WO2006134951A1 - Long-term culture proliferation method for fat-derived stem cell - Google Patents

Long-term culture proliferation method for fat-derived stem cell Download PDF

Info

Publication number
WO2006134951A1
WO2006134951A1 PCT/JP2006/311882 JP2006311882W WO2006134951A1 WO 2006134951 A1 WO2006134951 A1 WO 2006134951A1 JP 2006311882 W JP2006311882 W JP 2006311882W WO 2006134951 A1 WO2006134951 A1 WO 2006134951A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
stem cells
cell
adipose
derived stem
Prior art date
Application number
PCT/JP2006/311882
Other languages
French (fr)
Japanese (ja)
Inventor
Kotaro Yoshimura
Daisuke Matsumoto
Tomokuni Shigeura
Original Assignee
Biomaster, Inc.
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 Biomaster, Inc. filed Critical Biomaster, Inc.
Priority to JP2007521313A priority Critical patent/JPWO2006134951A1/en
Publication of WO2006134951A1 publication Critical patent/WO2006134951A1/en

Links

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
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

  • the present invention relates to a method for culturing undifferentiated pluripotent stem cells, and an isolated or cultured pluripotent stem cell.
  • the present invention relates to a novel long-term culture growth method for adipose-derived stem cells, and adipose-derived stem cells obtained using the same.
  • Pluripotent stem cells can proliferate and repeat passages, and have the ability to differentiate into various types of mature cells (eg, bone cells, cardiomyocytes, blood cells, skin cells, adipocytes, bone marrow cells, etc. Pluripotency). It is considered that tissue and organs can be repaired and regenerated by culturing pluripotent stem cells removed from a living body, further inducing differentiation, and then returning to the living body again. As pluripotent stem cells, embryonic stem cells (ES cells), mesenchymal stem cells, etc. are generally known.
  • ES cells embryonic stem cells
  • mesenchymal stem cells etc.
  • Stromal vascular fraction cells isolated from adipose tissue are known to be adipogenic precursors with a fibroblast-like morphology (Van et al. J Clin Invest 1976; 58 : 699-704: Non-patent document 1), which has been called adipose precursor cells, vascular stromal cells, and the like.
  • adherent stromal cells isolated from adipose tissue are referred to as adipose—derived stromal (or stem) cells (ASC).
  • ASC derived stromal (or stem) cells
  • Monoclonal nanole Zuk et al. Mol Biol Cell 2002; 13: 4279-4295: Non-patent literature 2
  • Polyclonal nanole Zuk et al.
  • Biochem Biophys Res Commun 2002 only in the mesenchymal line such as Mizuno et al. Plast Reconstr Surg 2002; 109: 199—209: Non-patent document 6) 294: 371-379: Non-patent document 7), angiogenesis (Planat- Benard et al. Circulation 2004; 109: 656-663: non-patent document 8), and hematopoietic (Cousin et al. Biochem Biophys Res) Commun 2003; 301: 1016-1022: Non-patent literature 9) It has also been shown by numerous in vitro and in vivo studies.
  • Adipose-derived stem cells can be used clinically without cell culture because a sufficient number of ASCs can be obtained by treating a large volume (eg, 500 ml ⁇ ) of lipoaspirate. Can be done. The use of freshly isolated cells can lead to higher safety and effectiveness in actual treatment.
  • newly isolated ASCs are found in bone lesions (Lendeckel et al. J Craniomaxilofac Surg 2004; 3 2: 370—373: Non-patent literature 10) and rectal vaginal fistulas (Garcia— Olmo et al. Int J Colorectal Dis 2003; 18: 451-454: Non-patent literature11), or used in some clinical trials, such as soft tissue augmentation.
  • Liposuction is one of the most popular cosmetic surgery, and it is estimated that over 800,000 procedures are performed every year worldwide. Liposuction is clinically capable of obtaining large volumes of adipose tissue and is considered a typical method for recovering ASC. However, at present, lipoaspirates have not been well studied for use in clinical situations.
  • a lipoaspirate is composed of a fat portion and a liquid portion.
  • fat part is meant aspirated adipose tissue that has been “chopped” by the interaction of force neutrons and reduced pressure.
  • the liquid part means a liquid sucked together with the fat part. Liquid is mainly (1
  • Patent Document 1 International Publication No. WO2005 / 035738 Pamphlet
  • Patent Document 2 International Publication No. WO2005 / 042730 Pamphlet
  • Non-Patent Document 1 Van et al. J Clin Invest 1976; 58: 699—704
  • Non-patent literature 2 Zuk et al. Mol Biol Cell 2002; 13: 4279-4295
  • Non-patent literature 3 Zuk et al. Tissue Eng 2001; 7: 211-228
  • Non-Patent Document 4 Erickson et al. Biochem Biophys Res Commun 2002; 29
  • Patent Document 5 Halvorsen et al. Int J Obes Relat Metab Disord 2000; 24 Suppl 4: S41-44
  • Non-Patent Document 6 Mizuno et al. Plast Reconstr Surg 2002; 109: 199-20 9
  • Non-Patent Document 7 Saff ord et al. Biochem Biophys Res Commun 2002; 29 4: 371-379
  • Non-patent literature 8 Planat— Benard et al. Circulation 2004; 109: 656-663
  • Non-patent literature 9 Cousin et al. Biochem Biophys Res Commun 2003; 30 1: 1016-1022
  • Non-Patent Document 10 Lendeckel et al. J Craniomaxilofac Surg 2004; 32: 370 -373
  • Non-Patent Document 11 Garcia— Olmo et al. Int J Colorectal Dis 2003; 18:45 1-454
  • the present inventors have performed detailed characterization as a cell source not only for the fat portion of the lipoaspirate but also for the liquid portion. As a result, it was found that the liquid partial force of lipoaspirate, and a considerable number of fat-derived stem cells (ASC) can be isolated. [0010] Furthermore, the present inventors conducted a comparative experiment between newly isolated ASC and cultured ASC. As a result, unexpectedly, by using the in vitro culture conditions of the present inventors, ASC that maintained the expression ability of CD34 and CD105 even after long-term culture of ASC (for example, 20 weeks) was liposuctioned. It was found that it can be obtained from both the fat and liquid parts of the product. In addition, ASCs obtained from the fat portion and the liquid portion were cultured in a differentiation-inducing medium, and it was confirmed that they had similar differentiation ability along the adipogenic, cartilage-forming, and osteogenic strains.
  • the present invention has been completed based on these findings, and has the following characteristics: isolated pluripotent stem cell, cultured pluripotent stem cell, cultured adipose-derived stem cell, fat Provided are a method for preparing a stem cell, and use of the stem cell in regenerative medicine.
  • An isolated pluripotent stem cell that can express a cell surface marker of CD34, CD105, or both.
  • a cultured pluripotent stem cell that is capable of expressing CD34, CD105, or both cell surface markers.
  • a cultured adipose-derived stem cell that can express cell surface markers of CD34, CD105, or both.
  • a method for preparing CD34 + and / or CD105 + adipose-derived stem cells comprising a step of culturing newly isolated adipose-derived stem cells.
  • the culture medium for the culture is
  • the culture is a
  • the step of isolating the fat-derived stem cells comprises
  • the culture medium for the culture is
  • a fat-derived stem cell prepared by the method according to any one of (17) to (35) above, which expresses a cell surface marker of CD34, CD105, or both of them.
