WO2011071085A1 - Procédé pour produire des cellules induites pour être différenciées à partir de cellules souches pluripotentes - Google Patents

Procédé pour produire des cellules induites pour être différenciées à partir de cellules souches pluripotentes Download PDF

Info

Publication number
WO2011071085A1
WO2011071085A1 PCT/JP2010/072044 JP2010072044W WO2011071085A1 WO 2011071085 A1 WO2011071085 A1 WO 2011071085A1 JP 2010072044 W JP2010072044 W JP 2010072044W WO 2011071085 A1 WO2011071085 A1 WO 2011071085A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
cell
pluripotent stem
derived
ips
Prior art date
Application number
PCT/JP2010/072044
Other languages
English (en)
Japanese (ja)
Inventor
啓光 中内
聡英 紙谷
奈穂 鈴木
慶一 伊藤
聡 山▲崎▼
Original Assignee
国立大学法人東京大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 国立大学法人東京大学 filed Critical 国立大学法人東京大学
Priority to JP2011545228A priority Critical patent/JP5896457B2/ja
Publication of WO2011071085A1 publication Critical patent/WO2011071085A1/fr

Links

Images

Classifications

    • 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
    • C12N5/067—Hepatocytes
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous 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
    • 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
    • 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
    • C12N5/0676—Pancreatic cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/12—Animals modified by administration of exogenous cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0331—Animal model for proliferative diseases
    • 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
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
    • C12N2502/1394—Bone marrow stromal cells; whole marrow
    • 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
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic 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
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells

