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WO2005100549A1 - Procédé de prolifération de cellules souches spécifiques d’un organe et appareil de prolifération pour celui-ci - Google Patents

Procédé de prolifération de cellules souches spécifiques d’un organe et appareil de prolifération pour celui-ci Download PDF

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Publication number
WO2005100549A1
WO2005100549A1 PCT/JP2005/006424 JP2005006424W WO2005100549A1 WO 2005100549 A1 WO2005100549 A1 WO 2005100549A1 JP 2005006424 W JP2005006424 W JP 2005006424W WO 2005100549 A1 WO2005100549 A1 WO 2005100549A1
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Prior art keywords
organ
stem cells
cells
specific stem
vascular endothelial
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English (en)
Japanese (ja)
Inventor
Nobuyuki Takakura
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Kanazawa Institute of Technology (KIT)
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Kanazawa Institute of Technology (KIT)
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/34—Internal compartments or partitions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/08—Chemical, biochemical or biological means, e.g. plasma jet, co-culture
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature

Definitions

  • the present invention relates to a method and apparatus for growing organ-specific stem cells, which makes it possible to grow organ-specific stem cells such as hematopoietic stem cells in vitro.
  • Stem cells are thought to be undifferentiated and self-replicate in the area of the body that is collectively referred to as the ecology niche (also called Nishobuchi).
  • the ecology niche also called Nishobuchi
  • the cells constituting the niche adhere to the stem cells and maintain the undifferentiated state of the stem cells, but their self-renewal and proliferation are suppressed.
  • stem cells are thought to be in a so-called quiescent phase in which division has stopped. Since stem cells can be applied to various regenerative medicines, various attempts have been made with respect to in vitro stem cell culture technology.
  • Currently, cells that can differentiate into multi-germ layers in ES cells, adult stem cells, etc. Techniques for in vitro proliferation at the level are being established, however, it is not possible to induce proliferation in vitro for organ-specific stem cells.
  • Patent Document 1 Japanese Patent Application No. 200 1—38 3 73 7
  • Patent Document 2 Japanese Patent Application No. 2000—6 1 5 73 7 Disclosure of the Invention
  • an object of the present invention is to provide a proliferation method and a proliferation apparatus that enable the target organ-specific stem cells to grow at the level of stem cells.
  • the present inventors have conducted intensive studies to achieve the above-mentioned object, and explained that the existing concept that stem cells maintain undifferentiation in a niche region in a living body and are in a quiescent state explains all phenomena. Stem cells can be undifferentiated in niches in vivo. We succeeded in finding an environment in which it can grow while maintaining it. That is, the present invention includes the following.
  • a method for expanding organ-specific stem cells which comprises culturing the organ-specific stem cells and vascular endothelial cells in a state where they are loosely adhered.
  • organ-specific stem cells are hematopoietic stem cells.
  • a second culture unit for culturing vascular endothelial cells A second culture unit for culturing vascular endothelial cells
  • An apparatus for culturing organ-specific stem cells comprising:
  • the porous member is a hollow fiber having porosity on an outer peripheral surface, wherein the first culturing unit is inside the hollow fiber, and the second culturing unit is outside the hollow fiber.
  • organ-specific stem cell according to (7), further comprising: an organ-specific stem cell supply channel connected to the first culture unit; and a vascular endothelial cell supply channel connected to the second culture unit.
  • Stem cell expansion device
  • FIG. 1 is a cross-sectional view showing an example of an organ-specific stem cell culture apparatus to which the present invention is applied. .
  • FIG. 2 is a fragmentary cross-sectional view schematically showing the state of adhesion between an organ-specific stem cell and a vascular endothelial cell in an organ-specific stem cell culture device.
  • FIG. 3 is a perspective view showing another example of an organ-specific stem cell culture apparatus to which the present invention is applied.
  • FIG. 4 is a perspective view showing still another example of the organ-specific stem cell culturing apparatus to which the present invention is applied.
  • FIG. 5 is a cross-sectional view of a principal part schematically showing an adhesion state between an organ-specific stem cell and a vascular endothelial cell in the culture device shown in FIG.
  • FIG. 6 is a photograph of the vicinity of the umbilical mesenteric artery in a fetal mouse immunostained with a TIE2 antibody, c-Kit antibody or Flk-1 antibody.