  • a regenerative medical material comprising the cell according to any one of (1) to (6) above or (36) above.
  • a method for producing a material for regenerative medicine which comprises the step of using the cells according to (1) to (6) above or any one of (1) to (6) above or (36).
  • a composition comprising pluripotent stem cells, the composition comprising at least 10% pluripotent stem cells capable of expressing CD34, CD105, or both cell surface markers.
  • composition according to (41), wherein the composition comprises at least 10% of pluripotent stem cells capable of expressing a cell surface marker of CD34.
  • a method for preparing a composition comprising CD34 + and / or CD105 + adipose-derived stem cells comprising the step of A) culturing newly isolated adipose-derived stem cells. (46) The method according to (45), wherein the adipose-derived stem cells are cultured for at least 2 weeks.
  • step of obtaining the lipoaspirate further includes a step of separating the liquid portion.
  • the isolated or cultured pluripotent stem cell of the present invention can express a clinically important surface marker such as CD34 or CD105, fat regeneration, bone regeneration, myocardial regeneration, blood vessel It is advantageous for use in regenerative medicine such as regenerative and refractory ulcers.
  • CD34 is widely considered to be a marker that represents the undifferentiated state of stem cells, and both positive cells with CD105, a mesenchymal marker, are expected to be effective for mesenchymal cell * tissue regeneration. .
  • the human-derived ASC obtained by the method for preparing ASC of the present invention can express clinically important surface markers such as CD34 and CD105 even after long-term culture growth.
  • ASC having such properties can provide an advantageous material for application to regenerative medicine such as fat regeneration, bone regeneration, myocardial regeneration, angiogenesis, and refractory ulcer.
  • the method for preparing pluripotent stem cells of the present invention can prepare a large number of pluripotent stem cells capable of expressing clinically important surface markers such as CD34 and CD105. Materials that can be used for the present invention can be advantageously provided.
  • suctioned fat is a tissue that is routinely discarded, but can be effectively used according to the present invention. In addition, it provides the advantage of effective use of previously discarded liquid parts.
  • FIG. 1 is a photograph and a graph showing isolated fat-derived stromal cells (ASC) of a lipoaspirate.
  • FIG. 2 is a photograph and graph showing the results of cell differentiation analysis using adherent PLA cells or adherent LAF cells at passage 3-5.
  • FIG. 3 is a graph and photographs ( a and b) showing CD34 and CD45 expression of newly isolated LAF cells.
  • Fig. 3-2 is a continuation of Fig. 3-1.
  • FIG. 4 is a diagram ( a to n) showing a comparison of flow cytometry data of PLA cells and LAF cells newly isolated or cultured for 2 weeks.
  • Figure 4-4 is a continuation of Figure 4-3.
  • Figure 4-10 is a continuation of Figure 4-9.
  • Figure 4-11 is a continuation of Figure 4-10.
  • Figure 4-12 is a continuation of Figure 4-11.
  • Figure 4-13 Figure 4-13 is a continuation of Figure 4-12.
  • Figure 4-14 Figure 4-14 is a continuation of Figure 4-13.
  • FIG. 5 is a diagram (at) showing a continuous change in the expression of representative cell surface markers of newly isolated or cultured PLA cells and LAF cells.
  • Fig. 5--7 Fig. 5— • 7 is a continuation of Fig. 5-6.
  • Fig. 5--10 is a continuation of Fig. 5_9.
  • Figure 5--11 is a continuation of Figure 5-10.
  • Figure 5--12 is a continuation of Figure 5-11.
  • Figure 5--13 is a continuation of Figure 5-12.
  • Figure 5--14 is a continuation of Figure 5-13.
  • Figure 5--15 is a continuation of Figure 5-14.
  • Fig. 5--16 is a continuation of Fig. 5-15.
  • Figure 5--17 is a continuation of Figure 5-16.
  • Fig. 5--18 is a continuation of Fig. 5-17.
  • Figure 5--19 is a continuation of Figure 5-18.
  • Fig. 5--20 is a continuation of Fig. 5-19.
  • FIG. 6 A graph showing changes in the ratio of CD34 positive cells in newly isolated PLA cells and LAF cells up to 20 weeks.
  • FIG. 7 is a graph showing changes in the ratio of CD105 positive cells in newly isolated PLA cells and LAF cells up to 20 weeks.
  • cell is used herein in its broadest sense in the art, and has an internal self-regenerative capability, has genetic information and its expression mechanism, and is an organism such as a cell. Is a structural unit of tissue of multicellular organisms that is enveloped in a membrane structure that also isolates external forces. In the method of the present invention, any cell can be used as a subject.
  • the number of cells used in the present invention can be counted with an optical microscope. When counting using an optical microscope, the number of nuclei is counted. Tissue is made into tissue section slices and then hematoxylin-eosin (HE) stained to identify extracellular matrix (eg, elastin or collagen) and cell-derived nuclei.
  • HE hematoxylin-eosin
  • the cells used herein may be naturally occurring cells or artificially modified cells (eg, fusion cells, genetically modified cells, etc.).
  • Examples of cell sources include a single cell culture; a normally grown embryo of a transgenic animal, blood, or body tissue; or a cell mixture such as cells from a normally grown cell line. However, it is not limited to them. Such sources themselves can be used as cells.
  • fat cells and corresponding substances may be any substance as long as the organism has fat cells or cells corresponding thereto.
  • Organisms e.g. metallunagi, shark eels, cartilaginous fish, teleosts, amphibians, reptiles, birds, mammals, etc.
  • mammals e.g. metallunagi, shark eels, cartilaginous fish, teleosts, amphibians, reptiles, birds, mammals, etc.
  • mammals e.g. metallunagi, shark eels, cartilaginous fish, teleosts, amphibians, reptiles, birds, mammals, etc.
  • mammals e.g.
  • Wings, wings, carnivores, carnivores, long-nosed animals, odd-hoofed animals, even-hoofed animals, rodents, scales, sea cattle, cetaceans, primates, rodents, maggots) Can be derived from In one embodiment, cells from a primate (eg, chimpanzee, dihonkey, human) are used. Most preferably, human-derived cells are used, but the invention is not so limited.
  • a primate eg, chimpanzee, dihonkey, human
  • stem cell refers to a precursor (or progenitor cell) of a differentiated cell having unipotency, pluripotency, or totipotency.
  • Stem cells can be differentiated in response to specific stimuli. Typically, stem cells can regenerate damaged tissue.
  • Stem cells used herein can be embryonic stem (ES) cells, tissue stem cells (also referred to as tissue stem cells, tissue-specific stem cells or somatic stem cells), or other progenitor cells. Not. As long as it has the above-mentioned ability, the stem cell may be an artificially produced cell (for example, a fusion cell described herein, a reprogrammed cell, etc.).
  • Embryonic stem cells are pluripotent stem cells derived from early embryos. Embryonic stem cells were first established in 1981 and have been applied since 1989 to the production of knockout mice. In 1998, human embryonic stem cells were established and recently used in regenerative medicine. Tissue stem cells, unlike embryonic stem cells, have a relatively limited level of differentiation potential. Tissue stem cells are present in tissues and have an undifferentiated intracellular structure. Tissue stem cells have a higher nucleus / cytoplasm ratio and a few intracellular organelles. Most tissue stem cells are pluripotent and maintain proliferative capacity over the life of an individual with a slow cell cycle. As used herein, stem cells are preferably tissue stem cells, although embryonic stem cells can also be used depending on the situation.