Definitions

  • the present invention relates to a method for producing target cells differentiated from pluripotent stem cells, and more specifically, a step of administering a differentiation inducer to target cells to the non-human mammal, and transplanted pluripotent stem cells Growing the animal for a period of time sufficient to form teratomas in the living body of the non-human mammal and inducing differentiation of the pluripotent stem cells into target cells, and from the non-human mammal to the mammal And a method for producing a target cell induced to differentiate from a pluripotent stem cell, and a target cell obtained by the production method.
  • Stem cells are undifferentiated cells that have both self-renewal ability and pluripotency.
  • ES cells and iPS cells form teratomas (benign tumors) when administered to a living body, including three germ layers (digestive tract, liver, pancreas, bladder, lung, tonsils, pharynx, parathyroid gland, etc.) Including mesoderm system, nerve cells and skin cells, inner ear, eyes, mammary gland, nails, teeth, spinal cord and brain, such as germ layer, blood cells and muscle cells, bone cells, heart, gonadal, urinary system, fat, spleen Because it contains fetal tissues and mature tissue structures derived from the ectoderm system, which becomes the nervous system, etc., it must be versatile enough to differentiate into all cell lines including germ cells in vivo. Are known (Non-Patent Documents 1 to 4).
  • HSC transplantable hematopoietic stem cells
  • HoxB4 transplantable hematopoietic stem cells
  • the present invention has been made in view of the above-described problems of the prior art, and is capable of efficiently inducing differentiation of pluripotent stem cells into desired functional cells, and an object of differentiation induction from pluripotent stem cells. It aims at providing the production method of a cell.
  • the present inventors utilize the teratoma-forming ability of pluripotent stem cells and control it in vivo, so that the desired functional cells and tissues, A method for creating an organ has been found and the present invention has been completed.
  • the target differentiated cells derived from the iPS cells are formed in the formed teratoma or in the mouse body.
  • transplanting the differentiated cells induced in this way to a dysfunctional animal restores its function, and it is clear that the induced differentiated cells have almost the same function as the original cells. did.
  • pluripotent stem cells are administered to various treated individuals, such as organ-deficient animals or immunodeficient animals, so that the target cells (including target tissues and target organs) can be more efficiently and transspecies. It also showed that it becomes possible to obtain.
  • pluripotent stem cells that have been induced to differentiate to some extent from universal cells such as ES cells and iPS cells, a teratoma containing more target cells and the like can be formed.
  • the inventors have also found that the influence can be suppressed, and have completed the present invention.
  • the present invention provides the following inventions. (1) transplanting pluripotent stem cells derived from a mammal individual to a non-human mammal; Administering to the non-human mammal a differentiation-inducing agent for target cells; Growing the animal for a time sufficient for the transplanted pluripotent stem cells to form teratomas in the living body of the non-human mammal, and inducing differentiation of the pluripotent stem cells into target cells; Recovering a target cell derived from the mammal individual from the non-human mammal; A method for producing a target cell induced to differentiate from a pluripotent stem cell.
  • the pluripotent stem cell is an induced pluripotent stem (iPS) cell prepared using a somatic cell of the mammal individual.
  • the pluripotent stem cell is an embryonic stem (ES) cell prepared from a fertilized egg derived from the mammal.
  • the pluripotent stem cell is transplanted into at least one tissue selected from the group consisting of subcutaneous, testis, and renal capsule of the non-human mammal (1) to (3) The method described in 1.
  • pancreatic cells are pancreatic Langerhans islet cells.
  • the target cell is a hematopoietic cell, and the hematopoietic cell is recovered from the bone marrow of the non-human mammal.
  • the pluripotent stem cell is a Lnk-deficient cell.
  • the step of inducing differentiation of the pluripotent stem cell into a target cell is performed in the presence of co-cultured cells.
  • the co-cultured cells are OP-9 cells.
  • the present invention has made it possible to efficiently induce differentiation of target cells from pluripotent stem cells. Moreover, unlike in vitro differentiation-inducing systems, differentiation induction into cells having almost the same functions as the original cells (for example, cells capable of restoring the functions when transplanted into dysfunctional animals). Became possible.
  • Murine iPS cells are transplanted into nude mice, differentiation inducers such as cytokines are administered to the nude mice, and teratomas are formed in the living body of the nude mice, differentiation induced hepatocytes, pancreatic cells, or hematopoietic system
  • differentiation inducers such as cytokines
  • teratomas are formed in the living body of the nude mice, differentiation induced hepatocytes, pancreatic cells, or hematopoietic system
  • FIG. 5 is a plot diagram showing that ⁇ Kit + Sca-1 + (KSL) exists.
  • Schematic diagram showing transplantation of mouse-derived bone marrow cells in which teratoma is formed into wild type C57 / BL6 mice, and by peripheral cytoplasm in wild type C57 / BL6 mice 4 weeks after transplantation by flow cytometer analysis.
  • FIG. 1 It is a plot figure which shows that the various hematopoietic system cell derived from an iPS cell exists.
  • a vector encoding a suicide gene (TK gene) used to remove cells other than target cells (hepatocytes) mixed in the teratoma, and Cre under the control of the Alb promoter which is a marker gene for hepatocytes.
  • TK gene suicide gene
  • It is the microscope picture of a cell line which shows that the screening system by a suicide gene is effective in removal of cells other than the target cell mixed in the teratoma.
  • FIG. 2 is a schematic diagram showing the process of establishing iPS cells from Lnk ⁇ / ⁇ GFP transgenic (Tg) mice or GFP Tg mice. It is a microscope picture which shows the result of the immunostaining of Nanog and SSEA-1 in a Lnk ⁇ / ⁇ GFP iPS cell. In the figure, the scale bar indicates 100 ⁇ m. It is a photograph showing a chimeric mouse prepared from Lnk ⁇ / ⁇ GFP iPS cells.
  • FIG. 5 is a photograph showing nude mice in which teratomas derived from Lnk ⁇ / ⁇ GFP iPS cells are formed.
  • FIG. 2 is a photograph showing teratomas derived from Lnk ⁇ / ⁇ GFP iPS cells collected from nude mice.
  • the scale bar indicates 1.25 cm.
  • HSC hematopoietic stem cell
  • FIG. 1 It is a graph which shows the ratio of the GFP + cell derived from iPS cell in the peripheral blood and the bone marrow of the mouse
  • 12 weeks after iPS cell injection 5 teratoma-forming mice under each condition were analyzed.
  • the vertical axis on the left indicates the ratio of GFP + cells in CD45 + cells (GFP + / CD45 + cells (%)) in the peripheral blood of teratoma-forming nude mice.
  • the right vertical axis shows the ratio of GFP + cells in KSL cells (GFP + / KSLcells (%)) in the bone marrow of teratoma-forming nude mice.
  • the left side shows the result of analyzing peripheral blood
  • the right side shows the result of analyzing bone marrow.
  • Error bars represent standard deviation. Those marked with three asterisks (***) indicate a significantly different value (p ⁇ 0.001) compared to untreated (None, iPS cells only) injected samples.
  • FIG. 2 is a photomicrograph showing the results of analyzing colonies derived from iPS cells formed by dividing GFP + CD34 ⁇ KSL cells derived from teratoma-forming mouse bone marrow cells one by one and adding a cytokine that induces hematopoietic differentiation.
  • the left side shows the result of phase difference observation
  • the right side shows the result of fluorescence observation.
  • a graph showing the results of evaluation of the quantity and type of CFC-nmEM by dividing 100 GFP + CD34 ⁇ KSL cells derived from teratoma-forming mouse bone marrow one by one, adding a cytokine that induces hematopoietic differentiation, and culturing for 10 days. is there.
  • CFC-nmEM indicates the number of colony forming cells-neutrophil / macrophage / erythroblast / megakaryocyte (colony-forming units-neutrophil / macrophage / erythroblast / megakaryocyte).
  • “nm” on the horizontal axis represents a colony composed of two strains of neutrophil / macrophage
  • “nmM” represents a colony composed of three strains of neutrophil / macrophage / megakaryocyte
  • “nmE” represents neutrophil.
  • NmEM indicates a colony consisting of 4 strains of neutrophil / macrophage / erythroblast / megakaryocyte.
  • FIG. 6 is a micrograph of a cytospin sample of a CD34 ⁇ KSL cell-derived colony observed by leuco staining (Leukostein).
  • GM represents granulocytes / macrophages
  • Meg represents megakaryocytes
  • E represents erythroblasts.
  • It is a graph which shows the result of a bone marrow transplantation assay. That is, GFP derived from Lnk ⁇ / ⁇ GFP iPS cells in the peripheral blood of a primary mouse transplanted with bone marrow cells derived from teratoma-forming mice into irradiated mice and a secondary transplant recipient mouse 12 weeks after the primary transplantation.
  • HPC hematopoietic progenitor cells
  • HSC hematopoietic stem cells
  • HSC hematopoietic stem cells
  • HPC hematopoietic progenitor cells
  • HSC hematopoietic stem cells
  • results were obtained by dividing 100 GFP + CD34 ⁇ KSL cells derived from teratoma-forming mouse bone marrow one by one, adding cytokines that induce hematopoietic differentiation, culturing, and culturing for 10 days to evaluate the quantity and type of CFC-nmEM. It is a graph to show.
  • CFC-nmEM indicates the number of colony forming cells-neutrophil / macrophage / erythroblast / megakaryocyte (colony-forming units-neutrophil / macrophage / erythroblast / megakaryocyte).
  • “nm” on the horizontal axis represents a colony composed of two strains of neutrophil / macrophage
  • “nmM” represents a colony composed of three strains of neutrophil / macrophage / megakaryocyte
  • “nmE” represents neutrophil.
  • NmEM indicates a colony consisting of 4 strains of neutrophil / macrophage / erythroblast / megakaryocyte.
  • FIG. 3 is a schematic diagram showing a method for producing m ⁇ c-iPS cells into T cells in X-SCID mice.
  • iPS cells were established from X-SCID mice, and m ⁇ c gene was introduced into the iPS cells to prepare m ⁇ c-iPS cells. Then, m ⁇ c-iPS cells and OP9 cells were injected into X-SCID mice for teratoma formation. It is an electrophoresis photograph showing the PCR analysis result of m ⁇ c-iPS cells.
  • m ⁇ c-iPS cells (m ⁇ c-iPSC) # 1 to 5 are obtained by introducing 3 factors (Oct3 / 4, Klf4, and Sox2) into cells derived from X-SCID mice having IL-2Rg mutation.
  • Cell. 4F B6 iPS cells (4F B6 iPS iPSC) are cells having a c-Myc transgene and having no common gamma chain ( ⁇ c) mutation. Pure water (Water) represents a negative control.
  • 2 is an electrophoresis photograph showing the results of analyzing the expression of an ES cell marker gene in m ⁇ c-iPS cells by RT-PCR. Pure water (Water) and MEF represent negative controls.
  • ES cells (ESC) represent a positive control.
  • FIG. 4 is a plot and histogram showing expression of GFP and mouse gamma chain ( ⁇ c) in m ⁇ c-iPS cell # 4 (m ⁇ c-iPSC # 4).
  • B6 iPS cells (B6 iPSC) represent a negative control.
  • the filled histogram on the right side shows the results for m ⁇ c-iPSC # 4, and the white histogram on the left side shows the results for the negative control (B6 iPS cells).
  • the numerical value indicates the ratio (%) of GFP + cells in each cell.
  • M indicates that it is derived from a mouse
  • h indicates that it is derived from a human.
  • It is a microscope picture which shows the result of having investigated the expression of CD45, CD34, and osteocalcin in the teratoma section derived from a human iPS cell by immunostaining.
  • “Merge” indicates a superposition of these three expressions.