  • FIG. 7 is a photograph showing the results of verification of activation of TIE2 and induction of cell adhesion by angiopoietin-1. '
  • FIG. 8 is a photograph showing the vicinity of the umbilical mesenteric artery in a wild type mouse and a TIE2 gene knockout mouse.
  • FIG. 9A shows the result of histological observation in fetal liver, and is a photograph showing a site where hematopoietic stem cells are proliferating in a nested network composed of vascular pericytes.
  • FIG. 9B is an enlarged photograph of a part of FIG. 9A.
  • FIG. 9C is a photograph showing the results of histological observation in adult bone marrow and showing a site where hematopoietic stem cells proliferate in the vascular lumen.
  • FIG. 10 is a photograph showing a site where hematopoietic stem cells proliferate in the umbilical mesenteric artery of a transgenic mouse transfected with a constitutively activated mouse TIE2 gene.
  • FIG. 11 is a photograph showing the results of examining the cell adhesion between bEnd3 expressing IS constitutively activated TIE2 or wild-type TIE2 and Ba / F3 cells expressing constitutively activated TIE2.
  • FIG. 12 is a photograph showing that hematopoietic stem cells self-replicate in a state where hematopoietic stem cells and vascular endothelial cells are loosely adhered.
  • Figures 13A and 13B are photographs showing that even hematopoietic stem cells recovered from human cord blood proliferated by creating a loosely adherent state with vascular endothelial cells.
  • an organ-specific stem cell is an adult stem cell having a function of differentiating into a specific organ.
  • organ-specific stem cells include hematopoietic stem cells, neural stem cells, vascular stem cells, myocardial stem Z precursor cells, neural stem cells, hepatic stem cells, and the like.
  • hematopoietic stem cells When hematopoietic stem cells are used as organ-specific stem cells, hematopoietic stem cells can be collected from the whole body, including, for example, bone marrow cells collected from bone marrow, umbilical cord blood, peripheral blood, fetal liver, and fetal spleen. More specifically, hematopoietic stem cells are isolated as lin-negative, c-kit-positive, and CD34-positive (or negative) cells from umbilical cord blood or nucleus cell components collected from bone marrow or peripheral blood using flow cytometry or the like. be able to.
  • vascular endothelial cells can be collected from the whole body, including, for example, a bone marrow cell group collected from bone marrow, umbilical cord blood, umbilical vein, and umbilical artery. More specifically, vascular endothelial cells can be isolated as CD45-negative and CD31-positive cells from a bone marrow cell group collected from bone marrow using flow cytometry or the like.
  • the organ-specific stem cells and vascular endothelial cells may be derived from any animal, and include, for example, human, monkey, mouse, rat and the like.
  • organ-specific stem cells obtained by the expansion method according to the present invention are used in regenerative medicine
  • organ-specific stem cells and vascular endothelial cells collected from patients undergoing regenerative medicine are required.
  • vesicles are used.
  • the organ-specific stem cells are slowly adhered to the vascular endothelial cells.
  • Loose adhesion refers to histological observation that hematopoietic stem cells proliferate near vascular endothelial cells without completely dissociating from vascular endothelial cells. Means the presence of targeted stem cells. Further, histologically loose adhesion can also be defined as a state in which organ-specific stem cells and vascular endothelial cells adhere via pseudopods.
  • the organ-specific stem cells and the vascular endothelial cells In a state where the organ-specific stem cells and the vascular endothelial cells are loosely adhered to each other, the organ-specific stem cells and the vascular endothelial cells have at least a distance that can be influenced by molecules secreted by the organ-specific stem cells and the vascular endothelial cells. . More specifically, when the organ-specific stem cells and the vascular endothelial cells are loosely adhered to each other, the distance between the organ-specific stem cells and the vascular endothelial fibroblasts is 1 to: ⁇ ⁇ . Is preferred. More specifically, by positioning vascular endothelial cells outside the hollow fiber and organ-specific stem cells inside the hollow fiber, the organ-specific stem cells can be slowly adhered to the vascular endothelial cells.
  • the culture conditions for growing the organ-specific stem cells are generally medium used for animal cell culture and various conditions (temperature, pH, C0 2, etc. conditions) was left or modifications of its not particularly limited Can be applied.
  • Examples of the culture solution include a DMEM culture solution, a MEM culture solution, an ⁇ -MEM culture solution, an RPMI culture solution, and a DMEM / F12 culture solution.
  • those obtained by adding an appropriate amount of bovine serum to these culture solutions may be used.