  • tissue stem cells When classified according to the site of origin, tissue stem cells are classified into, for example, the skin system, digestive system, myeloid system, nervous system and the like.
  • skin tissue stem cells include epidermal stem cells and hair follicle stem cells.
  • digestive tissue stem cells include knee (common) stem cells and liver stem cells.
  • myeloid tissue stem cells include hematopoietic stem cells and mesenchymal stem cells.
  • Neural tissue stem cells include neural stem cells and retinal stem cells.
  • progenitor cell refers to an undifferentiated parent cell that does not express a differentiation trait when it is clear that a cell that is a progeny expresses a specific differentiation trait.
  • the progenitor cell includes progenitor cells.
  • progenitor cells obtained by stem cell differentiation can respond to “differentiated cells” when viewed from the stem cells.
  • PLA asspirated fat-derived cells
  • PLA aspirate cells.
  • PLA processed lipoaspirate cells after their recovered origin (for example, Zuk et al Mol Biol Cell 2002; 13: 4279-4295). Therefore, similarly in this specification, cells isolated from the fat portion of the lipoaspirate are referred to as PLA cells. Moreover, in this specification, the cell isolated from the liquid part of the lipoaspirate is called LAF Qiposucti on aspirate fluid (cell).
  • adipose-derived progenitor cell refers to stem cells and other progenitor cells obtained by liposuction, and other progenitor cells are obtained by this liposuction. Such as peripheral blood-derived stem cells or vascular stromal cells (preadipocytes).
  • An adipose-derived progenitor cell means any pluripotent progenitor cell or a population of unipotent progenitor cells obtained by adipose tissue or liposuction.
  • adipose-derived vascular stromal cells predipocytes, adipose-derived stromal cells
  • adipose-derived stem cells adipose stem cells
  • endothelial progenitor cells hematopoietic stem cells, and the like.
  • a number of technical capabilities for isolating such stem cells such as Nakats uji et al., “Stem Cell / Clone Research Protocol”, Yo dosha (2001); WO00Z53795; W003Z022988; W ⁇ Known as described in 0l / 62901. These documents are incorporated herein by reference in their relevant parts.
  • the term “adipose-derived progenitor cells” "Means all adipose tissue-derived stem cells including adipose tissue-derived stem cells obtained by these known isolation methods.
  • progenitor cells include unipotent undifferentiated cells as well as pluripotent undifferentiated cells.
  • stem cell includes progenitor cells.
  • PLA asspirated fat-derived cells
  • Adipose-derived precursor cells include PLA (aspirated fat-derived cells) and aspirate cells.
  • ASCs adipose-derived stem cells
  • the origin of cells is classified into ectoderm, endoderm and mesoderm.
  • Stem cells of ectoderm origin are mainly present in the brain, including neural stem cells.
  • Endoderm-derived cells are mainly present in the bone marrow, and include vascular stem cells and their differentiated cells, hematopoietic stem cells and their differentiated cells, mesenchymal stem cells and their differentiated cells, and the like.
  • Cells derived from mesoderm are mainly present in organs, and include hepatic stem cells and differentiated cells thereof, knee stem cells and differentiated cells thereof.
  • somatic cells may be derived from any germ layer.
  • mesenchymal stem cells can be used.
  • meenchymal stem cell refers to a stem cell found in mesenchyme.
  • meenchymal stem cell may be abbreviated as “MSC”.
  • MSC mesenchymal stem cell
  • Mesenchyme refers to a population of free cells with stellate or irregular protrusions found in each stage of multicellular animal development that fill the gaps between epithelial tissues.
  • Mesenchyme also refers to tissue formed with intracellular adhesion factors that bind to cells.
  • Mesenchymal stem cells have the ability to proliferate and differentiate into bone cells, chondrocytes, muscle cells, stromal cells, tendon cells, and adipocytes.
  • Mesenchymal stem cells are used for culturing or proliferating bone marrow cells and the like collected from patient force or differentiating them into chondrocytes or osteoblasts. Mesenchymal stem cells also have alveolar bone; It is used as a reconstruction material for bone, cartilage, or joints with arthropathy. There is a great demand for mesenchymal stem cells. Mesenchymal stem cells can also differentiate into blood cells and lymphoid cells. Therefore, there is an increasing demand for mesenchymal stem cells.
  • composition derived from liposuction refers to any substance that is produced when liposuction is performed.
  • liposuction-derived materials include adipose tissue and lipoaspirate.
  • lipoaspirate refers to a liquid that is generated when liposuction is performed.
  • Such lipoaspirates are: (1) liquid aspirated together at the time of liposuction (for example, containing tomesent fluid and blood) or (2) washing the aspirated fat with a solution (for example, physiological saline) This refers to the waste liquid that is generated during the process.
  • adipocyte is located between tissues or forms adipose tissue as a group of loose connective tissue or capillaries, It refers to cells that contain large amounts of lipids. Examples of fat cells include yellow adipocytes and brown adipocytes. These cells can be used equivalently herein. Intracellular fat can be easily detected using Sudan III or osmium tetroxide.
  • tissue refers to a collection of cells having substantially the same function and / or morphology in a multicellular organism.
  • a “tissue” is usually a collection of cells of the same origin, but can be a collection of cells of different origins as long as the cells have the same function and / or morphology.
  • the stem cells of the present invention are used for tissue regeneration, an aggregate strength of cells of two or more different origins can be constructed.
  • the tissue forms part of an organ. Animal tissues are classified into epithelial tissues, connective tissues, muscle tissues, nerve tissues, etc. based on morphological, functional or developmental basis. Plants are roughly divided into meristems and permanent tissues depending on the stage of development of the constituent cells. Alternatively, the tissue can be distinguished into a single tissue and a composite tissue depending on the type of constituent cells. In this way, organizations are divided into various categories. Any tissue can be contemplated as a target to be treated herein.
  • any organ can be a target (such as a treatment) of the present invention.
  • Pairs targeted by the present invention The tissue or cell can be from any organ.
  • organ refers to an independent structure located in a specific part of an individual organism that performs a specific function. In multicellular organisms (eg, animals, plants), an organ is composed of a plurality of tissues spatially arranged in a specific manner, and each tissue is composed of a large number of cells.
  • An example of such an organ is an organ related to the vascular system.
  • organs targeted by the present invention include skin, blood vessels, cornea, kidney, heart, liver, umbilical cord, intestine, nerve, lung, placenta, knee, brain, extremity, retina, etc. But not limited to these.
  • Any organ can be used as a target herein.
  • mesenchymal tissues eg, fat, bone, ligaments, etc.
  • mesenchymal tissues can be targeted, but are not limited to these.
  • the term "corresponding to a desired site” related to cells, tissues, S-ware, etc. means that when the purpose is transplantation or regeneration in the present invention, Forces obtained from the target site (for example, heart-derived cells in the case of the heart) or cells having substantially the same properties as cells existing in the target site (for example, differentiated into heart cells) Cells). Therefore, cells corresponding to a desired site can be confirmed by having substantially the same characteristics (for example, cell surface markers).
  • graft means a tissue piece, cell, composition, medicine, etc. of the present invention that is transferred into the body.
