., 156: 3207-3214, 1996).
  • Phycoerythrin-conjugated Sea-1 antibody (Pharmingen), arophycosinine-conjugated anti-c-KIT antibody (Lifetech Oriental), and the following six types of biotin-specific differentiation antigens as differentiation markers (Lin) Antibody, CD45R / B220 (RA-36B2), CD4 (thigh-5), CD8 (53-6.72), Gr-1 (RB6-8C5), TER119 (more than 5 antibodies
  • the body was purchased from Pharmingen, SanDiego) and Mac-1 (Ml / 70. 15.1) (Serotec, O ford, UK) was added to the cell suspension and allowed to react for 20 minutes on ice. .
  • the suspension was suspended in the staining buffer, Texas Red-conjugated streptavidin (Life Technologies) was added, and the plate was left on ice for 20 minutes. After washing twice, the cells were subjected to cell sorting (Becton Dickinson, FACSVantage) to differentiate hematopoietic stem cells (c-KIT + Sca-1 +) positive for differentiation antigen negative, Sea-1 antibody positive and c-KIT positive. Lin—hematopoietic stem cells).
  • FACS was set using bone marrow cells stained only with phycoerythrin-conjugated rat immunoglobulin G2a (Cedarlane), allophycocynin-conjugated rat immunoglobulin G2b (Pharmingen), or Texas red-conjugated streptavidin as a negative control. Then, only cells that were specifically stained with the antibody were collected.
  • AGM_s3 was seeded at 5 ⁇ 10 3 each in a 24-well culture dish, cultured in a MEM medium containing 10% FCS for 2 days, and grown until the cells covered the entire bottom of the culture dish.
  • 100 mouse-bone marrow-derived C-KIT + Sca-1 + Lin-hematopoietic stem cells were overlaid and cultured in 1 ml of MEM medium containing 10% FCS.
  • a further 1 ml of the same medium was added.
  • C-KIT + Sca-1 + Lin collected at the start of co-culture and a part of hematopoietic stem cells and the co-cultured cells were evaluated for hematopoietic stem cells as follows, and the proliferation status of hematopoietic stem cells was analyzed. did.
  • the cells cultured in the above co-culture system were appropriately diluted, applied to a 1 ml methylcellulose culture system, and analyzed in triplicate.
  • the methylcellulose culture system is 0.9 ° / ⁇ on ⁇ -medium (Flow Labo ratories). Methylcellulose (Shin-Etsu Chemical), 30% fetal serum (HyClone), 1% crystallized deionized serum albumin fraction V (Sigma), 0.05 mM 2-mercaptoethanol (Eastman), lOOng / ml mouse SCF, 20ng / ml mouse IL-3, 2units / ml human EP0, lOOng / ml human IL-6, 10ng / ml human G-CSF, 4ng / ml human TP0
  • the test was performed on a culture dish (Falcon # 1008). Observe under a microscope the colonies that have emerged after 10 days of culture, and analyze the properties of the emerging cells. did.
  • Fig. 4 shows the results.
  • the multiplication factor is defined as the number of colony-forming cells contained in 100 mouse C-KIT + Sca_l + Lin_ hematopoietic stem cells at the start of coculture and the number of colonies formed after coculture. did. CFU-GEMM containing erythroid colonies increased remarkably, confirming that mouse hematopoietic stem cells proliferated by co-culture with AGM-derived stroma cell line.
  • mice 8 weeks old, male were transplanted from the tail vein. On day 12 after the transplantation, the spleen was removed from the mouse, and the number of colonies formed in the spleen (CFU-S12: day 12 spleen colony-for ming unit) was calculated.
  • mice transplanted with 100 c-KIT + Sca-1 + Lin-hematopoietic stem cells 3 ⁇ 1 spleen colonies were formed in mice transplanted with 100 c-KIT + Sca-1 + Lin-hematopoietic stem cells.
  • 4 ⁇ 1 spleen colonies were detected in mice transplanted after diluting 1-well cells recovered from the co-culture system to 1/6. Therefore, it is calculated that from the initial 100 C-KIT + Sca-1 + Lin-hematopoietic stem cells, about 24 (4 ⁇ 6) spleen colony forming cells had proliferated. That is, spleen colony forming cells grew about 8-fold by co-culture.
  • human umbilical cord blood cells containing human hematopoietic stem cells were co-cultured with AGM-s3 and transferred to immunodeficient mice. Transplanted.
  • survival after co-culture of pluripotent human hematopoietic stem cells and hematopoietic progenitor cells capable of differentiating into myeloid and lymphoid lineages ⁇ Proliferation was examined.
  • NOD / Shi-scid mice 1 ⁇ 10 6 mononuclear cells from human umbilical cord blood were transplanted into NOD / Shi-scid mice immediately after isolation and after co-culture with AGM-s3 for 4 weeks.