  • 2 is a photomicrograph showing the results of examining the expression of CD45, CD34, and VE-cadherin by immunostaining in teratoma sections derived from human iPS cells.
  • “Merge” indicates a superposition of these three expressions.
  • DAPI + merge shows the expression of CD45 and GFP and the result of DAPI staining superimposed, and the arrow shows GFP + CD45 + cells derived from GFP iPS cells.
  • the scale bar indicates 150 ⁇ m.
  • FIG. 2 is a photomicrograph showing the results of immunostaining of CD45, c-Kit, and HSC niche marker: VE-cadherin in teratoma.
  • “DAPI + merge” indicates a superposition of these three expressions and the results of DAPI staining, and the arrow indicates CD45 + c-Kit + cells.
  • the scale bar indicates 75 ⁇ m. It is the photograph which shows the teratoma formed in the mouse
  • the present invention (A) transplanting pluripotent stem cells derived from a mammal individual to a non-human mammal; (B) administering to the non-human mammal an agent for inducing differentiation into a target cell; (C) growing the animal for a time sufficient for the transplanted pluripotent stem cells to form a teratoma in the living body of the non-human mammal, and inducing differentiation of the pluripotent stem cells into target cells; (D) recovering a target cell derived from the mammal individual from the non-human mammal; A method for producing target cells induced to differentiate from pluripotent stem cells.
  • the “mammal” is not particularly limited, and examples thereof include humans, mice, rats, cows, horses, pigs, sheep, monkeys, dogs, and cats.
  • the “non-human mammal” is not particularly limited, and examples thereof include mice, rats, cows, horses, pigs, sheep, monkeys, dogs, and cats.
  • the “target cell” according to the present invention is one or a plurality of cells selected from various cells constituting the mammal individual, for example, hematopoietic cells, hepatocytes, pancreas Examples include cells, intestinal cells, thymocytes, and bone / chondrocytes.
  • the “target cell” according to the present invention includes various cell groups (for example, tissues and organs) constituting the mammal individual.
  • hematopoietic cell means a hematopoietic stem cell (HSC), a hematopoietic progenitor cell (HPC), and a cell obtained by differentiation from HSC or HPC, that is, erythroblast, myeloid cell, megakaryocyte system, lymphocyte. And the like, and examples thereof include HSC, HPC, neutrophils, macrophages, erythroblasts, and megakaryocytes.
  • pancreatic cell means a cell constituting the pancreas, that is, an exocrine cell constituting the exocrine part, a Langerhans islet cell constituting the Langerhans islet (endocrine part), and the like, for example, an acinar cell, A Examples include cells ( ⁇ cells), B cells ( ⁇ cells), D cells ( ⁇ cells), and PP cells.
  • the “pluripotent stem cell” to be differentiated in the present invention is a cell having pluripotency capable of differentiating into various cells constituting the mammal individual and self-replicating ability, for example, an embryonic stem cell (Embryonic stem cells (ES cells), induced pluripotent stem cells (Induced pluripotent stem cells, iPS cells), embryonic tumor cells (Embryonic carcinoma cells, EC cells), embryonic germ cells (EmbrycellG cells) Universal cells such as multipotent germline stem cell (mGS cell), fertilized egg, inner cell mass (inner cell mass, ICM), ectoderm progenitor cells, endoderm progenitor cells, mesoderm
  • mGS cell multipotent germline stem cell
  • ICM inner cell mass
  • ectoderm progenitor cells endoderm progenitor cells
  • mesoderm mesoderm
  • progenitor cells, differentiable cell series include stem cells / progenitor cells are limited.
  • iPS cells prepared using somatic cells of the individual mammal as the pluripotent stem cells according to the present invention.
  • the “iPS cell” is a cell that has acquired ES cell (Embryonic stem cells) -like differentiation pluripotency by introducing a cell reprogramming factor into the somatic cell of the individual mammal.
  • the “cell reprogramming factor” used in the present invention when introduced into a somatic cell, imparts pluripotency to the somatic cell alone or in cooperation with other pluripotent factors. It can be any factor that can be used, and is not particularly limited.
  • Oct3 / 4 c-Myc, Sox2, Klf4, Klf5, LIN28, Nanog, ECAT1, ESG1, Fbx15, Eras, ECAT7, ECAT8, Gdf3, Sox15
  • It is preferably at least one protein selected from the group consisting of ECAT15-1, ECAT15-2, Fthl17, Sal14, Rex1, Utf1, Tcl1, Stella, ⁇ -catenin, Stat3 and Grb2.
  • Oct3 / 4, Sox2 and Klf4 (3 factors) excluding c-Myc into the somatic cells.
  • pluripotent stem cell for example, the stem cell / progenitor cell in which the differentiable cell line is limited
  • pluripotent stem cells are seeded in a culture dish coated with Collagen-Type4 as shown in the Examples below, and activin A, BMP4, etc. And then culturing in a serum-free medium supplemented with activin A, EGF, FGF4, and the like.
  • Non-Patent Documents 5 to 8 for example, it is generally difficult to induce differentiation of pluripotent stem cells into functional cells without genetic modification.
  • functional cells induced to differentiate can be efficiently obtained even using pluripotent stem cells that are not genetically modified.
  • pluripotent stem cells that have been genetically modified can also be used as pluripotent stem cells according to the present invention from the viewpoint of increasing the number of target cells produced and enhancing the function of the target cells.
  • genetic modification is not particularly limited, and known techniques can be appropriately employed. For example, in the case of enhancing the function or expression of a protein that induces an increase in the number of target cells produced or enhancement of function, a method of introducing a gene encoding the protein into a pluripotent stem cell as a transgene, And a method for introducing into the pluripotent stem cell a mutation that enhances the expression of the gene in the DNA sequence of the expression control region of the gene.
  • a method of knocking out or knocking in a gene encoding the protein in pluripotent stem cells for example, a method of knocking out or knocking in a gene encoding the protein in pluripotent stem cells.
  • a method of introducing a mutation that suppresses the expression of the gene into the DNA sequence of the gene expression regulatory region, or a transgene encoding siRNA or shRNA for the gene is introduced into the pluripotent stem cell. A method is mentioned.
  • the target cell is a hematopoietic cell
  • a mouse lacking the Lnk protein overproduces a hematopoietic stem cell (HSC) having a high self-renewal capacity, and the Lnk protein is deficient as shown in Examples described later.
  • HSC hematopoietic stem cell
  • the pluripotent stem cell-derived HSC has a very high replication ability, it is preferable to use Lnk-deficient cells as the pluripotent stem cells according to the present invention.
  • the Lnk protein is an intercellular adapter protein that is known as a factor that negatively regulates the TPO / c-mpl pathway in HSC and the like.
  • the protein (gene) specified by NP_005466 (No. NM_005474) is mentioned, and Access No. Examples include proteins (genes) specified by NP_032533 (No. NM_008507).
  • the “Lnk deficient cell” is a cell deficient in the expression and / or function of the Lnk protein, and the “Lnk deficient cell” is, for example, as described above, knocking out the Lnk gene, It can be prepared by introducing siRNA against the Lnk gene.
  • a method for transplanting the pluripotent stem cells into a non-human mammal is not particularly limited, and a known technique can be appropriately used.
  • the tissue for transplanting the pluripotent stem cells into a non-human mammal is not particularly limited, and examples thereof include subcutaneous, testis, renal capsule, and bone marrow. Among these, it is preferable to transplant subcutaneously from the viewpoint that it is frequently used as a place where teratomas derived from mouse iPS cells and the like are formed. In addition, when transplanting human iPS cells into a non-human mammal, the frequency of teratoma formation is low even when injected into mice or the like subcutaneously, and from the viewpoint that it is a tissue with more blood flow, It is preferable to transplant to the testis.
  • transplantation into bone marrow is preferable. Furthermore, from the viewpoint that the donor-derived tissue and the recipient tissue are not easily mixed, it is preferable to transplant to a tissue (ectopic) other than the tissue in which the target cell exists.
  • the pluripotent stem cells when the pluripotent stem cells are transplanted into a non-human mammal, the pluripotent stem cells may be used by mixing with other components.
  • Such other components are not particularly limited, and include physiological saline such as phosphate buffered saline (PBS), medium, buffer, and preservative.
  • PBS phosphate buffered saline
  • co-cultured cell means a cell that can be used in a culture system for pluripotent stem cells to prepare culture conditions for inducing proliferation or differentiation of pluripotent stem cells.
  • Cells stromal cells, and more specifically OP-9 cells, PA6 cells, MEF (mouse fetal fibroblasts), NIH3T3 cells, M15 cells, and 10T / 2 cells.
  • the “co-culture cells” according to the present invention can increase the induction efficiency of the pluripotent stem cells to the target cells by mixing with the pluripotent stem cells.
  • OP-9 cells are preferably used.
  • TGF- at least selected from the group consisting of ⁇ , epidermal growth factor, insulin-like growth factor, fibroblast growth factor, tissue plasminogen activator 3, 4 and other growth factors naturally produced in EHS tumors
  • One component is preferably mixed in the pluripotent stem cell.
  • the non-human mammal to which the pluripotent stem cells are transplanted is not particularly limited, and includes the aforementioned mice, etc., but a space for inducing target cells derived from pluripotent stem cells is secured.
  • an animal lacking the ability to form target cells is preferable.
  • the target cell differentiated from the pluripotent stem cell is produced. From the viewpoint of being able to do so, it is preferably an immunodeficient animal.
  • the "differentiation inducer to target cells" administered to the non-human mammal is not particularly limited as long as it is a physiologically active substance that works when differentiation induction from pluripotent stem cells to target cells is performed, Examples thereof include polypeptides and proteins (cytokines, hormones, enzymes, etc.), vitamins, saccharides, fatty acids, amino acids, nucleic acids, minerals and the like.
  • the “differentiation inducer to target cells” is more specifically, when the target cells are hepatocytes, retinoic acid, fibroblast growth factor 1 (FGF1), FGF4, hepatocyte proliferation Factor (HGF), oncostatin M (OsM), and when the target cell is a pancreatic cell, activin A, epidermal growth factor (EGF), Noggin, insulin-like growth factor-2 (IGF-2), When nicotinamide is used and the target cell is a hematopoietic cell, stem cell factor (SCF) and thrombopoietin (TPO) are used. When the target cell is an enteric cell, activin A and bone morphogenetic factor 4 ( BMP4), EGF, FGF4, NOGGIN.
  • BMP4 bone morphogenetic factor 4
  • pancreatic Langerhans islet cells in the differentiation induction of pancreatic Langerhans islet cells, first, activin A or the like is added to iPS cells to induce differentiation into endoderm progenitor cells, and EGF, After inducing endoderm cells committed to the pancreas by adding bFGF and Noggin, nicotinamide or IGF-2 is added to the endoderm cells to induce differentiation of the pancreas into Langerhans islet cells.
  • the differentiation stage at which the “differentiation inducer” acts varies depending on the type of differentiation inducer.
  • the predetermined period for administering the differentiation-inducing agent to the non-human mammal is a period sufficient for each differentiation-inducing agent to act and induce the desired differentiation.
  • Each differentiation-inducing agent may be administered to cells transiently or continuously as long as each target differentiation can be achieved. From the viewpoint of mimicking the development of an original organ, it is preferable to administer the differentiation inducer continuously.