  • the amount of bovine serum added is not particularly limited, and is appropriately set according to the origin and type of the cells. Preferably, 0% to 20%, more preferably about 5% to 10% bovine serum is added. Nutridoma (Boehringer), human serum, etc.
  • Conditions such as culturing temperature and C0 2 is appropriately set depending on the nature of the cells used, generally 4 to 6% C0 2, is performed at 33 to 37 ° C.
  • the culture period of the cells is not particularly limited, and the culture may be performed while appropriately changing the medium until the growth of the organ-specific stem cells J5 is completed.
  • the organ-specific stem cells may be expanded by the expansion method according to the present invention, and differentiation may be induced in the expanded organ-specific stem cells.
  • a cytokine that promotes differentiation and proliferation of cells may be appropriately added to the culture solution.
  • tokine examples include EGF family such as EGF, HB-EGF, FGF and HGF, TGF family such as TGF-a, jS and BMP, and IL !; ⁇ 17 IL families, kinetics such as GM-CSF, M-CSF, G-CSF, Epo, TPO, SCF, etc., VEGF family such as VEGF-A, PDGF family such as PDGF-BB, and ephrin family such as ephrin B Lee, LIF, TNFa, etc.
  • EGF family such as EGF, HB-EGF, FGF and HGF
  • TGF family such as TGF-a, jS and BMP, and IL !
  • ⁇ 17 IL families kinetics such as GM-CSF, M-CSF, G-CSF, Epo, TPO, SCF, etc.
  • VEGF family such as VEGF-A
  • PDGF family such as PDGF-BB
  • cytokine ⁇ cells The amount of added cytokine is appropriately determined depending on the properties of cytokine ⁇ cells to be used.
  • hematopoietic stem cells can be obtained by site cytokines such as IL-1-17 IL family, GM-CSF, M-CSF, G-CSF, ⁇ , TP0, and SCF. To blood cells.
  • the apparatus for growing organ-specific stem cells according to the present invention can maintain the state in which the organ-specific stem cells are allowed to adhere slowly to vascular endothelial cells, enabling the growth of the organ-specific stem cells as described above. It has a configuration. That is, the proliferation device according to the present invention is a device for culturing the organ-specific stem cells while maintaining the state in which the organ-specific stem cells are slowly adhered to the vascular endothelial cells.
  • the breeding apparatus shown in FIG. 1 can be mentioned.
  • This proliferation apparatus comprises a first culture unit 1 for culturing organ-specific stem cells, a second culture unit 2 for culturing vascular endothelial cells, a first culture unit 1 and a second culture unit. And a porous member 3 that partitions the space between the two.
  • the growth apparatus shown in FIG. 1 includes a supply path for supplying a culture solution containing organ-specific stem cells into the first culture unit 1, and vascular endothelial cells in the second culture unit 2 It is desirable to provide a supply path for supplying the culture solution.
  • a container having a predetermined thickness such as a culture dish generally used for culturing animal cells, can be used.
  • a membrane having pores 4 having a diameter of 0.1 to 30 ⁇ , preferably 1 to 10111, and more preferably 2 to 5 ⁇ m can be used.
  • the porous member 3 is from 1 to 200 111, preferably from 2 to
  • the thickness is 5 to 10 ⁇ .
  • the porous member 3 includes polyethylene, cenorellose diacetate, polyatarilonit linole, polysnoreon, polymethylmethallate, polyphenylene ether, Examples include, but are not limited to, polycarbonate, polylactic acid, polycaprolactone, polyhydroxybutyric acid, polyimide, and the like.
  • the surface of the porous member 3 is preferably coated with a cell matrix component.
  • a cell matrix component By coating the cell matrix component, adhesion of organ-specific stem cells and vascular endothelial cells to the surface of the porous member 3 can be promoted.
  • the cell matrix component is not particularly limited, and vitronectin and / or fibronectin can be used.
  • a culture solution containing organ-specific stem cells is supplied to the first culture unit 1, and vascular endothelium is supplied to the second culture unit 2.
  • the organ-specific stem cells supplied to the first culturing unit 1 and the vascular endothelial cells supplied to the second culturing unit 2 gradually adhere via the porous member 3. That is, as schematically shown in FIG. 2, the vascular endothelial cells 6 cause their cytoplasm to protrude from the pores 4 into the first culture part 1 and slowly adhere to the organ-specific stem cells 5. It will be.
  • the organ-specific stem cells in the first culturing unit 1 can be expanded by setting the culture conditions described in the above “Method for culturing organ-specific stem cells”.