  • the conditions for cell preparation and co-culture were performed in the same manner as in Example 1.
  • Transplantation of cells into NOD / Shi-scid mice is performed by transplanting cells from the tail vein of 8-10 week old NOD / Shi-scid (Central Laboratory Animal Research Institute) irradiated with 300 Rad of gamma rays. went.
  • anti-asialo GM-1 antibody 2 (g / ml) (Wako Pure Chemical Industries) 3001 was administered immediately before transplantation and 11 days after transplantation.
  • bone marrow cells were collected from mice and stained with various anti-human blood cell marker antibodies that specifically recognize human cells, thereby engrafting human blood cells. Bone marrow cells collected from mice into which cells had not been transplanted were stained in the same manner.These staining techniques and the analysis using FACS described below were performed according to the method described in Example 1. The same was done.
  • CD45 which is a pancreatic cell marker
  • FITC-labeled anti-human CD45 antibody Becton Dickinson
  • mouse bone marrow cells after transplantation.
  • FITC-labeled mouse IgGl (Becton Dickinson) was used as a negative control for FACS.
  • no cells that responded to the anti-human CD45 antibody were detected in the bone marrow cells of mice to which the cells had not been transplanted.
  • monocytes immediately after separation, 6.2 ° /. Of the bone marrow cells were human cells.
  • mice In mouse bone marrow transplanted with mononuclear cells co-cultured with AGM-s3, 5.2% of the cells were human cells. This result suggests that hematopoietic stem cells or hematopoietic progenitor cells were included in the transplanted human hematopoietic cells, and thus the appearance of human hematopoietic cells was observed for 5 weeks after transplantation into immunodeficient mice.
  • Cy5-labeled phycoerythrin (PECy5) -labeled CD45 antibody and antibodies against various differentiation antigens that is, phycoerythrin (PE) -labeled anti-CD13, anti-CD33, anti-CD14, and anti-CD14 antibodies.
  • Double staining was performed using the CD19 antibody, FITC-labeled anti-CD10 antibody, or anti-CD34 antibody.
  • the anti-CD34 antibody was purchased from Ioki unoteck, and all other antibodies were purchased from Becton Dickinson.
  • CD45-positive cells were gated using FACS and the expression of these differentiation antigens on human blood cells was analyzed.
  • CD34-positive cells were 8 ° / CD45-positive cells. It was confirmed that they existed at the frequency of The results indicate that the young hematopoietic cells were maintained in the co-culture with AGM-s3 for 4 weeks and maintained the young trait after transplantation into mice. Furthermore, of the CD45-positive cells, the cells positive for the antigens CD13, CD33, and CD14, which are differentiation markers for myeloid cells, were 29% and 38 ° / 38%, respectively. Was 12%. As for lymphoid cells, the presence of CD19-positive cells, a B cell marker, was confirmed at a frequency of 58%.
  • PBS phosphate buffered saline
  • FCS Hyclone
  • ImM EDTA ImM EDTA
  • a biotinylated antibody against the differentiation antigen marker i.e., an anti-CD4 antibody, an anti-CD8 antibody, an anti-CDllb antibody, an anti-Gr-1 antibody, an anti-B220 antibody, and an anti-Ter9 antibody to this cell suspension, and add ice. It was left in the room for 30 minutes. Then, after washing twice with the staining buffer, a magnetic beads (avidin magnet beads, Perseptive) coated with avidin were added, and the mixture was left on ice for 30 minutes. After washing twice with the staining buffer again, magnet beads were collected using a magnet to remove cells expressing the differentiation antigen, and a differentiation antigen-negative cell group (LirT cells) was obtained.
  • a biotinylated antibody against the differentiation antigen marker i.e., an anti-CD4 antibody, an anti-CD8 antibody, an anti-CDllb antibody, an anti-Gr-1 antibody, an anti-B220 antibody, and an anti-Ter9 antibody to this
  • Lin add FITC-labeled anti-CD34 antibody, phycoerythrin (PE) -labeled anti-Sea-1 antibody, Texas Red-labeled avidin, and arophycocynin (APC) -labeled anti-c-KIT antibody to the cells. For 30 minutes. After washing twice with a staining buffer, hematopoietic stem cell fractions (CD34 negative to weakly positive, Sea-1 positive, C-KIT positive cells) were selected using a cell sorter (FA CSVantage, Becton Dickinson).