  • the following methods, forms and amounts for introduction into a treatment site can be used: the medicament of the present invention can be directly injected into an injured site, applied and sutured, inserted, etc. Is done.
  • the combination of adipose-derived progenitor cells and differentiated cells of the invention can be administered either simultaneously (eg, simultaneously as a mixture, separately but simultaneously or concurrently); or sequentially.
  • “Combination” administration further includes the separate administration of one of the compounds or agents given first, followed by the second.
  • the term "recipient” refers to an individual that receives cells to be transplanted and the like, and is also referred to as "host”. In contrast, an individual that provides cells to be transplanted is called a “donor”. Recipients and donors can be the same or different.
  • the cells used in the present invention may be autologous (autologous), allogeneic (non-autologous), or heterologous. From the viewpoint of rejection, self-derived cells are preferred. If rejection is not an issue, allogeneic cells can be used.
  • disease, disorder or abnormal condition resulting from the loss of differentiated cells refers to any disease, disorder or abnormal condition involving differentiated cells.
  • differentiated cells are preferably mesenchymal cells, but are not limited thereto.
  • diseases and disorders targeted by the present invention may be those of the circulatory system (such as blood cells).
  • diseases or disorders include anemia (eg, aplastic anemia (particularly severe aplastic anemia), renal anemia, cancerous anemia, secondary anemia, refractory anemia, etc.), cancer Or tumors (eg, leukemia) and their chemotherapy Post-placement hematopoietic failure, thrombocytopenia, acute myeloid leukemia (especially the first remission phase (High-risk group), second remission phase and beyond), acute lymphoblastic leukemia (especially the first remission phase, 2nd remission phase and later), chronic myelogenous leukemia (especially chronic phase, transitional phase), malignant lymphoma (especially 1st remission phase (High-risk group), 2nd remission phase and later), frequent occurrence Myeloma (especially early after onset); heart failure, angina pectoris, myocardial infarction, arrhythmia
  • anemia eg,
  • the diseases and disorders targeted by the present invention may be those of the nervous system.
  • diseases or disorders include, but are not limited to, dementia, stroke and its sequelae, brain tumors, spinal cord injury.
  • the diseases and disorders targeted by the present invention may be those of the immune system.
  • diseases or disorders include, but are not limited to, force S, including T cell deficiency, leukemia and the like.
  • diseases and disorders targeted by the present invention may be those of the musculoskeletal and skeletal systems.
  • diseases or disorders include fractures, osteoporosis, joint dislocation, subluxation, sprains, ligament damage, osteoarthritis, osteosarcoma, Ewing sarcoma, muscular dystrophy, osteogenesis imperfecta, osteochondral dysfunction Examples include, but are not limited to, dysplasia.
  • the diseases and disorders targeted by the present invention may be of the skin system.
  • diseases or disorders include, but are not limited to, alopecia, melanoma, cutaneous malignant lymphoma, angiosarcoma, histiocytosis, blistering, pustulosis, dermatitis, eczema.
  • the diseases and disorders targeted by the present invention may be of the endocrine system.
  • diseases or disorders include hypothalamic Z pituitary disease, thyroid disease, parathyroid (parathyroid) disease, adrenal cortex / medullary disease, abnormal glucose metabolism, abnormal lipid metabolism, abnormal protein metabolism, nucleic acid Metabolic disorders, inborn errors of metabolism (phenylketonuria, galactosemia, homocystinuria, maple syrup urine), abuminemia, Ascorbic acid synthesis deficiency, hyperbilirubinemia, hyperpyrurubinuria, kallikrein deficiency, mast cell deficiency, diabetes insipidus, abnormal sonopressin secretion, gonorrhea, Wolman's disease (acid lipase deficiency), Powers including mucopolysaccharidosis type VI, but not limited to.
  • diseases and disorders targeted by the present invention may be of the respiratory system.
  • diseases or disorders include, but are not limited to, pulmonary diseases (eg, pneumonia, lung cancer, etc.), bronchial diseases and the like.
  • diseases and disorders targeted by the present invention may be of the digestive system.
  • diseases or disorders include esophageal diseases (eg, esophageal cancer), gastric / duodenal diseases (eg, gastric cancer, duodenal cancer), small intestine / colon diseases (eg, colon polyps, colon cancer, Rectal cancer, etc.), biliary tract disease, liver disease (eg, cirrhosis, hepatitis (A, B, C, D, E, etc.), fulminant hepatitis, chronic hepatitis, primary liver cancer, alcoholic liver Disorders, drug-induced liver disorders, etc.), spleen diseases (acute knee inflammation, chronic knee inflammation, knee cancer, cystic knee disease, etc.), peritoneal / abdominal wall / diaphragm diseases (hernia, etc.), Hirschsprung disease, etc. Power is not limited to these.
  • the diseases and disorders targeted by the present invention may be of the urinary system.
  • diseases or disorders include renal diseases (e.g., renal failure, primary glomerular disease, renal vascular disorder, tubular dysfunction, interstitial renal disease, renal disorder due to systemic disease, renal cancer, etc. ), Bladder diseases (eg, cystitis, bladder cancer, etc.), but are not limited thereto.
  • diseases and disorders targeted by the present invention may be of the reproductive system.
  • diseases or disorders include male genital diseases (eg, male infertility, benign prostatic hyperplasia, prostate cancer, testicular cancer), female genital diseases (eg, female infertility, ovarian dysfunction, uterine fibroids, uterus) Adenomyosis, uterine cancer, endometriosis, ovarian cancer, chorionic disease, etc.).
  • amount effective for diagnosis, prevention, treatment or prognosis refers to a medically effective in diagnosis, prevention, treatment (or therapy) or prognosis, respectively. An amount that is recognized as being. Such amounts are well known in the art. It can be determined by one skilled in the art by taking into account various parameters using surgery.
  • the present invention may be used in cosmetic treatment, treatment or improvement.
  • cosmetic purposes include cosmetic treatment for post-surgical or post-traumatic and congenital deformities as well as purely healthy cosmetic purposes.
  • the present invention includes, for example, breast tissue augmentation (breast augmentation), tissue augmentation for upper and lower eyelid depression, and tissue enlargement to facial atrophy, tissue atrophy after facial paralysis, funnel chest, etc. It can be applied to surgery, but is not limited thereto.
  • the present invention can be applied to auricular chondrogenesis for auricular deformation / deformation such as nasal plastic surgery, rhinoplasty, genioplasty (tissue augmentation), forehead plasticity (tissue augmentation), and microtia.
  • auricular chondrogenesis for auricular deformation / deformation such as nasal plastic surgery, rhinoplasty, genioplasty (tissue augmentation), forehead plasticity (tissue augmentation), and microtia.
  • nasal plastic surgery rhinoplasty
  • genioplasty tissue augmentation
  • the medicament may further comprise a pharmaceutically acceptable carrier. Any pharmaceutically acceptable carrier known in the art can be used in the medicament of the present invention.
  • Pharmaceutically acceptable carriers or suitable formulation materials include antioxidants, preservatives, colorants, flavors, diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffering agents. Power, including, but not limited to, delivery vehicles, and / or pharmaceutical adjuvants.
  • the medicament of the present invention is administered in the form of a composition comprising the cells of the present invention and other active ingredients together with one or more physiologically acceptable carriers, excipients or diluents.