  • the multiplication apparatus shown in FIG. 3 has a configuration in which a plurality of 'first culturing units la and lb' and a plurality of second culturing units 2a, 2b and 2c are alternately overlapped.
  • the porous member 3 is provided between the second culture part 2a and the first culture part la, between the second culture part 2b and the first culture part la, and between the second culture part 2b and the first culture part la.
  • the second culturing unit 2c and the first culturing unit 1b are provided between the first culturing unit 1b and the second culturing unit 2c.
  • the growth apparatus shown in FIG. 3 can supply a culture solution or the like independently to the plurality of first culture units 1a, 1b and the plurality of second culture units 2a, 2b, 2c.
  • the apparatus further includes a supply path 7 and a discharge path 8 connected to the plurality of first culture units 1a, 1b and the plurality of second culture units 2a, 2b, 2c, respectively.
  • organ-specific stem cells can be proliferated in the same manner as the proliferating apparatus shown in FIG.
  • multiple culture sections la and 1 In b different types of organ-specific stem cells can be proliferated, or the same type of organ-specific stem cells can be proliferated.
  • FIG. 4 is a diagram schematically showing the breeding apparatus, and the configuration other than the plurality of hollow fibers 10 is omitted.
  • hollow fiber 10 composed of a porous member
  • examples of the hollow fiber 10 composed of a porous member include polyethylene, cellulose diacetate, polyacrylonitrile, polysulfone, polymethyl methacrylate, polyphenylene ether, polycarbonate, polylactic acid, polycaprolactone, and polyhydroxy.
  • Butyric acid, polyimide, and the like can be used, but are not limited thereto.
  • the proliferation device shown in FIG. 4 immerses a region excluding both ends of a plurality of hollow fibers 10 into a culture solution containing vascular endothelial cells, A culture solution containing specific stem cells can be supplied and used.
  • the propagation device shown in FIG. 4 may include a tubular member having a shape sufficient to insert the entirety of the plurality of hollow fibers 10.
  • the proliferation device is configured such that the vascular endothelial cell is formed between the inner surface of the tubular member and the outer peripheral surface of the plurality of hollow fibers 10 with the plurality of hollow fibers 10 inserted inside the tubular member.
  • a culture solution containing an organ-specific stem meniscus from one end of the plurality of hollow fibers 10 can be supplied and used.
  • the organ-specific stem cells supplied into the plurality of hollow fibers 10 and the vascular endothelial cells supplied to the gap formed by the plurality of hollow fibers 10 are the outer surfaces of the hollow fibers. (Porous member) to adhere gently. That is, as schematically shown in FIG. 5, the vascular endothelial cell 6 causes its cytoplasm to protrude from the hole 4 into the hollow fiber, and slowly adheres to the organ-specific stem cell 5. In this state, the organ-specific stem cells in the hollow fiber can be proliferated by setting the culture conditions described in the above “Method for culturing organ-specific stem cells”.
  • the manufacturing method and manufacturing apparatus of the organ-specific stem cell which concerns on this invention, the proliferation of the organ-specific stem cell in in vitro which was difficult in the past becomes possible. It is also possible to induce the differentiation of the obtained organ-specific stem cells to obtain desired organ cells (blood cells, neural cells, etc.). The obtained organ cells or organ-specific stem cells can be used for regenerative medicine.
  • organ-specific stem cells or vascular endothelial cells used as the material are those of a mammal to be transplanted
  • organ-specific stem cells or organs that can be transplanted without causing rejection in the mammal will be used.
  • Cells can be obtained. That is, the organ-specific stem cells and organ cells obtained by the method of the present invention can be suitably used for regenerative medicine in which rejection is prevented. Therefore, the heart regeneration according to the present invention makes a great contribution to the medical industry.
  • hematopoietic stem cells in the fetal period firstly enter the umbilical mesenteric artery, the only artery connecting the fetus and the yolk sac. It has occurred. Then, the hematopoietic stem cells adhere to vascular endothelial cells in the artery to form a cell mass.
  • the receptor-type tyrosine kinase TI E2 which is expressed on the membrane of hematopoietic stem cells, is activated by its ligand, angiopoietin-l, to induce cell adhesion of hematopoietic stem cells to collagen fibronectin. Is done.
  • angiopoietin-l ligand that stimulates cell adhesion of hematopoietic stem cells to collagen fibronectin.