  • ⁇ MEM medium As a basal medium for co-culturing hematopoietic stem cells and AGM_s3, ⁇ MEM medium (GIBC0) supplemented with 10% FCS (Hyclone) was used. After seeding 5 ⁇ 10 4 AGM-s3 cells in a 48-well plate, the cells were cultured for 3 days. On this AGM-s3, 50 hematopoietic stem cells separated by a cell sorter were added 50 by 50, and cultured for 0 and 1 week.
  • the cells were washed once with PBS (GIBC0), and a trypsin solution (GIBC0) was added. After standing at 37 ° C for 5 minutes, a cell culture medium was added to collect the cells. The recovered cells were added to a 96-well plate, and Lin-cells prepared from the bone marrow of C57BL / 6N (Nippon Steel Slipper) were added to each well as rescue cells to support the short-term survival of irradiated mice. Added four . After suspending the cells in each well, the cells were transplanted from the tail vein into C57BL / 6N (Nippon Cirrus River 1) that had been subjected to lethal dose of X-ray (9.5 Gy).
  • C57BL / 6N Nippon Cirrus River 1
  • Example 5 Examination of survival or proliferation promoting activity on mouse embryonic hematopoietic stem cell fraction
  • the basic procedure of the cell fractionation method described below is described in Seaberg, LA, "Weir's Handbook of Experimental Immunology, 5th edition, Blackwell science. Inc. 1997; Seiji Takatsu, "Basic Technologies for Immune Research,” Yodosha, 1995. All antibodies used for cell separation were purchased from Pharmingen.
  • Lpep mice Male and female C57BL-Ly5. Lpep mice (aged 8 years or older) (bred to Clea Japan) were mated, and fetuses were removed from female mice 14 days after mating. The fetal liver was aseptically separated under a stereomicroscope, and the liver cells were dispersed through a 23G needle and suspended in PBS. This liver cell suspension was overlaid on an equal volume of Lymphoprep (Nycomed), and centrifuged at 1500 rpm, 20 ° C for 10 minutes to collect cells collected at the interface between Lymphoprep and the upper layer. Cell staining buffer
  • PE phycoerythrin
  • APC arophycocynin
  • Texas Red-labeled avidin to the above Lin-cells and leave on ice for 30 minutes did.
  • FACSVantage, Becton a cell sorter (FACSVantage, Becton). (Dickinson) to sort hematopoietic stem cell fractions (Sea-1 positive, c-KIT positive, differentiated antigen negative cells).
  • a basal medium for co-culturing the above hematopoietic stem cells and AGM-S3 a medium obtained by adding 10% FCS (Hyclone) to ⁇ medium (GIBC0) was used. After seeding 5 ⁇ 10 4 AGM-s3 cells in a 48-well plate, the cells were cultured for 3 days. The AGM- s3 on, was subjected to isolated hematopoietic stem cells by 10 3 added 0 and 4 days co-culture by Celso Ichita scratch. After the culture, the cells were washed once with PBS, and a trypsin solution (GIBC0) was added. After incubation at 37 ° C for 5 minutes, a cell culture medium was added to collect the cells.
  • FCS Hyclone
  • the collected cells were added to a 96-well plate, and Sea-1 positive prepared from the bone marrow of C57BL / 6N (Nippon Charles River) as rescue cells to support the short-term survival of irradiated mice.
  • C-KIT-positive, a differentiation antigen-negative cells were added 10 three in each Uniru. After suspending the cells in each well, the cells were transplanted from the tail vein into C57BL / 6N (Nippon Cirrus River) irradiated with a lethal dose of X-ray (9.5 Gy).
  • Ly5.1 antigen positive cells ie, the percentage of blood cells derived from transplanted cells, was determined by flow cytometry. It was calculated by one.
  • the results are shown in FIG. As is evident from this figure, the percentage of cells derived from cultured hematopoietic stem cells in peripheral blood of recipient mice 1 and 2 months after transplantation was higher in AGM-s3 cultured hematopoietic stem cells. -It was found to be equal to or higher than the group (Input) transplanted with hematopoietic stem cells at the start of co-culture with s3. Considering these results, it is considered that the present invention makes it possible to survive or proliferate fetal hematopoietic stem cells or hematopoietic progenitor cells. INDUSTRIAL APPLICABILITY
  • the stromal cell line of the present invention can support the proliferation or survival of hematopoietic stem cells and hematopoietic progenitor cells.
  • hematopoietic stem cells and hematopoietic progenitor cells can be grown.
  • the hematopoietic stem cells and hematopoietic progenitor cells grown by the method of the present invention can be suitably used as a transplant for blood cell transplantation and a target cell for gene therapy.