  • a suitable vehicle can be water for injection, physiological solution, or artificial cerebrospinal fluid, which can be supplemented with other substances commonly used in compositions for parenteral delivery. is there.
  • Acceptable carriers, excipients or stabilizers used herein are preferably non-toxic to the recipient and are preferably inert at the dosages and concentrations used. And preferably, phosphate, kenate, or other organic acids; ascorbic acid, hytocopherol; low molecular weight polypeptide; protein (eg, serum albumin, gelatin or immunoglobulin); hydrophilic polymer Amino acids (eg glycine, gnoretamine, asparagine, arginine or lysine); monosaccharides, disaccharides and other carbohydrates (including glucose, mannose or dextrin); chelating agents (eg EDTA) Sugar alcohols (eg mannito Or salt-forming counterions (eg, sodium); and / or non-ionic surfactants (eg, Tween, pluronic, or polyethylene glycol (PEG)) It is not limited to these.
  • phosphate, kenate, or other organic acids eglycine, gno
  • suitable carriers include neutral buffered saline or saline mixed with serum albumin.
  • the product is formulated as a lyophilizer using a suitable excipient (eg, sucrose).
  • suitable excipient eg, sucrose
  • Other standard carriers, diluents and excipients may be included as desired.
  • Other exemplary compositions include Tris buffer at pH 7.0-8.5 or acetate buffer at pH 4.0-5.5, which may further include sorbitol or a suitable replacement thereof. .
  • Veterinary drug compositions, quasi-drugs, marine drug compositions, food compositions and cosmetic compositions can also be produced by known techniques.
  • the cells of the present invention can be combined with a pharmaceutically acceptable carrier as necessary and administered parenterally as liquid preparations such as injections, suspensions, solutions, sprays and the like.
  • pharmaceutically acceptable carriers include excipients, lubricants, binders, disintegrants, disintegrators, absorption enhancers, absorbents, humectants, solvents, solubilizers, suspending agents. , Tonicity agents, buffering agents, soothing agents and the like.
  • Product additives such as preservatives, antioxidants, colorants, sweeteners and the like may be used as needed.
  • the composition of the present invention can be blended with substances other than the polynucleotide, polypeptide and the like of the present invention.
  • parenteral routes of administration examples include, but are not limited to, intravenous, intramuscular, subcutaneous, intradermal, mucosal, rectal, vaginal, topical, and transdermal.
  • the medicament used in the present invention may be in the form of a pharmaceutically acceptable aqueous solution free of pyrogens.
  • the preparation of such pharmaceutically acceptable compositions is within the skill of the art by considering pH, isotonicity, stability, and the like.
  • the pharmaceutical composition of the present invention may further contain a colorant, a preservative, a fragrance, a flavoring agent, a sweetener and the like, and other agents.
  • the amount of the composition used in the treatment method of the present invention depends on the purpose of use, target disease (type, severity, etc.), patient age, weight, sex, medical history, cell morphology or type, and the like. Thought Therefore, it can be easily determined by those skilled in the art.
  • the frequency with which the treatment method of the present invention is applied to a subject (or patient) also depends on the purpose of use, target disease (type, severity, etc.), patient age, weight, gender, medical history, treatment history, etc. In view of this, it can be easily determined by one skilled in the art. Examples of frequencies include daily to once every few months (eg once a week to once a month). Preferably, the administration is performed once a week to once a month while observing the course.
  • the dosage can be determined by estimating the amount required by the area to be treated.
  • the term "instruction" diagnoses a method of administering a medicine or a method for diagnosis, etc., such as a doctor, a patient, etc. Described for a person (eg, a doctor, patient, etc.) or a person being diagnosed.
  • This instruction describes a word indicating an appropriate method for administering the diagnostic agent, medicine and the like of the present invention.
  • This instruction is prepared according to the format prescribed by the national supervisory authority (for example, the Ministry of Health, Labor and Welfare in Japan and the Food and Drug Administration (FDA) in the United States, etc. in the US). It will be clearly stated that it has been approved. Instructions are so-called package inserts, usually provided on paper. This instruction is not limited to this, and may be provided in the form of, for example, an electronic medium (for example, an electronic medium provided on the Internet (for example, a website), an electronic mail).
  • the end of treatment according to the methods of the present invention may be determined by the results of standard laboratory tests using commercially available accessories or equipment or diseases associated with the intended treatment (eg, bone disease, heart It can be supported by the disappearance of the clinical symptoms characteristic of the disease, neurological disease, etc. or the restoration of the cosmetic state (eg, recovery of appearance). Treatment can be resumed by a recurrence of a disease (eg, neurological disease) associated with a deficiency of differentiated cells or a cosmetic condition injury.
  • a disease eg, neurological disease
  • the present invention also provides a pharmaceutical package or kit comprising one or more containers filled with one or more pharmaceutical compositions.
  • a notice in the form of government agencies regulating the manufacture, use or sale of pharmaceuticals or biological products may optionally be attached to such containers, which may be produced, used or sold for human administration. Represents approval by the government agency.
  • the kit can include an injectable device.
  • Toxicity studies have used various animal models (eg, mouse, rabbit, or non-rodent) Can be done. In these animal models, general conditions, body weight, food consumption, water consumption, etc., and tests such as blood tests, blood chemistry tests, urine tests, and ophthalmological tests are performed. Various tests are well known in the art, and include, but are not limited to, for example, the following forces.
  • Hematological examination red blood cell count, white blood cell count, platelet count, hemoglobin content, hematocrit, blood image
  • Percentage of leukocytes are measured.
  • blood cell composition is measured as follows: (1) Drug is administered to mice (untreated control mice should also be measured); (2) Each treatment group A blood sample is obtained periodically from one mouse through the tail vein; and (3) the sample is obtained from the red blood cell and white blood cell counts, the blood cell composition and the lymphocytes and polymorphonuclear cells. Analyze percentages. Each dosing regimen and control indicates whether toxicity is present.
  • Blood chemistry tests serum (plasma) protein, albumin, A / C ratio, protein fraction, glucose, cholesterol, triglyceride, pyrilbin, urea nitrogen, creatinine, transaminase (ASAT (GOT), ALAT ( GPT)), alkaline phosphatase, electrolytes (sodium, potassium, chlorine, calcium, inorganic phosphorus, etc.).
  • Urinalysis measurement of urine volume, pH, protein, sugar, ketone body, bilirubin, occult blood, sediment, specific gravity or osmotic pressure, electrolyte (sodium, potassium, etc.).
  • Ophthalmological examination Using the naked eye and an ophthalmoscope, the anterior segment, intermediate translucent body, and fundus are analyzed.
  • the fat portion (aspiration fat) of the lipoaspirate can be isolated from the fat portion (aspiration fat) of the lipoaspirate. Specifically, for example, (1) The aspirated fat is thoroughly washed with physiological saline using a 1 liter separatory funnel; (2) The upper aspirated fat is sufficiently removed from the lower physiological saline. Check that it has been separated, and discard the lower layer. This procedure is repeated until the saline is substantially clear when viewed with the naked eye; (3) Add 0.075% collagenase / PBS equal to the amount of aspirated fat.