  • TIE2 knockout mice adhesion of hematopoietic stem cells was not observed in the umbilical mesenteric artery where fetal hematopoietic stem cells adhere to vascular endothelial cells and cluster formation is observed. This indicates that TIE2 is involved in the adhesion of hematopoietic stem cells to vascular endothelial cells via the function of cell adhesion to Matrittas (see FIG. 8).
  • fetal liver which is a hematopoietic tissue
  • Histological observation of fetal liver revealed that hematopoietic stem cells were surrounded by vascular endothelial cells, as shown in Figs. 9A and 9B. It was revealed that there are sites where stem cells are proliferating. Histological analysis revealed that hematopoietic stem cells did not adhere firmly to endothelial cells in the areas shown in Figures 9A and B, but proliferated in a pseudopod-like structure. became. Analysis of the localization of hematopoietic stem cells in adult bone marrow revealed the sites where hematopoietic stem cells proliferate in the vascular lumen in some vascular regions, as shown in Figure 9C.
  • Example 2 based on the findings obtained in Example 1, the effect of the strength of adhesion between hematopoietic stem cells and vascular endothelial cells on the proliferation of hematopoietic stem cells was examined.
  • a constitutively activated mouse TIE2 gene was prepared as follows. In wild-type mouse TIE2, a mutant mouse TIE2 gene was constructed so that the 849th Arg located in the region immediately below the membrane was changed to Trp. Constitutively active dani-type mouse TIE2 can form a dimer without angiopoietin-1 binding, and is constantly activated.
  • transgenic mice were prepared in which the obtained constitutively activated mouse TIE2 gene was expressed under the control of the TIE2 promoter.
  • the mouse died of anemia on the 10th day of pregnancy.
  • Embryos of day 9 of pregnancy were embedded in wax, and sections were cut against CD31 expressed at this stage on both hematopoietic stem cells and vascular endothelial cells.
  • Immunostaining with an antibody Pieringen.
  • a site where hematopoietic stem cells proliferate in the umbilical mesenteric artery is observed (Fig.
  • CD31-positive lk stem cells had a morphologically flat shape and were firmly adhered to vascular endothelial cells, and the number of hematopoietic stem cells was decreased compared to that of normal mice (Fig. 10, right).
  • vascular endothelial cells must be present in the vicinity of hematopoietic stem cells in order to proliferate hematopoietic stem cells.However, when hematopoietic stem cells and vascular endothelial cells are firmly adhered to, hematopoietic stem a Found to inhibit.
  • Example 2 based on the results of Example 2, the induction of adhesion between a blood cell line and a vascular endothelial cell line by a constantly activated ⁇ 2 in vitro was examined.
  • the constitutively activated TIE2 gene and the wild-type TIE2 gene prepared in Example 2 were incorporated into a pMy retrovirus vector, and a Winores solution was prepared according to a conventional method.
  • bEnd3 vascular endothelial cell line
  • Type TIE2 was expressed.
  • the cells were cultured in a culture dish having a diameter of 3 cm.
  • the Ba / F3 Itoda cells expressing constitutively active I ⁇ TIE2 was Rabenore to a red fluorescent color with PKH26 (Sigma Corp.), were plated 10 4 on the previous bEnd3 cells.
  • the cells floating in the culture dish were collected, and the adhesion of the red fluorescently labeled Ba / F3 cells was observed with a fluorescence microscope (IX-70: manufactured by Olympus Corporation).
  • Figure 11 shows the results.
  • Figure 11 right shows the result of seeding Ba / F3 cells expressing constitutively activated TIE2 on bEnd3 cells expressing wild-type TIE2
  • Figure 11 left shows bEnd3 cells expressing constitutively activated TIE2
  • the results of seeding Ba / F3 cells expressing constitutively activated TIE2 are shown above.
  • TIE2 is also expressed in skin cells, it was revealed that angiopoietin-1 secreted by hematopoietic stem cells also activated TIE2 expressed in vascular endothelial cells. Further, it was revealed that it is a favorable condition that both TIE2 expressed in hematopoietic stem cells and TIE2 expressed in vascular endothelial cells are activated to proliferate hematopoietic stem cells.
  • bone marrow cells were collected from the bone marrow of 8-week-old C57B1 / 6 mice using a conventional method.
  • the bone marrow cells were stained with CD45 and CD31 antibodies (both from Pharmingen), and CD45-negative CD31-positive vascular endothelial cells were collected by flow cytometry (Epics coulter).
  • FIG. 12 shows the results.