  • the site for administering the differentiation-inducing agent in the non-human mammal is not particularly limited, but is preferably subcutaneous from the viewpoint that the procedure required for administration is simple.
  • the method for administering the differentiation inducer to the non-human mammal is not particularly limited, and a known method (for example, a method using an osmotic pump) can be appropriately selected and used.
  • the transplanted pluripotent stem cells are allowed to grow for a sufficient period of time to form teratomas in the living body of the non-human mammal, and the pluripotent stem cells are induced to differentiate into target cells.
  • the process will be described.
  • a sufficient time for the transplanted pluripotent stem cells to form teratomas in the living body of the non-human mammal is preferably 4 to 12 weeks after the transplantation, More preferably, it is 8-12 weeks. If the time is less than the lower limit, the transplanted pluripotent stem cells tend not to be sufficiently induced to differentiate into target cells. On the other hand, if the time exceeds the upper limit, the health of the non-human mammal due to teratoma increase It tends to adversely affect the condition.
  • the step of inducing differentiation of the pluripotent stem cell derived from the animal individual according to the present invention into the target cell cells other than the target cell to be mixed are removed from the viewpoint of efficiently inducing differentiation of only the target cell. It is preferable to perform the operation to do.
  • the “cell other than the target cell” is not particularly limited.
  • a pluripotent stem cell undifferentiated cell
  • Cells derived from pluripotent stem cells that have differentiated into other cells are derived from pluripotent stem cells that have differentiated into other cell lineages).
  • the operation for removing cells other than the target cells to be mixed is not particularly limited.
  • a “suicide gene” used to cause a suicide gene to function in cells other than the target cell is cell death (apoptosis) in cells expressing the protein under conditions that allow the protein encoded by the gene to function.
  • cell death apoptosis
  • Necrosis etc.
  • TK thymidine kinase
  • HSVtk herpesvirus thymidine kinase
  • cytosine deaminase gene EG11326 codA 355395.356678 E.
  • coli uracil phosphoribosyltransferase gene (EG11332 up 26618894.26618268 E.c) li), guanine phosphoribosyl transferase (gpt) gene (EG10414 gpt 255977..256435 E.coli), a nitro reductase gene.
  • gpt guanine phosphoribosyl transferase
  • TK is a microorganism-derived metabolic enzyme gene, which metabolizes ganciclovir (GCV) to produce ganciclovir 5'-triphosphate.
  • GCV ganciclovir
  • This ganciclovir 5'-triphosphate inhibits DNA synthesis, thereby inducing cell death of cells expressing TK. That is, by adding GCV, the condition is such that TK can function, and cell death is induced in cells in which TK is expressed.
  • the method of ⁇ working the suicide gene '' there is no particular limitation as to the method of ⁇ working the suicide gene '', and any method can be used as long as the protein encoded by the suicide gene can function.
  • Examples include a method in which a TK gene is introduced into a pluripotent stem cell and the TK gene is removed only by a cell differentiated into a target cell by a Cre-LoxP system, as shown in the Examples described later. More specifically, when the target cell is a hepatocyte, as shown in FIG. 9, a viral vector in which an HSV-derived TK gene is sandwiched between LoxP sequences, and control of an Alb promoter that is a marker gene for hepatocytes.
  • a viral vector expressing Cre recombinase is prepared below. After the cells are infected with the two viruses, differentiation induction into hepatocytes is performed in the present invention. Thereafter, by administering GCV, only Alb-producing cells from which the TK gene has been removed by the expression of Cre can survive, and cell death of undifferentiated cells or cells differentiated into other cell lineages can be induced. In the same manner as described above, even when the target cell is a cell other than a hepatocyte, by selecting a promoter specific to the target cell (for example, when a pancreatic lineage cell is used as the target cell). By selecting the Pdxl promoter, cells other than the target cell can be removed.
  • the pluripotent stem cells can be used as promoters of undifferentiated marker genes (Nanog, Oct3 / 4 gene, etc.).
  • undifferentiated marker genes Nag, Oct3 / 4 gene, etc.
  • TK which is a suicide gene, has been introduced, and inducing differentiation of the pluripotent stem cells into target cells according to the present invention, then selectively removing only undifferentiated cells by administering GCV. Can do.
  • the method for recovering the target cells derived from the individual mammal is not particularly limited, and examples thereof include a method for recovering from the teratoma formed in the living body of the non-human mammal.
  • the target cell is a hematopoietic cell, as shown in the examples below, the hematopoietic cell induced to differentiate by the method of the present invention surprisingly moves from the teratoma and engrafts in the bone marrow. It can also be recovered from the bone marrow of the non-human mammal.
  • the present invention also provides target cells obtained by the above-described method. That is, the present invention also provides one or a plurality of cells selected from various cells constituting the mammalian individual, which are induced to differentiate from pluripotent stem cells by the method described above. Furthermore, a tissue or organ composed of one or a plurality of cells selected from various cells constituting the mammal individual is also provided.
  • target cells are not particularly limited, and examples thereof include hematopoietic cells, hepatocytes, pancreatic cells, intestinal cells, thymocytes, and bone / chondrocytes.
  • ⁇ Cell> As mouse iPS cells, three genes of Oct3 / 4, Sox2 and Klf4 were introduced into the tail tip fibroblasts (TTF, tail tip fibroblast) of Kusterrorism Orange transgenic mice (129 / Sv mice derived) using a retroviral vector. Established and Lnk knockout mice (derived from C57BL / 6 mice) were used by introducing the above 3 genes with a lentiviral vector. In addition, it was confirmed before the experiment that both the cell lines can produce chimeric mice as blastocyst hosts for wild-type mice.
  • E14.1 KSR medium Mouse iPS cells were cultured in E14.1 KSR medium by co-culture with mouse fetal fibroblasts (MEF) treated with mitomycin C.
  • the composition of the E14.1 KSR medium is as follows. Dulbecco's modified Eagle medium (DMEM, manufactured by Invitrogen), 15% knockout serum substitute (KSR, manufactured by Invitrogen) as an additive, 2 mM L-glutamine-penicillin-streptomycin (manufactured by Invitrogen), 1 ⁇ non-essential amino acid ( 1 mM HEPES (manufactured by Invitrogen), 0.1 mM 2-mercaptoethanol (manufactured by Gibco), 1000 IU / ml leukemia inhibitory factor (Leukemia Inhibitory Factor, LIF).
  • DMEM Dulbecco's modified Eagle medium
  • KSR knockout serum substitute
  • 2 mM L-glutamine-penicillin-streptomycin manufactured by In
  • ⁇ Teratoma formation and differentiation into target cells Mouse iPS cells were peeled off from the dish by trypsin treatment and suspended in PBS at about 5 ⁇ 10 6 cells / 50 ⁇ L and injected subcutaneously into KSN / Slc-nu / nu mice. The day of iPS cell injection was set to “day 1”, and cytokine administration was started on the same day. A total amount of 100 ⁇ L of cytokine was placed in alzet micro-osmotic pump model 1002 (manufactured by DURECT Corporation), and a pump was implanted subcutaneously into the mouse.
  • alzet micro-osmotic pump model 1002 manufactured by DURECT Corporation
  • cytokine type, dose, and administration timing are shown in FIG.
  • KSN / Slc-nu / nu indicates a nude mouse in the KSN background
  • the names of cytokines and the like injected into the mouse are as follows.
  • RA Retinoic Acid (retinoic acid)
  • FGF1 Fibroblast Growth Factor 1 (fibroblast growth factor 1)
  • FGF4 Fibroblast Growth Factor 4 (fibroblast growth factor 4)
  • HGF Hepatocyte Growth Factor (Hepatocyte Growth Factor)
  • OsM Oncostatin M (Oncostatin M, a cytokine belonging to leukemia inhibitory factor, has multiple functions)
  • Activin A Activin A
  • EGF Epidermal Growth Factor (epidermal growth factor)
  • bFGF basic FGF (basic fibroblast growth factor) Nicotinamide (Nicotinamide)
  • IGF-2 Inslin Like Growth Factor-2 (insulin-like growth factor-2)
  • OP9 Mouse bone marrow stromal cell line
  • SCF Stem Cell Factor (stem cell factor)
  • TPO Thrombopoietin (thrombopoietin, a hematopoietic factor involved in the proliferation and
  • C. T.A A frozen section was prepared by embedding with a compound (manufactured by Tissue-TeK). Sections were fixed with 4% paraformaldehyde (PFA), treated with acetone, blocked with MAXBlock Blocking Medium (registered trademark, manufactured by Active Motif) for 1 hour at room temperature, washed twice with PBS and applied with primary antibody. The reaction was allowed to proceed overnight at 4 ° C.
  • PFA paraformaldehyde
  • MAXBlock Blocking Medium registered trademark, manufactured by Active Motif
  • Primary antibodies include goat anti-mouse ALB Ab, Rabbit anti-mouse CK19 Ab (manufactured by Invitrogen), Rat anti-mouse CYP7A1 Ab (manufactured by Santa Cruz), mouse anti-mouseIns It was. Next, after washing 3 times with PBS, the reaction was allowed to proceed with the secondary antibody for 1 hour at room temperature. As secondary antibodies, donkey anti-goat IgG Alexa 647, goat anti-rabbit IgG Alexa 488, goat anti-rat IgG Alexa 488, and goat anti-mouse IgG Alexa 488 (manufactured by Invitrogen) were used.
  • Indocyanine green adsorption reaction Indocyanine green (Indocyanine green, manufactured by Sigma) was dissolved in 5 mg / ml DMSO (manufactured by Sigma) and prepared with PBS to 1 mg / ml. 500 ⁇ L of this solution was intravenously injected into mice, and after 30 minutes, the teratomas and liver were excised and the color was observed. Alternatively, the teratoma and liver excised from the mouse were immersed in an indocyanine green solution, and the color was observed after incubation at 37 ° C. for 30 minutes.
  • Flow cytometry analysis> For determination of blood cell differentiation ability, peripheral blood and bone marrow cells were analyzed by flow cytometry (FACS Aria).
  • the antibodies used were anti-mouse CD45-APC, anti-mouse CD4, CD8, Gr-1, Mac-1, B220, IL-7R-Biotin, anti-Streptavidin-APC / Cy7, anti-mouse Sca-1- Pacific Blue, anti-mouse c-Kit-APC, anti-mouse CD3-PE / Cy5, anti-mouse B220- Pacific Blue, anti-mouse Gr-1-APC (manufactured by Anti-mouse Mac-1-APC) ) was used.
  • Peripheral blood was collected from the mouse's inferior vein and analyzed by reacting with antibodies after hemolysis. Bone marrow cells were collected from the femur and tibia of mice and reacted with antibodies for analysis.
  • Example 1 ⁇ Differentiation into the liver> As described above, frozen sections were prepared from teratomas differentiated into hepatocytes and immunostained with albumin, CK19, and CYP7A1, which are hepatocyte markers. The obtained results are shown in FIGS. As is clear from the results shown in FIG. 2 and FIG. 3, only the teratoma that received differentiation induction by cytokines was confirmed to express the same marker as that of hepatocytes.
  • Example 2 ⁇ Differentiation into pancreas> As described above, frozen sections were prepared from teratomas differentiated into pancreatic cells, and immunostaining for insulin, which is a marker for pancreatic islets of Langerhans, was performed. The obtained results are shown in FIG. As is clear from the results shown in FIG. 5, only the teratoma that received differentiation induction by cytokines was confirmed to express insulin similar to pancreatic cells.
  • Example 3 ⁇ Differentiation into blood cells and hematopoietic stem cells>
  • blood cells derived from Lnk ⁇ / ⁇ iPS cells were confirmed in the blood of the nude mouse (see FIG. 6).
  • the ratio of blood cells derived from iPS cells was higher under the conditions of SCF + TPO + OP9 than SCF + TPO.