  • adipose-derived progenitor cells can be isolated from the liquid portion of lipoaspirate (lipoaspirate), for example as follows: (1) preparing the liquid portion of lipoaspirate; (2) Centrifuge the liquid portion to obtain the cell fraction; (3) density gradient centrifuge the cell fraction to separate the cells based on specific gravity; and (4) specific gravity over red blood cells. Collect cells from the cell layer.
  • the liquid part of the aspirate can be prepared using saline or Ringer's injection. This centrifugation can be performed at a speed of about 800 X g or less, or about 400 X g or more.
  • the density gradient centrifugation is performed at a speed of about 370 Xg to: 1,100 X g.
  • This density gradient centrifugation is performed using a medium having a specific gravity (20 ° C.) of about 1.076 g / ml to: 1.078 gZml.
  • the medium used in this density gradient centrifugation can be Ficoll TM, Percoll TM or sucrose.
  • the specific gravity of the recovered cell layer is approximately 1. It can be in the range of 050-1. This cell layer can be collected using a pipette.
  • the liposuction method can be performed using techniques well known to those skilled in the art.
  • devices used for the fat suction method include, but are not limited to, KSEI degreasing and suction machine monkey pump SAL 76-A (KASEY Medical Co., Ltd., Hongo 3-19-6, Bunkyo-ku, Tokyo).
  • a liquid such as a saline solution containing 0.0001% adrenaline is injected into the adipose tissue by an amount equal to twice the expected amount of aspirated fat, and then a force nucleus (made of metal) having an inner diameter of 2 to 3 mm.
  • suction tube suction is performed at a negative pressure of about 250 to 900 mmHg.
  • physiological saline is usually used as the liquid used for washing the aspirated fat cells.
  • any liquid other than physiological saline can be used as long as it does not adversely affect the stem cells to be isolated.
  • any isotonic solution can be used in the present invention, such as, for example, Ringer's solution and mammalian culture media (eg, DMEM, MEM, Ml 99, MCDB153, etc.).
  • the cell fraction and other components for example, plasma, physiological saline injected at the time of surgery, anesthetic, hemostatic, destroyed Any conditions for separating the fat cell force (leaked lipid component) can be used. For example, centrifugation at 400 to 800 X g for 5 to 15 minutes can be used.
  • the isolated cell fraction can be subjected to density gradient centrifugation.
  • Any medium that can be used for density gradient centrifugation can be used as this medium.
  • a preferred medium may have a specific gravity of 1.076 to 1.078 g / ml.
  • the preferred medium pH is between 4.5 and 7.5.
  • the preferred endotoxin level in the medium is 0.12 EUZml or less.
  • Typical media include sucrose and ficoll (copolymer of sucrose and epichlorohydrin). , And Percoll (a colloidal silica product with a polyvinylpyrrolidone coating).
  • Ficollore is commercially available under the names Ficoll-Paque PLUS (Falmacia Biotech Co., Ltd., Tokyo), Histopaque-1077 (Sigma Aldrich Japan Co., Ltd., Tokyo).
  • Percoll is marketed under the name PercolK Sigma Aldrich Japan, Tokyo).
  • Centrifugation by specific gravity is performed when Ficoll—Paque PLUS is used as a medium, 400 X g, 30-40 component force S, and when Histopaque—1077 is used as a medium, 37 Ocg, approximately 30 minutes is used.
  • conditions include, but are not limited to, 800 xg, about 20 minutes or 1 100 xg, about 10 minutes.
  • red blood cells When centrifugation by specific gravity is used, the most abundant cells are usually red blood cells, which can be confirmed as a red cell layer. Stem cells are lighter in specific gravity than red blood cells, and therefore, when separating stem cells, a cell layer lighter than red blood cells is collected. Preferably, a cell layer with a specific gravity in the range of 1 ⁇ 050 to 1.075 is collected.
  • the specific gravity of the cells is roughly determined by preparing density gradient centrifugation media such as Percoll and Readygrad in sodium chloride solution or sucrose solution, adding density marker beads together with the collected cells, and centrifuging. However, it can be tested by checking which of the 5 to 10 layers separated by the beads is in which layer.
  • density gradient centrifugation media such as Percoll and Readygrad in sodium chloride solution or sucrose solution
  • the cells separated into layers can be collected using, for example, a pipette.
  • a cell separator for example, blood component separation apparatus A STEC204, Amco Co., Ltd.
  • a STEC204 for example, blood component separation apparatus A STEC204, Amco Co., Ltd.
  • the conditions under which adipose tissue is separated from cells can be those under which adipose tissue is separated from cells and facilitates cell isolation.
  • Such conditions include, but are not limited to, for example, the degradation of the extracellular matrix, such as collagenase available from Wako (collagenase for cell dispersion, 032-10534).
  • collagenase available from Wako (collagenase for cell dispersion, 032-10534).
  • collagenase is dissolved in PBS at a concentration of 0.075%, and the solution is warmed to 37 ° C and added in an amount equivalent to fat.
  • An agitator is used to stir the sampnore at 37 ° C and 80 rpm for 30 minutes.
  • the method of the present invention is used to treat blood cells in order to achieve a high degree of stem cell purity.
  • the step of removing is further included.
  • blood cells can be removed by subsequent culturing on a dish and passage of this cell population to generate more than 80% (preferably more than 90%) stem cells. This is also a surprising effect that was not expected from the prior art.
  • Such blood cells can be removed by a blood cell removal step including a step of adding a component that degrades blood cells.
  • the present inventors showed the ability to express CD34 and CD105 even after long-term culture (eg, 20 weeks) under the culture conditions used by the present inventors. It was found that the maintained ASC (hereinafter including both PLA and LAF cells unless otherwise specified) is obtained from both the fat and liquid portions of the lipoaspirate.
  • CD34 is the most established stem cell marker, and CD34 + cells are expected to be clinically applicable.
  • CD105 is known as a mesenchymal stem cell-related marker, and is thought to contribute to the differentiation of ASC into strains of mesenchymal origin such as fat, cartilage, and bone.
  • the present invention provides a clinically useful human-derived cultured ASC and a novel method for preparing such ASC.
  • This method typically includes the following steps:
  • human donor means a human who provides fat. Typically, humans (including men and women) undergoing liposuction, and more typically healthy women undergoing liposuction for purposes such as improving body shape. Donor undergoing liposuction It is preferable that sufficient informed consent is provided.
  • lipoaspirate means an aspirant obtained by liposuction.
  • Liposuction usually involves the insertion of a suction tube called force neulet into the thick part of the body's subcutaneous fat (eg, abdomen, waist, thigh, etc.) for the purpose of improving body shape, etc. Is.
  • the lipoaspirate is composed of a “fat part” and a “liquid part”.
  • Fat portion typically refers to aspirated adipose tissue that has been “chopped” by the interaction of force neuules and reduced pressure.
  • the “liquid part” means the liquid sucked together with the fat part.
  • the fluid is primarily derived from (1) saline previously injected into the site to promote liposuction without damaging nerves and blood vessels, (2) peripheral blood, and (3) adipose tissue Cell or tissue fragment.
  • saline previously injected into the site to promote liposuction without damaging nerves and blood vessels
  • peripheral blood and (3) adipose tissue Cell or tissue fragment.
  • either the fat portion or the liquid portion of the lipoaspirate can be used.
  • Isolation of adipose-derived stem cells from the resulting lipoaspirate is typically performed using the method of Zuk et al. (Zuk et al. Tissu Eng 2001; 7: 211-228) or a modification thereof. It can be done as follows.