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Abstract

L'invention concerne une lignée cellulaire isolée et établie à partir d'une région foetale mammifère AGM, et capable de soutenir la prolifération ou la survie de cellules souches hématopoïétiques et de cellules parentes hématopoïétiques. L'invention concerne également des greffes contenant ces cellules souches et parentes hématopoïétiques cultivées ensemble avec la lignée cellulaire ci-dessus, et ayant donc proliféré, des compositions de thérapie génique contenant les cellules souches ou parentes hématopoïétiques cultivées ensemble avec la lignée cellulaire ci-dessus et portant un gène étranger transféré dans celles-ci. L'invention concerne encore un procédé de support de la prolifération ou de la survie de cellules souches ou parentes hématopoïétiques, consistant à faire une culture de cellules impliquant au moins des cellules souches ou parentes hématopoïétiques, ou des fractions de celles-ci, avec la lignée cellulaire ci-dessus, un procédé de transplantation de cellules souches hématopoïétiques consistant à transplanter des cellules souches ou parentes hématopoïétiques cultivées avec la lignée cellulaire ci-dessus, et ayant donc proliféré. L'invention concerne enfin un procédé de thérapie génique consistant à transférer un gène étranger dans des cellules souches ou parentes hématopoïétiques cultivées ensemble avec la lignée cellulaire ci-dessus et ayant donc proliféré, et à transplanter les cellules portant ainsi le gène qu'on leur a transféré.
PCT/JP1998/003209 1997-07-16 1998-07-16 Cellules de stroma derivees d'agm WO1999003980A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU82437/98A AU8243798A (en) 1997-07-16 1998-07-16 Agm-derived stroma cells

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP9/191216 1997-07-16
JP19121697 1997-07-16

Publications (1)

Publication Number Publication Date
WO1999003980A1 true WO1999003980A1 (fr) 1999-01-28

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PCT/JP1998/003209 WO1999003980A1 (fr) 1997-07-16 1998-07-16 Cellules de stroma derivees d'agm

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AU (1) AU8243798A (fr)
WO (1) WO1999003980A1 (fr)