  • hematopoietic progenitor cells Lineage-c-Kit + Sca-1 + (KSL) derived from iPS cells were confirmed in the bone marrow (see FIG. 7). Further, when this bone marrow cell was transplanted into a wild type C57 / BL6 mouse and peripheral blood after 4 weeks was analyzed, it was confirmed that almost 100% of the blood was derived from iPS cells and differentiated into various cells. (See Figure 8). That is, it was suggested that iPS cells differentiated into hematopoietic stem cells in nude mice and home to bone marrow.
  • hematopoietic stem cells and progenitor cells deficient in the protein Lnk that negatively regulates the functions of hematopoietic stem cells and progenitor cells. Furthermore, by using ES cells and iPS cells deficient in the protein Lnk that negatively regulates the functions of hematopoietic stem cells and progenitor cells, a larger amount of hematopoietic stem cells and progenitor cells was amplified compared to normal ES cells iPS cells. .
  • Example 4 Removal of undifferentiated cells to be mixed and method of removing other than target cells>
  • a problem of tumor formation due to pluripotency may occur.
  • transplanting differentiation-induced cells in vitro there is a risk that the mixed undifferentiated cells form a tumor. Therefore, in the present invention, it is preferable to use a screening system that allows only the target differentiated cells to survive.
  • TK Thymidine Kinase
  • GCV ganciclovir
  • This ganciclovir 5'-triphosphate inhibits DNA synthesis, thereby inducing cell death of cells expressing TK.
  • a virus-derived TK gene is introduced into an ES cell or iPS cell, and a system in which the TK gene is removed by the Cre-LoxP system only in cells that have differentiated into target cells is constructed.
  • a retroviral vector in which an HSV-derived TK gene is sandwiched between LoxP sequences and a lentiviral vector that expresses Cre recombinase under the control of the Alb promoter, which is a marker gene for hepatocytes are prepared.
  • a VSV-G envelope for virus production, it can be used for both mouse ES / iPS cells and human ES / iPS cells (see FIG. 9).
  • hepatocyte differentiation is induced as described above.
  • GCV it is considered that only Alb-producing cells from which the TK gene has been removed by the expression of Cre can survive, and cell death of undifferentiated cells or cells differentiated into other cell lineages can be induced.
  • genes such as Cre recombinase and TK under the control of the Alb promoter were introduced into HuH7 cells (cell line derived from human liver cancer) and NIH3T3 cells (mouse fetal epithelial cell line) using the viral vector shown in FIG.
  • HuH7 cells cell line derived from human liver cancer
  • NIH3T3 cells mouse fetal epithelial cell line
  • the present method can be suitably used in the present invention.
  • the establishment of this system makes it possible to survive only differentiated cells of interest such as Alb-expressing cells by administering GCV after transplanting undifferentiated cells into the living body.
  • Example 3 the method for producing hematopoietic stem cells and hematopoietic progenitor cells of the present invention shown in Example 3 and the functions of these cells obtained by the method were analyzed in more detail.
  • Examples 5 to 8 the following materials were used and the following methods were used.
  • ⁇ Mouse> C57BL / 6 (B6) mice, KSN / Slc nude mice, and green fluorescent protein (GFP) transgenic mice were purchased from Japan SLC Corporation. Lnk ⁇ / ⁇ GFP transgenic mice were bred and maintained at the laboratory animal research facility of the Institute of Medical Science, University of Tokyo. In addition, the production and evaluation of X-SCID mice having a genetic background of B6 were carried out according to the description in “Ohbo, K et al., Blood, 1996, Vol. 87, pages 956-967”. Furthermore, NOD / SCID mice were purchased from Nippon Claire Co., Ltd. NOD / SCID / JAK3-deficient mice were purchased from Sankyo Lab Service Co., Ltd. In addition, the care of these mice was performed according to the guidance regarding the recombinant DNA experiments and experimental animals of the University of Tokyo.
  • Mouse iPS cells include Lnk ⁇ / ⁇ GFP transgenic C57BL / 6 (B6) mice, or tail tip fibroblasts (TTF, tail tip fibroblasts) derived from GFP transgenic B6 mice, Oct3 / 4, Sox2, and Klf4. These three genes were introduced by lentivirus all-in-one vector and re-programmed and used. The characteristics of the obtained iPS cells were confirmed as shown in FIGS. 11 to 16 described later.
  • mouse iPS cells maintained an undifferentiated state by co-culture with mouse fetal fibroblasts (MEF).
  • the composition of the medium used for the co-culture is as follows. Dulbecco's modified Eagle medium (DMEM, manufactured by GIBCO), 15% knockout serum substitute (Knockout SR, manufactured by GIBCO), 20 mM HEPES buffer solution (manufactured by Invitrogen), 0.1 mM MEM non-essential amino acid solution (Manufactured by Invitrogen), 0.1 mM L-glutamine (manufactured by Invitrogen), 100 U / ml penicillin, 100 ⁇ g / ml streptomycin (manufactured by Sigma-Aldrich), 0.1 mM 2-mercaptoethanol (manufactured by GIBCO), and 1000 U / Ml ESGRO (manufactured by GIBCO). The medium was changed daily and the cells were passaged every 2-3 days to avoid abnormal
  • human iPS cells those established by reprogramming by introducing 3 genes of Oct3 / 4, Sox2 and Klf4 into normal human epidermal keratinocytes (Lonza) by using a lentiviral vector were used.
  • human iPS cells maintained an undifferentiated state by co-culture with MEF.
  • the composition of the human iPS cell culture medium used for co-culture is as follows.
  • Dulbecco's Modified Eagle Medium-F12 (Sigma-Aldrich), 20% Knockout SR (GIBCO), 0.1 mM MEM non-essential amino acid solution (Invitrogen), 0.2 mM L-glutamine (additives) Invitrogen), 0.1 mM 2-mercaptoethanol (GIBCO), and 5 ng / ml bFGF (Peprotech).
  • the medium was changed every day and the cells were passaged every 7 days.
  • OP9 cells were maintained in a growth medium consisting of minimal essential medium ⁇ ( ⁇ -MEM, manufactured by Invitrogen) supplemented with 20% fetal calf serum (FBS, manufactured by HyClone).
  • ⁇ -MEM minimal essential medium ⁇
  • FBS fetal calf serum
  • Tissue sections were evaluated by paraffin-embedding teratoma tissue and staining with hematoxylin and eosin (H & E).
  • Fluorescent immunostaining of Nanog and SSEA-1 was performed using anti-mouse Nanog antibody (diluted to 1/100, Cosmo Bio) and anti-mouse SSEA-1 antibody (diluted to 1/100, Abcam) And then Alexa Fluor 546-labeled anti-rabbit IgG antibody (diluted to 1/300, manufactured by Invitrogen), and allophycocyanin (APC) -labeled anti-mouse IgM antibody (1/100) Used by diluting and incubating with eBioscience). The counterstaining of the nuclei was performed using DAPI (manufactured by Sigma-Aldrich) according to the manufacturer's instructions. Subsequently, the sections immunofluorescently stained were visualized with a microscope (BX-51) and a digital camera system (DP-71) (both from Olympus) and photographed.
  • DAPI manufactured by Sigma-Aldrich
  • the teratoma tissue is rapidly frozen using dry ice, embedded in Optimal Cutting Temperature (OCT) compound (Sakura Finetek), and using CM3050 cryostat (Leica Microsystems), Prepared in 7-8 ⁇ m sections. These tissue sections were fixed with ethanol and immunostained. That is, each section was incubated with the primary antibody for 24 hours at 4 ° C. and then with the secondary antibody for 30 minutes at room temperature.
  • OCT Optimal Cutting Temperature
  • the primary antibodies are anti-mouse CD45 antibody (diluted to 1/50, used by BD Bioscience), Alexa Fluor488 labeled anti-mouse CD117 (c-Kit) antibody (diluted to 1/10, used by BioLegend) ), Anti-mouse osteocalcin antibody (Ostecalcin, BGLAP) (diluted to 1/200, LifeSpan Biosciences), and anti-mouse VE-cadherin antibody (diluted to 1/200, used, Abcam) ) was used.
  • Alexa Fluor 546-labeled goat-derived anti-rat IgG antibody and Alexa Fluor 647-labeled goat-derived anti-rabbit IgG antibody both manufactured by Invitrogen were used.
  • the section was sealed with a fluorescent staining mounting medium (manufactured by Dako) containing 4,6-diamidino-2-phenylindole (DAPI) for counterstaining the nucleus, and both TCS SP2 and AOBS were encapsulated. Observation was carried out with a focal laser scanning microscope (manufactured by Leica Microsystems).
  • a fluorescent staining mounting medium manufactured by Dako
  • DAPI 4,6-diamidino-2-phenylindole
  • iPS cells were trypsinized with a 0.25% trypsin-EDTA solution (GIBCO). Trypsinized iPS cells and MEFs were replated on non-coating dishes and incubated for 30 minutes to remove MEFs. To create a chimeric mouse, 10 iPS cells were injected into blastocysts derived from ICR mice, and the blastocysts injected with iPS cells were transplanted into the uterus of pseudopregnant mice.
  • GEBCO trypsin-EDTA solution
  • HSC hematopoietic stem cells
  • Hematopoietic cytokines containing 200 ng stem cell factor (SCF, manufactured by Peprotech) and 200 ng thrombopoietin (TPO, manufactured by Peprotech) were placed in a micro osmotic pump (manufactured by ALZET), and the pump was implanted subcutaneously for 2 weeks.
  • iPS cells were transplanted with 1 ⁇ 10 6 OP9 stromal cells.
  • the hematopoietic cytokine and the OP9 stromal cells were administered.
  • differentiation induction from human iPS cells to HSC is as follows. That is, 1 ⁇ 10 6 human iPS cells and 5 ⁇ 10 5 OP9 stromal cells were injected into the testis of NOD / SCID mice (5-7 weeks old). Furthermore, hematopoietic cytokines containing 200 ng human SCF (Peprotech) and 200 ng TPO (Peprotech) were placed in a micro-osmotic pump (ALZET), and the pump was implanted subcutaneously for 2 weeks.
  • AZAT micro-osmotic pump
  • HSC hematopoietic stem cells
  • mice Peripheral blood cells and spleen cells of mice were APC-labeled anti-CD45 antibody (BD Biosciences), APC-Cy7-labeled anti-CD34 antibody (manufactured by eBioscience), Pacific Blue-labeled anti-CD45R / B220 antibody (manufactured by eBioscience), phycoerythrin (Phycoerythrin (PE) -Cy7-labeled anti-Gr-1 antibody (BioLegend) and PE-Cy7-labeled anti-Mac-1 antibody (BioLegend) were used for staining.
  • APC-labeled anti-CD45 antibody BD Biosciences
  • APC-Cy7-labeled anti-CD34 antibody manufactured by eBioscience
  • Pacific Blue-labeled anti-CD45R / B220 antibody manufactured by eBioscience
  • phycoerythrin Phycoerythrin (PE) -Cy7-labeled anti-Gr-1
  • the mouse bone marrow cells in which the teratoma was formed were analyzed according to the description of Osawa, M, et al., Science, 1996, 273, 242-245. That is, the bone marrow cells are biotinylated anti-Gr-1 antibody, anti-Mac-1 antibody, anti-CD45R / B220 antibody, anti-CD4 antibody, anti-CD8 antibody, anti-IL-7R antibody, and anti-TER119 antibody (manufactured by eBioscience). ).
  • the cells were stained with Alexa Fluor 700-labeled anti-CD34 antibody, Pacific Blue-labeled anti-Sca-1, and APC-labeled anti-c-Kit antibody (all manufactured by eBioscience).
  • the biotinylated antibody was detected using streptavidin-APC-Cy7 (manufactured by eBioscience).
  • Four-color analysis and sorting were performed by FACSAria (Becton Dickinson).
  • HSC hematopoietic stem cells
  • Bone marrow cells of teratoma-formed mice were treated with APC-labeled anti-CD45 antibody (BD Biosciences), Pacific Blue-labeled anti-human CD45 antibody (BioLegend), and FITC-labeled anti-human CD34 antibody (BD Biosciences). Stained.
  • Peripheral blood of recipient mice was APC-labeled anti-mouse CD45 antibody (BD Biosciences), Pacific Blue-labeled anti-human CD45 antibody (BioLegend), Alexa Fluor 488-labeled anti-human CD3 antibody (BD Biosciences), APC- Staining was performed using an H7-labeled anti-human CD19 antibody (BD, Biosciences) and a PE-Cy7-labeled anti-human CD33 antibody (BD, Biosciences).
  • ⁇ Single cell culture> Purified CD34 ⁇ KSL cells derived from iPS cells were seeded in 96-well plates containing 200 ⁇ L of medium in each well for single clones.
  • the composition of the used culture medium is as follows. S-clone SF-O3 medium (manufactured by Sanko Junyaku Co., Ltd.), as additives, 1% bovine serum albumin (BSA), mouse SCF (50 ng / mL), mouse TPO (50 ng / mL), mouse IL-3 ( 10 ng / mL), and mouse EPO (1 U / mL) (all manufactured by PeproTech). The cells were cultured at 37 ° C.
  • S-clone SF-O3 medium manufactured by Sanko Junyaku Co., Ltd.