  • the material from the fat portion is first treated with an enzyme such as collagenase to dissociate the cells from the extracellular matrix.
  • an enzyme such as collagenase to dissociate the cells from the extracellular matrix.
  • mature adipocytes, connective tissue, and the like are separated from the pellet by centrifugation, and the collected pellet is treated with an erythrocyte lysis buffer, and then erythrocytes and non-cellular components are removed through a filter (for example, 100 ⁇ m in pore diameter).
  • the target ASC ie, PLA cells
  • the resulting ASC may be further filtered to remove impurities.
  • the liquid from the liquid portion is directly centrifuged to recover the pellet.
  • the pellet is treated with erythrocyte lysis buffer and then passed through a filter to remove erythrocytes and non-cellular components.
  • the target ASC ie, LAF cells
  • the target ASC is concentrated and isolated from the filtrate that has passed through the filter by density gradient centrifugation.
  • ASC preparation method of the present invention ASC isolated from lipoaspirate is further cultured and expanded.
  • the procedure for culturing is in accordance with conventional methods (see, for example, the New Chemistry Laboratory (18) “Cell Culture Technology”, edited by the Japanese Biochemical Society, 1st edition, 1st edition, 1990, Tokyo Kagaku Dojin).
  • Cells can be cultured in suspension or on a fixed culture medium.
  • the cells are attached to a substrate (eg, glass or plastic petri dish, flask, plate, etc.) and cultured in vitro.
  • a substrate eg, glass or plastic petri dish, flask, plate, etc.
  • a product which has been surface-treated so that cells can be easily adhered may be used.
  • any extracellular matrix eg, collagen, hyaluronic acid, gelatin, poly-L-lysine, polyglycolic acid, (1) __force prolatatatone etc.
  • an extracellular matrix is coated on the adhesion surface of the culture vessel.
  • the extracellular matrix may be coated on the adhesive surface of the culture vessel prior to use in a manner known to those skilled in the art, or a product pre-coated with the extracellular matrix can be obtained by a manufacturer known to those skilled in the art. Power may also be obtained commercially.
  • a medium used for cell culture is composed of a basic medium and an additive.
  • the basic medium consists of amino acids such as L-arginine, L-glutamine, L-histidine, L-isoleucine, inorganic salts such as CaCl ⁇ 20, KC1, NaCl,
  • M-199 medium (Morgan et al. Proc • Soc. Exp. Biol. 1950; 73: 1) is typically used as the basic medium.
  • M-199 medium is commonly used as a basic medium mainly for culturing mammalian normal cells, and is commercially available from suppliers (eg, Invitrogen Corporation) force, powder or liquid well known to those skilled in the art. It's terrible.
  • Other basic media that can be used in the ASC preparation method of the present invention include Eagle's minimum essential medium (MEM), Eagle's basic medium (BME), Dulbecco's modified Eagle medium (DMEM), and Ham's F12 medium.
  • nutrients required for cell culture preferably, nutrients required for cell culture Additives that serve as sources such as or are added to the basal medium for the purpose of stimulating or maintaining cell growth and / or viability.
  • additives include serum, cell growth factor, antibiotics and the like.
  • the types of "serum” that can be used in the present invention include urine fetal serum, urine neonatal serum, pupal serum, horse serum, human serum and the like. Is preferred. Preferably, non-immobilized serum is used. Serum can be used by being added to the medium at a concentration of about 2 to 25%, and is usually added to the medium at a concentration of about 5 to 20%. More preferably, the serum concentration is about 7 to about 15%, and most preferably about 10%.
  • Examples of "cell growth factor” that can be used in the present invention include fibroblast growth factor
  • FGF acidic FGF, basic FGF
  • PDGF_AB platelet-derived growth factor
  • IGF-1 insulin-like growth factor 1
  • EGF epidermal growth factor
  • HGF hepatocyte growth factor
  • GDNF glia Cell line neurotrophic factor
  • NF neurotrophic factor
  • BMP bone morphogenetic factor
  • TGF transforming growth factor
  • EGF epidermal growth factor
  • VEGF vascular endothelial cell proliferation Factor
  • acidic fibroblast growth factor acidic FGF
  • heparin and the like can be added to enhance the action of acidic FGF.
  • the cell growth factor can be used in the medium at a concentration of about 0.05 to 20 ng / ml, preferably 0.1 to about 10 ng / ml, more preferably about 1 to 5 ng / ml. Can be used at a concentration of about 2 ng / ml.
  • heparin can be added to the medium at a concentration of about 0.:!
  • the antibiotic for example, penicillin, streptomycin, gentamicin and the like can be used, but as the antibiotic used in the present invention, penicillin and streptomycin are particularly preferable.
  • the antibiotic may be used at a concentration of about 20 to 450 IU or 20 to 450 mg / ml, preferably a concentration of about 50 to 400 IU or 50 to 400 mgZml, more preferably 50 to 200 IU or 50 to Concentration on the order of 200 mgZml, even more preferred 70-: 150 IU or a concentration on the order of 70-150 mgZml Degrees, most preferably at concentrations as high as 100 IU or 100 mg / ml.
  • EGF EpidermalGrowthFactor
  • amino acids for example, arginine, cysteine, Gnoretamine, glycine, histidine, isoleucine, leucine, lysine, methionine,
  • a typical example of the culture medium used in the present invention is 5-20% urine fetal serum, 50-200 IU penicillin, 50-200 mg Zml sprepmycin,:!-10 ⁇ gZ M-199 medium supplemented with ml heparin, and 0.5 to: 10 ng / ml acidic fibroblast growth factor.
  • the period required for culturing the isolated adipose-derived stem cells is at least 1 week, preferably at least 2 weeks, more preferably at least 3 weeks, more preferably At least 4 weeks, more preferably at least 5 weeks, more preferably at least 6 weeks, more preferably at least 7 weeks, more preferably at least 8 weeks, more preferably at least 9 weeks, more preferably At least 10 weeks, most preferably at least 20 weeks.
  • medium exchange is performed at least twice a week and cell passage is performed at least once a week.
  • the medium is usually replaced with one having the same composition, but the medium is not limited to this, and the medium is cultivated using a new medium having a changed composition (for example, a new medium having a changed serum concentration, cellular growth factor concentration, etc.) You may exchange land.
  • the culture can be performed under any conditions suitable for mammalian cell culture, but preferably at a temperature of about 37 ° C (eg, 35-38 ° C), for example, Performed in a humid atmosphere under a 5% CO atmosphere.
  • the ASC culture method used in the present invention can be used not only for adipose-derived stem cells isolated from a liposuction material obtained by liposuction, but also for other methods (eg, It can be understood by those skilled in the art that it can also be used for adipose-derived stem cells isolated from fat obtained by general surgical excision. Moreover, it is not limited to human-derived adipose-derived stem cells, but derived from other mammals (for example, pigs, monkeys, chimpanzees, inu, ushi, magpies, rats, mice, etc.), birds, reptiles, etc. It can be understood by those skilled in the art that the method of the present invention can be applied to the adipose-derived stem cells.
  • the present invention provides a method for preparing CD34 + and / or CD105 + adipose-derived stem cells, comprising the step of culturing newly isolated adipose-derived stem cells.