  • FIG. 12 left is a phase contrast microscope image
  • FIG. 12 right is a fluorescence microscope image.
  • hematopoietic stem cells could self-replicate to 2-3 cells by culturing for 2 to 3 days in a state where 5t blood stem cells and vascular endothelial cells were loosely adhered.
  • the intravascular cell line MSSI31 was cultured in a culture medium containing 10% bovine serum using RPMI1640 (manufactured by Sigma) as a basic medium on a normal culture dish to obtain a confluent state.
  • MSS31 transfected with constitutively activated TIE2 was cultured under the same conditions to be in a confluent state.
  • mononuclear cells were fractionated from human cord blood using Ficoll (manufactured by Amersham).
  • Ficoll manufactured by Amersham
  • Lin-negative c-kit-positive CD34-positive cells using human Lin antibody (manufactured by Coulter), antibodies against c-Kit and CD34 (all manufactured by Pharmingen). Epics coulter).
  • FIGS. 13A and 13B The results are shown in FIGS. 13A and 13B.
  • FIG. 13A left is a phase contrast microscope image when wild-type MSS31 is used, and FIG.
  • FIG. 13A right is a fluorescence microscope image when wild-type MSS31 is used.
  • FIG. 13B left is a phase contrast microscopy image when the constitutively activated ⁇ 2 introduced MSS31 is used, and
  • FIG. 13B right is a fluorescence microscopic image when the constitutively activated ⁇ 2 introduced MSS31 is used.
  • the present invention it is possible to provide a novel method and apparatus for expanding organ-specific stem cells such as hematopoietic stem cells. According to the present invention, it is possible to establish a system for enriching organ-specific stem cells in vitro, and it is possible to widely use organ-specific stem cells in regenerative medicine. Therefore, the present invention makes a great contribution especially to the regenerative medicine industry.

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Abstract

Une prolifération au niveau des cellules souches de cellules souches spécifiques à un organe cible. Il est proposé un procédé de prolifération de cellules souches spécifiques à un organe, comprenant la culture de cellules souches spécifiques à un organe et de cellules vasculaires endothéliales causant une faible adhérence des cellules entre elles.
PCT/JP2005/006424 2004-04-14 2005-03-25 Procédé de prolifération de cellules souches spécifiques d’un organe et appareil de prolifération pour celui-ci Ceased WO2005100549A1 (fr)

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JP2012044908A (ja) * 2010-08-25 2012-03-08 Nomura Unison Co Ltd 細胞培養用中空糸モジュールおよび細胞培養方法
WO2012032646A1 (fr) * 2010-09-10 2012-03-15 株式会社島津製作所 Dispositif de culture cellulaire et procédé de culture cellulaire
CN103849567A (zh) * 2012-12-06 2014-06-11 中国科学院大连化学物理研究所 非接触共培养诱导干细胞体外三维定向分化的生物反应器
JP2016505266A (ja) * 2013-01-15 2016-02-25 コーネル ユニヴァーシティー 確定因子によるヒト内皮の造血性多系列前駆細胞へのリプログラミング

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WO2009139177A1 (fr) * 2008-05-15 2009-11-19 国立大学法人大阪大学 Procédé d'induction de plaquettes sanguines
EP2298865A4 (fr) * 2008-05-15 2012-06-27 Univ Osaka Procédé d'induction de plaquettes sanguines
US8535943B2 (en) 2008-05-15 2013-09-17 Osaka University Blood platelet induction method
JP2012044908A (ja) * 2010-08-25 2012-03-08 Nomura Unison Co Ltd 細胞培養用中空糸モジュールおよび細胞培養方法
WO2012032646A1 (fr) * 2010-09-10 2012-03-15 株式会社島津製作所 Dispositif de culture cellulaire et procédé de culture cellulaire
JP5700460B2 (ja) * 2010-09-10 2015-04-15 株式会社島津製作所 細胞培養デバイス及び細胞培養方法
CN103849567A (zh) * 2012-12-06 2014-06-11 中国科学院大连化学物理研究所 非接触共培养诱导干细胞体外三维定向分化的生物反应器
JP2016505266A (ja) * 2013-01-15 2016-02-25 コーネル ユニヴァーシティー 確定因子によるヒト内皮の造血性多系列前駆細胞へのリプログラミング
US10113149B2 (en) 2013-01-15 2018-10-30 Cornell University Reprogramming of human endothelium into hematopoietic multi-lineage progenitors by defined factors

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