  • BSA bovine serum albumin
  • mouse SCF 50 ng / mL
  • mouse TPO 50 ng / mL
  • mouse IL-3 10 ng / m
  • hema color registered trademark, manufactured by MERCK
  • ⁇ Bone marrow transplantation assay> For Lnk ⁇ / ⁇ GFP iPS cell-derived HSCs, 1 ⁇ 10 7 BM cells of mice with teratoma formation were transplanted into lethal radiation treated (9.5 Gy) wild type B6 recipient mice. Then, 4 weeks and 12 weeks after transplantation, PB cells of recipient mice were analyzed by flow cytometry.
  • GFP + CD34 ⁇ KSL cells were selected from the primary recipient mouse and secondary to another recipient mouse (secondary recipient mouse) along with 2 ⁇ 10 5 B6 bone marrow cells. Transplanted as a transplant.
  • mice with teratoma formed were transplanted into radiation-treated (2 Gy) NOD / SCID recipient mice or NOD / SCID / JAK3-deficient recipient mice. did. And 8 weeks after transplantation, PB cells of recipient mice were analyzed by flow cytometry.
  • iPS cells were first established from Lnk ⁇ / ⁇ GFP transgenic mice. That is, as shown in FIG. 11, the TTF of Lnk ⁇ / ⁇ GFP transgenic mice was reprogrammed by introducing 3 factors (Oct3 / 4, Klf4, and Sox2) using a lentiviral all-in-one vector. did.
  • the obtained iPS cells (Lnk ⁇ / ⁇ GFP iPS cells) were confirmed to express GFP, Nanog, and SSEA-1 by immunofluorescence analysis (see FIG. 12).
  • Lnk ⁇ / ⁇ GFP iPS cells have the ability to form teratomas in nude mice and can contribute to chimeric mice by injecting into blastocysts, confirming that they have pluripotency. (See FIGS. 13-16).
  • Lnk ⁇ / ⁇ GFP iPS cells were analyzed using KSN / Slc nude mice were injected subcutaneously and induced to differentiate into HSC under the following conditions (see FIG. 17).
  • Condition 1 As a control, iPS cells were injected into nude mice.
  • Condition 2 Hematopoietic cytokines (SCF and TPO) were placed in a micro osmotic pump and administered continuously for 2 weeks.
  • Condition 3 OP9 stromal cell line was transplanted with iPS cells.
  • Condition 4 The hematopoietic cytokine and the OP9 stromal cell line were administered.
  • the ratio was highest when cytokine and OP9 cells were administered (the average value after 12 weeks after iPS cell introduction was the condition 1: 0. 002 ⁇ 0.01%, Condition 2: 1.02 ⁇ 1.15%, Condition 3: 0.87 ⁇ 0.79%, Condition 4: 4.26 ⁇ 3.79%).
  • hematopoietic cells defined as an immunophenotype including a BM primitive cell population is similar to the results described in Example 3 through Lnk ⁇ / ⁇ . It has also been found that this can be done from GFP iPS cells and that hematopoietic cytokines and co-cultured cells (eg, OP9 cells) promote this induction.
  • CFC-nmEM indicates the number of colony forming cells-neutrophil / macrophage / erythroblast / megakaryocyte (colony-forming units-neutrophil / macrophage / erythroblast / megakaryocyte). The obtained results are shown in FIGS.
  • CD34 ⁇ KSL cells formed large colonies containing nmEM colonies (see FIG. 22), and CD34 ⁇ KSL cells were neutrophils.
  • the formation of all blood cell lineages with spheres, macrophages, erythroblasts and megakaryocytes demonstrated that HSCs derived from Lnk ⁇ / ⁇ GFP iPS cells are multipotent, ie functional HSCs (See FIG. 23 and FIG. 24). .
  • HSPC hematopoietic stem / progenitor cells
  • hematopoietic cells derived from Lnk ⁇ / ⁇ GFP iPS cells having multi-lineage reconstruction ability were found. Detected frequently. Further, as is apparent from the results shown in FIG. 27, cells derived from Lnk ⁇ / ⁇ GFP iPS cells are bone marrow cells, and the average proportion of GFP + cells in the CD34 ⁇ KSL cell fraction (HSC cell fraction) is It was as high as 45%.
  • lethal bone marrow cells of primary recipient mice as shown in FIG. Transplanted into B6 mice (secondary recipient mice) irradiated with radiation (secondary transplantation). The obtained results are shown in FIG.
  • HSCs derived from Lnk ⁇ / ⁇ GFP iPS cells have normal hematopoietic ability. Became clear.
  • Example 6 ⁇ Production of functional HSC from GFP iPS cells not having Lnk mutation>
  • iPS cells GFP iPS cells
  • GFP iPS cell-derived cells were obtained in the peripheral blood, spleen, and bone marrow cells of primary recipient mice. It was detected that blood cells were engrafted. Furthermore, GFP + cells were also detected in the hematopoietic primitive cell fraction in the recipient bone marrow, including Lin ⁇ cells, KSL cells, and CD34 ⁇ KSL cells (see FIG. 34).
  • CD34 ⁇ KSL cells formed all the blood cell lineages with neutrophils, macrophages, erythroblasts, megakaryocytes, and thus derived from GFP iPS cells. It has been demonstrated that HSCs are pluripotent, ie functional HSCs.
  • the method of the present invention is a method capable of producing a functional HSC having long-term bone marrow reconstruction ability even from iPS cells without Lnk mutation.
  • X-linked severe combined immunodeficiency is one of severe combined immunodeficiencies (SCID) characterized by severe impairment in T cell and B cell immunity. Luo patients are known to have mutations in a gene encoding a common gamma chain ( ⁇ c) shared by many cytokine receptors that play important functions in the immune system. (See Buckley, RH, et al., Journal of Pediatrics, 1997, 130, 378-387).
  • hematopoietic progenitor cells that can be transplanted and can reconstruct the lymph-myeloid lineage can be prepared from iPS cells. Then, next, an attempt was made to apply the present invention to an X-SCID treatment model using gene therapy and disease-specific iPS cells.
  • iPS cells were prepared from X-SCID mice, mouse ⁇ C gene and the like were introduced via retrovirus, and then clone selection was performed, so that mouse ⁇ C was highly expressed.
  • Cell line (m ⁇ c-iPSC # 4) was established. (See Figures 37-39).
  • m ⁇ c-iPSC # 4 was injected together with OP9 cells subcutaneously in X-SCID mice. Twelve weeks after the injection of iPS cells, GFP + CD45 + cells derived from m ⁇ c-iPS cells (m ⁇ c-iPSC) were detected in the peripheral blood of one teratoma-formed X-SCID mouse ( (See FIG. 40).
  • HSPC hematopoietic stem / progenitor cells
  • a heterogeneous cell population (such as HSPC derived from a patient) into which a viral vector is randomly integrated must be transplanted, and there is a risk that the transplanted patient suffers from leukemia. It was.
  • the iPS cell into which the gene is introduced at a risk-free position on the genomic DNA can be selected, cloned and proliferated. Therefore, by using hematopoietic stem cells derived from iPS cells whose safety has been confirmed for transplantation, the present invention can also provide a gene therapy method capable of avoiding the risk of leukemia.
  • Example 8 ⁇ Induction of engraftable HSCs from human iPS cells via teratoma formation>
  • human iPS cells are injected subcutaneously into NOD / SCID mice as shown in FIG. 41 to produce teratomas.
  • HSC was induced by the same method as mouse HSC induction.
  • a human-derived cell population (mCD45 ⁇ hCD45 + ) was clearly detected in the peripheral blood of 1 of 15 mice examined, and 10 teratomas were found.
  • fractions of human-derived cells (hCD45 ⁇ hCD34 + , hCD45 dull hCD34 + , and hCD45 + hCD34 ⁇ ) could be detected in the mCD45 negative cell population.
  • the present invention can produce engraftable HSCs from human iPS cells without any genetic modification.
  • HSCs niche-like cells were present was examined.
  • HSC niche is a microenvironment necessary for maintaining the HSC.
  • the actual state of HSC has not yet been clarified (Calvi, LM, et al., Nature, 2003, 425, 841-846, Kiel, MJ, et al., Cell, 2005, 121.
  • BM-residential glial cells contribute to HSC niche formation.
  • osteocalcin expression as an osteoblast marker
  • VE-cadherin expression as an endothelial marker
  • GFAP glial fibrillary acidic protein
  • teratoma mCD45 ⁇ cells contained human iPS cell-derived cell fractions (hCD45 + hCD34 ⁇ , hCD45 + hCD34 + , hCD45 ⁇ hCD34 + ) (see FIG. 47). ).
  • HSC niche-like cells such as osteocalcin + cells, VE-cadherin + cells, and GFAP + cells can be confirmed (see FIG. 50). Furthermore, it was confirmed that CD45 + c-Kit + cells containing mouse iPS cell-derived HSC were present in the vicinity of VE-cadherin + cells and osteocalcin + cells (see FIGS. 51 and 52).
  • an environment corresponding to bone marrow is formed in the teratoma by producing various cells that can constitute the HSC niche in an environment suitable for hematopoietic differentiation, and as a result. It was suggested that HSC is produced.
  • Example 9 ⁇ Organogenesis in mouse individuals using germinal progenitor cells>
  • a method for organ transplantation by directly transplanting ES cells or iPS cells as described above into a mouse individual 1. Since pluripotent stem cells retain the differentiation ability of various cell lineages, cells other than the target cells may be formed. 2. When the teratoma-forming ability of the transplanted pluripotent stem cells is too high, there is a possibility that the health condition of the host is harmed by the increase in teratomas before the target cells are formed. This can be a problem.
  • ES cells and iPS cells are first induced to differentiate in vitro, and endoderm progenitors having high differentiation potential to endoderm system while maintaining pluripotency.
  • Cells were prepared and transplanted into mice. That is, it was performed according to the materials and methods described below. The obtained results are shown in FIGS.
  • E14tg2a a MEF-independent ES cell line
  • E14tg2a a MEF-independent ES cell line
  • GMEM Glasgow Modified Eagle Medium
  • FCS 1% L-glutamine-penicillin-streptomycin solution
  • SIGMA 1% L-glutamine-penicillin-streptomycin solution
  • GIBCO 1% L-glutamine-penicillin-streptomycin solution
  • GIBCO 0.1 mM non-essential amino acid
  • 1 mM Sodium pyruvate 0.1 mM 2-mercaptoethanol
  • LIF leukemia inhibitory factor
  • Collagen-Type4 coated culture dishes were seeded with E14tg2a at a density of 1 ⁇ 10 4 cells / ml. 20 ng / ml human activin A (manufactured by Peprotech) and 10 ng / ml human BMP4 (manufactured by Peprotech) were added to an SFO3 serum-free medium (manufactured by Sanko Junyaku Co., Ltd.) and cultured for 2 days.
  • the medium was changed to one in which 20 ng / ml human activin A (manufactured by Peprotech), 20 ng / ml mouse EGF (manufactured by Peprotech) and 10 ng / ml FGF4 (manufactured by SIGMA) were added to the SFO3 medium.
  • the culture was further continued for 5 days.
  • Matrigel is a soluble basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma rich in extracellular matrix protein.
  • the main components are laminin, collagen IV, heparan sulfate proteoglycan, And entactin / nidogen 1 and 2, which are naturally produced by TGF- ⁇ , epidermal growth factor, insulin-like growth factor, fibroblast growth factor, tissue plasminogen activator 3, 4 and EHS tumors Other growth factors.
  • the tissue specimen was washed twice with PBS and immersed in methanol (MtOH) cooled to ⁇ 20 ° C. for 10 minutes. After washing MtOH twice with PBS, blocking with 5% donkey serum (manufactured by SIGMA) / PBS was performed at room temperature for 30 minutes. Primary antibodies (anti-FoxA2 and CK19 antibodies) diluted in blocking buffer were added and allowed to react overnight at 4 ° C.
  • ES cells and teratomas obtained by injecting endoderm progenitor cells obtained by inducing differentiation from ES cells, and CK19-positive intestinal tract present in tissue sections. The number of like structures was counted.
  • pluripotent stem cells that have been induced to differentiate to some extent from universal cells such as ES cells, a teratoma containing more cells constituting the target organ can be formed, and a host due to an increase in teratoma It has become clear that the effects on the health status of can be suppressed.
  • pluripotent stem cells can be efficiently induced to differentiate into desired functional cells. Therefore, the method for producing target cells induced to differentiate from pluripotent stem cells of the present invention is useful in regenerative medicine, transplantation medicine, cell medicine and the like.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Hematology (AREA)
  • Immunology (AREA)
  • Environmental Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Animal Husbandry (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