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JP2002537849A (ja) * 1999-03-10 2002-11-12 ユニヴァーシティ オヴ ピッツバーグ オヴ ザ コモンウェルス システム オヴ ハイアー エデュケーション 脂肪由来の幹細胞および格子
WO2002100898A2 (fr) * 2001-06-11 2002-12-19 Kirin Beer Kabushiki Kaisha Polypeptide a activite entretenant la proliferation ou la survie des cellules souches hematopoietiques et des progeniteurs hematopoietiques, et adn codant ce polypeptide
WO2003014336A1 (fr) * 2001-08-07 2003-02-20 Kirin Beer Kabushiki Kaisha Procede de preparation de cellules souches hematopoietiques multipotentes
WO2003091280A1 (fr) * 2002-04-26 2003-11-06 Kirin Beer Kabushiki Kaisha Polypeptide a activite entretenant la proliferation ou la survie de cellules souches hematopoietiques ou de progeniteurs hematopoietiques, et adn codant ce polypeptide
WO2007040123A1 (fr) * 2005-09-30 2007-04-12 Nihon University Méthode pour détecter un précurseur de lymphocyte t et un précurseur de lymphocyte b humains
WO2012074106A1 (fr) * 2010-12-03 2012-06-07 国立大学法人京都大学 Procédé de production d'éosinophiles à partir de cellules souches pluripotentes
US8486700B2 (en) 1999-08-19 2013-07-16 Artecel Sciences Inc. Multiple mesodermal lineage differentiation potentials for adipose tissue-derived stromal cells and uses thereof

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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002537849A (ja) * 1999-03-10 2002-11-12 ユニヴァーシティ オヴ ピッツバーグ オヴ ザ コモンウェルス システム オヴ ハイアー エデュケーション 脂肪由来の幹細胞および格子
JP2015109834A (ja) * 1999-03-10 2015-06-18 ユニヴァーシティ オヴ ピッツバーグ オヴ ザ コモンウェルス システム オヴ ハイアー エデュケーション 脂肪由来の幹細胞および格子
US8486700B2 (en) 1999-08-19 2013-07-16 Artecel Sciences Inc. Multiple mesodermal lineage differentiation potentials for adipose tissue-derived stromal cells and uses thereof
US7320880B2 (en) 2001-06-11 2008-01-22 Nuvelo, Inc. Polypeptide having an activity to support proliferation or survival of hematopoietic stem cell and hematopoietic progenitor cell, and DNA coding for the same
WO2002100898A3 (fr) * 2001-06-11 2003-05-30 Kirin Brewery Polypeptide a activite entretenant la proliferation ou la survie des cellules souches hematopoietiques et des progeniteurs hematopoietiques, et adn codant ce polypeptide
WO2002100898A2 (fr) * 2001-06-11 2002-12-19 Kirin Beer Kabushiki Kaisha Polypeptide a activite entretenant la proliferation ou la survie des cellules souches hematopoietiques et des progeniteurs hematopoietiques, et adn codant ce polypeptide
WO2003014336A1 (fr) * 2001-08-07 2003-02-20 Kirin Beer Kabushiki Kaisha Procede de preparation de cellules souches hematopoietiques multipotentes
WO2003091280A1 (fr) * 2002-04-26 2003-11-06 Kirin Beer Kabushiki Kaisha Polypeptide a activite entretenant la proliferation ou la survie de cellules souches hematopoietiques ou de progeniteurs hematopoietiques, et adn codant ce polypeptide
US7439332B2 (en) 2002-04-26 2008-10-21 Kirin Pharma Kabushiki Kaisha Polypeptide having an activity to support proliferation or survival of hematopoietic stem or progenitor cells
WO2007040123A1 (fr) * 2005-09-30 2007-04-12 Nihon University Méthode pour détecter un précurseur de lymphocyte t et un précurseur de lymphocyte b humains
JPWO2007040123A1 (ja) * 2005-09-30 2009-04-16 学校法人日本大学 ヒトｔ前駆細胞およびｂ前駆細胞の検出方法
WO2012074106A1 (fr) * 2010-12-03 2012-06-07 国立大学法人京都大学 Procédé de production d'éosinophiles à partir de cellules souches pluripotentes
US9404082B2 (en) 2010-12-03 2016-08-02 Kyoto University Method for production of eosinophil from pluripotent stem cell
JP5995237B2 (ja) * 2010-12-03 2016-09-21 国立大学法人京都大学 多能性幹細胞からの好酸球の製造方法

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