La présente invention concerne un procédé pour produire des cellules cibles qui ont été induites pour être différenciées à partir de cellules souches pluripotentes, qui met en œuvre un procédé pour implanter chez un mammifère non-humain des cellules souches pluripotentes dérivées d'un mammifère, un procédé pour administrer au mammifère non-humain mentionné ci-dessus un agent pour induire la différenciation en cellules cibles, un procédé pour élever le mammifère non-humain mentionné ci-dessus assez longtemps pour que les cellules souches pluripotentes implantées forment des tératomes in vivo dans ledit mammifère et pour induire la différenciation des cellules souches pluripotentes mentionnées ci-dessus en cellules cibles, et un procédé pour récupérer chez le mammifère non-humain mentionné ci-dessus les cellules cibles dérivées du mammifère mentionné ci-dessus.
PCT/JP2010/072044 2009-12-08 2010-12-08 Procédé pour produire des cellules induites pour être différenciées à partir de cellules souches pluripotentes WO2011071085A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2011545228A JP5896457B2 (ja) 2009-12-08 2010-12-08 多能性幹細胞から分化誘導された細胞の生産方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US26756609P 2009-12-08 2009-12-08
US61/267,566 2009-12-08

Publications (1)

Publication Number Publication Date
WO2011071085A1 true WO2011071085A1 (fr) 2011-06-16

Family

ID=44145630

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2010/072044 WO2011071085A1 (fr) 2009-12-08 2010-12-08 Procédé pour produire des cellules induites pour être différenciées à partir de cellules souches pluripotentes

Country Status (2)

Country Link
JP (1) JP5896457B2 (fr)
WO (1) WO2011071085A1 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015133147A1 (fr) * 2014-03-05 2015-09-11 公益財団法人先端医療振興財団 Modèle animal humanisé pathologique et tissu de tératome
KR20150104689A (ko) * 2014-03-06 2015-09-16 건국대학교 산학협력단 만능줄기세포로부터 형성된 테라토마 유래 체내 신경줄기세포 생산 방법
EP2756092A4 (fr) * 2011-09-16 2015-10-28 Giovanni Amabile Procédés de fabrication de cellules, de tissus et d'anticorps
WO2016039278A1 (fr) * 2014-09-08 2016-03-17 株式会社オーガンテクノロジーズ Procédé de production de glande sécrétoire
WO2016039279A1 (fr) * 2014-09-08 2016-03-17 株式会社オーガンテクノロジーズ Procédé de production de peau avec toute son épaisseur dotée des organes auxiliaires de la peau
WO2016143836A1 (fr) * 2015-03-09 2016-09-15 株式会社メガカリオン Procédé pour la production de culture contenant des mégacaryocytes, et procédé de production de plaquettes l'employant
WO2016208532A1 (fr) * 2015-06-22 2016-12-29 全国農業協同組合連合会 Procédé de production d'un animal chimère de sang

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP7209619B2 (ja) * 2016-07-28 2023-01-20 エフ．ホフマン－ラ ロシュ アーゲー 非ヒト霊長類人工多能性幹細胞由来肝細胞およびその使用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006506047A (ja) * 2002-05-28 2006-02-23 ノボセル インコーポレイテッド インスリン産生細胞のための方法、成分並びに成長および分化因子
JP2008512095A (ja) * 2004-09-07 2008-04-24 財団法人実験動物中央研究所 コモンマーモセット胚性幹細胞株

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006506047A (ja) * 2002-05-28 2006-02-23 ノボセル インコーポレイテッド インスリン産生細胞のための方法、成分並びに成長および分化因子
JP2008512095A (ja) * 2004-09-07 2008-04-24 財団法人実験動物中央研究所 コモンマーモセット胚性幹細胞株

Non-Patent Citations (18)

* Cited by examiner, † Cited by third party
Title
BLUM, B. ET AL.: "Clonal analysis of human embryonic stem cell differentiation into teratomas", STEM CELLS, vol. 25, 2007, pages 1924 - 1930 *
DREWEL, D.: "SCF modulates organ distribution and hematopoietic engraftment of CB-derived pluripotent HPC transplanted in NOD/SCID mice", CYTOTHERAPY, vol. 8, no. 1, 2006, pages 70 - 78 *
FURTH, M.E. ET AL.: "Stem cell sources to treat diabetes", J.CELL.BIOCHEM., vol. 106, 7 January 2009 (2009-01-07), pages 507 - 511, XP009147430, DOI: doi:10.1002/jcb.22000 *
HAMAZAKI, T. ET AL.: "Hepatic maturation in differentiating embryonic stem cells in vitro", FEBS LETT., vol. 497, 2001, pages 15 - 19, XP004250029, DOI: doi:10.1016/S0014-5793(01)02423-1 *
HENTZE, H. ET AL.: "Teratoma formation by human embryonic stem cells : Evaluation of essential parameters for future safty studies", STEM CELL RESEARCH, vol. 2, May 2009 (2009-05-01), pages 198 - 210, XP026087757, DOI: doi:10.1016/j.scr.2009.02.002 *
HIROMITSU NAKAUCHI: "Saibo Chiryo kara Jisshitsu Zoki no Saisei Iryo e", CLINICIAN, vol. 56, no. 575, 1 January 2009 (2009-01-01), pages 17 - 21 *
KROON, E. ET AL.: "Pancreatic endoderm derived from human embryonic stem cells generates glucose-responsive insulin-secreting cells in vivo", NATURE BIOTECH., vol. 26, no. 4, 2008, pages 443 - 452, XP002561975, DOI: doi:10.1038/nbt1393 *
LAVON, N. ET AL.: "The effect of overexpression of Pdxl and Foxa2 on the differentiation of human embryonic stem cells into pancreatic cells", STEM CELLS, vol. 24, 2006, pages 1923 - 1930, XP055097084, DOI: doi:10.1634/stemcells.2005-0397 *
MCCULLAGH, P. ET AL.: "Interception of the development of self tolerance in fetal lambs", EUR.J.IMMUNOL., vol. 19, 1989, pages 1387 - 1392 *
SASAKI, K. ET AL.: "Hematopoietic microchimerism in sheep after in utero transplantation of cultured cynomolgous embryonic stem cells", TRANSPLANTATION, vol. 79, no. 1, 2005, pages 32 - 37 *
SEITA, J.: "Lnk negatively regulates self- renewal of hematopoietic stem cells by modifying thrombopoietin-mediated signal transduction", PROC.NATL.ACAD.SCI.USA, vol. 104, no. 7, 2007, pages 2349 - 2354 *
SHIRAKI, N. ET AL.: "Differentiation of mouse and human embryonic stem cells into hepatic lineages", GENES TO CELLS, vol. 13, 2008, pages 731 - 746, XP003026524, DOI: doi:10.1111/J.1365-2443.2008.01201.X *
TAKAHASHI, K. ET AL.: "Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors", CELL, vol. 126, 2006, pages 663 - 676 *
TAKIZAWA, H. ET AL.: "Transient blocking of Lnk-mediated pathways as a potential approach to promote engrafting ability of hematopoietic progenitor cells", INFLAMMATION AND REGENERATION, vol. 27, no. 1, January 2007 (2007-01-01), pages 59 - 64 *
THOMSON, J.A. ET AL.: "Embryonic stem cell lines derived human blastocysts", SCIENCE, vol. 282, 1998, pages 1145 - 1147, XP002933311, DOI: doi:10.1126/science.282.5391.1145 *
YAMAMOTO, H. ET AL.: "Differentiation of embryonic stem cells into hepatocytes: Biological functions and therapeutic application", HEPATOLOGY, vol. 37, 2003, pages 983 - 993, XP002321738, DOI: doi:10.1053/jhep.2003.50202 *
YUTAKA HANAZONO: "Hitsuji o Mochiita Saru Soshiki Sanseiho", BIO IND., vol. 25, no. 12, 2008, pages 76 - 80.6 *
ZHANG, D. ET AL.: "Highly efficient differentiation of human ES cells and iPS cells into mature pancreatic insulin- producing cells", CELL RESEARCH, vol. 19, no. 4, 3 March 2009 (2009-03-03), pages 429 - 438, XP055074920, DOI: doi:10.1038/cr.2009.28 *

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2756092A4 (fr) * 2011-09-16 2015-10-28 Giovanni Amabile Procédés de fabrication de cellules, de tissus et d'anticorps
WO2015133147A1 (fr) * 2014-03-05 2015-09-11 公益財団法人先端医療振興財団 Modèle animal humanisé pathologique et tissu de tératome
KR20150104689A (ko) * 2014-03-06 2015-09-16 건국대학교 산학협력단 만능줄기세포로부터 형성된 테라토마 유래 체내 신경줄기세포 생산 방법
KR101588110B1 (ko) 2014-03-06 2016-01-25 건국대학교 산학협력단 만능줄기세포로부터 형성된 테라토마 유래 체내 신경줄기세포 생산 방법
CN106794278A (zh) * 2014-09-08 2017-05-31 株式会社器官再生工学 产生具有皮肤附属器官的全厚皮肤的方法
WO2016039279A1 (fr) * 2014-09-08 2016-03-17 株式会社オーガンテクノロジーズ Procédé de production de peau avec toute son épaisseur dotée des organes auxiliaires de la peau
WO2016039278A1 (fr) * 2014-09-08 2016-03-17 株式会社オーガンテクノロジーズ Procédé de production de glande sécrétoire
EP3192533A4 (fr) * 2014-09-08 2018-05-30 Organ Technologies, Inc. Procédé de production de peau avec toute son épaisseur dotée des organes auxiliaires de la peau
WO2016143836A1 (fr) * 2015-03-09 2016-09-15 株式会社メガカリオン Procédé pour la production de culture contenant des mégacaryocytes, et procédé de production de plaquettes l'employant
JPWO2016143836A1 (ja) * 2015-03-09 2017-12-21 株式会社メガカリオン 巨核球を含む培養物の製造方法及びこれを用いた血小板の製造方法
US11976301B2 (en) 2015-03-09 2024-05-07 Megakaryon Corporation Method for producing culture containing megakaryocytes, and method for producing platelets using same
WO2016208532A1 (fr) * 2015-06-22 2016-12-29 全国農業協同組合連合会 Procédé de production d'un animal chimère de sang
CN107920499A (zh) * 2015-06-22 2018-04-17 全国农业协同组合连合会 血液嵌合动物的制作方法
JPWO2016208532A1 (ja) * 2015-06-22 2018-06-07 全国農業協同組合連合会 血液キメラ動物の作出法
EP3311660A4 (fr) * 2015-06-22 2018-11-14 National Federation of Agricultural Cooperative Associations Procédé de production d'un animal chimère de sang
US11432537B2 (en) 2015-06-22 2022-09-06 The University Of Tokyo Method for producing blood chimeric animal

Also Published As

Publication number Publication date
JPWO2011071085A1 (ja) 2013-04-22
JP5896457B2 (ja) 2016-03-30

Similar Documents

Publication Publication Date Title
JP7134317B2 (ja) 生体組織から単離できる多能性幹細胞
JP5896457B2 (ja) 多能性幹細胞から分化誘導された細胞の生産方法
US11261426B2 (en) Pluripotent stem cell that can be isolated from body tissue
JP2018531025A6 (ja) 多能性幹細胞の免疫細胞への分化を誘導する方法
JP2018531025A (ja) 多能性幹細胞の免疫細胞への分化を誘導する方法
WO2012133948A1 (fr) Composition pour thérapie cellulaire par allogreffe, ladite composition contenant une cellule souche pluripotente positive pour ssea-3 pouvant être isolée de tissu corporel
WO2012133942A1 (fr) Cellule souche pluripotente pouvant être isolée de tissu adipeux ou de cordon ombilical de corps biologique
JP2023502522A (ja) ヒト多能性幹細胞からバイオエンジニアリングされた胸腺オルガノイド
Li et al. Differentiation of embryonic stem cells in adult bone marrow
Muggeo A STEP-BY-STEP PROCESS TO GENERATE FUNCTIONAL OSTEOCLASTS FROM SITE SPECIFIC GENE-CORRECTED INDUCED PLURIPOTENT STEM CELLS: AN AUTOLOGOUS CELL THERAPY APPROACH TO TREAT AUTOSOMAL RECESSIVE OSTEOPETROSIS.
Banerjee et al. 598 Stem Cell Rev and Rep (2013) 9586–598
Chong Defining the origin of adult cardiac multipotent colony forming stem cells

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 10836005

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 2011545228

Country of ref document: JP

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 10836005

Country of ref document: EP

Kind code of ref document: A1