  • freshly isolated means that the cell is simply obtained by a suitable method of fat or lipoaspirate obtained from a donor (eg, a human donor). A cell that has been detached and has not been grown in culture.
  • the newly isolated adipose-derived stem cells are not limited to those isolated from lipoaspirate obtained by liposuction.
  • Adipose-derived stem cells are not limited to those derived from humans.
  • the present invention is also a composition comprising pluripotent stem cells, the composition comprising at least pluripotent stem cells capable of expressing CD34, CD105, or both cell surface markers.
  • a composition comprising 10% is provided.
  • the present invention can provide such a composition that can be stored for a long period of time.
  • this thread and adult product ⁇ 5-20 ⁇ / ⁇ ⁇ tsukidai pupa blood blue, 50-400 IU or ⁇ 50-400 mg / m 1 Antibiotic, 1-: 10 / ig / ml heparin, and 0.5-: 10 ng / ml acidic fibroblasts can be cultured (eg, 2-20 weeks) using a medium containing growth factors.
  • compositions of the invention may comprise at least 50% pluripotent stem cells capable of expressing CD34, CD105, or both cell surface markers. It has been difficult to obtain a composition containing such cells.
  • the composition of the present invention may comprise at least 50% of pluripotent stem cells capable of expressing a cell surface marker of CD105.
  • pluripotent stem cells capable of expressing a cell surface marker of CD105.
  • Such as mesenchymal stem cells No stem cells with properties have been provided so far. Such cells have a great effect in that they have a high proportion of undifferentiated cells.
  • composition of the invention may comprise at least 10% pluripotent stem cells capable of expressing a cell surface marker of CD34.
  • a mesenchymal stem cell having such characteristics has not been provided so far. Such cells have a great effect in that the ratio of undifferentiated cells is high.
  • the present invention also provides a method for preparing a composition comprising CD34 + and / or CD105 + adipose-derived stem cells, comprising A) culturing newly isolated adipose-derived stem cells A method comprising the steps is provided.
  • the fat-derived stem cells are preferably cultured for at least 2 weeks, but the method is not limited thereto.
  • adipose-derived stem cells may be cultured for 1 to 2 weeks. :! ⁇ 2 weeks of culturing provided a composition containing large amounts of CD34 positive cells (eg, 20% or 50%).
  • the adipose-derived stem cells are cultured for at least 10 weeks or at least 20 weeks.
  • the provision of a composition containing CD34 + and / or CD105 + adipose-derived stem cells even in long-term culture is an unexpected and remarkable effect.
  • culturing in the method for producing a composition of the present invention is 5 to 20% Ushiki pupa blood.
  • Blue, 50-400 IU or ⁇ or 50-400 mg / ml antibiotics:! ⁇ 10 ⁇ g / ml hexane phosphorous, and 0.5 ⁇ : 10 ng / ml medium containing acidic fibroblast growth factor Can be done.
  • the use of such a culture environment is expected to give the effect of long-term culture, so any environment may be used as long as the conditions are equivalent to this.
  • the fat-derived stem cells obtain a lipoaspirate from a human donor, and the step of isolating the fat-derived stem cells from the lipoaspirate
  • the lipoaspirate used in this method includes a liquid portion. It is surprising that a composition comprising CD34 + and / or CD105 + adipose derived stem cells could be prepared from the liquid part. [0113] Therefore, in the present invention, the step of obtaining the lipoaspirate further includes a step of separating the liquid portion. The step of separating the liquid portion can employ any of the methods detailed elsewhere herein.
  • Both PLA cells and LAF cells prepared by the ASC preparation method of the present invention can express CD34 and CD105 even after long-term culture. This property is very useful for clinical applications.
  • the present invention is the first to show that CD34 + ASC can be expanded over a long culture period.
  • CD34 + ASC obtained from lipoaspirate can be expanded from 10 4 to 10 7 times by culturing for 4 weeks. This fact further enhances the value of human ASC for clinical application in cell-based therapy.
  • the present invention also provides isolated pluripotent stem cells or cultured pluripotent stem cells, which can express CD34, CD105, or both as cell surface markers.
  • the present invention provides cultured adipose-derived stem cells that can express CD34, CD105, or both as cell surface markers.
  • the present invention provides a long term (eg, at least 2 weeks, more preferably at least 3 weeks, more preferably at least 4 weeks, more preferably at least 5 weeks, more preferably at least 6 weeks, more preferably at least 7 weeks). More preferably at least 8 weeks, more preferably at least 9 weeks, more preferably at least 10 weeks, even more preferably at least 20 weeks) human-derived fat-derived stems grown in culture.
  • the present invention in another aspect, also comprises the step of administering pluripotent stem cells to a subject, to treat a human or animal disease, or to regenerate a human, animal cell, tissue, or organ.
  • diseases include skin diseases such as refractory ulcer 'decubitus' burns, bone defects 'bone diseases with bone degeneration, cartilage deformation' defects ⁇ cartilage diseases with degeneration, fat atrophy ⁇ degeneration Fatty diseases, myocardial infarction ⁇ Cardiovascular diseases including dilated cardiomyopathy, muscle diseases including Duchenne muscular dystrophy, spinal cord injury * Cerebral infarction ⁇ Central nervous system diseases including Parkinson's disease, beauty Examples include tissue augmentation and anti-aging treatment.
  • the present invention can be used for regenerative medicine such as fat regeneration, bone regeneration, myocardial regeneration, angiogenesis, cartilage regeneration, bone marrow regeneration, nerve regeneration, and skeletal muscle regeneration.
  • the pluripotent stem cells of the present invention can be administered to a subject by various methods. For example, it can be transplanted by local injection into the wall of the gastrointestinal tract, administration systemically or to the portal circulatory system, or by any other practical means.
  • the use of pluripotent stem cells in the treatment of disorders such as human gastrointestinal disorders can be demonstrated by using animal models.
  • the present invention also includes the use of pluripotent stem cells prepared with different donor tissue powers for the host.
  • pluripotent stem cells prepared with different donor tissue powers for the host.
  • recipients can usually be immunosuppressed, for example, by the use of immunosuppressive drugs such as cyclosporine, or by local immunosuppressive strategies using locally applied immunosuppressive drugs.
  • the method of immunosuppression is not limited to these, and other methods (for example, gene replacement or knockout method using homologous recombination in stem cells) can also be used.
  • the expression of class 2HLA human leukocyte antigen
  • immunosuppressant Even if there is no use of ASC, the rejection of transplantation of non-self ASCs may not occur is expected.
  • the pluripotent stem cells of the invention can be delivered in a site-specific manner (ie, locally) or globally. Cells can be administered to a particular area using any method that maintains the integrity of the surrounding area, preferably by local injection. The cells can also be administered systemically or in the portal circulatory system.
  • the present invention also provides a drug containing the pluripotent stem cells of the present invention for use in the treatment of human or animal diseases or the regeneration of human or animal cells, tissues, or organs.
  • this drug is a growth factor that promotes the growth and differentiation of pluripotent stem cells (eg, basic or acidic fibroblast growth factor (bFGF; aFG F), platelet-derived growth factor (PDGF_AB , BB), insulin, insulin-like growth factor—1 (IGF_ 1), hepatocyte growth factor (HGF), glial cell line-derived neurotrophic factor (GDNF), neurotrophic factor (NF), hormone, cytosite in, bone Including formation factor (BMP), transforming growth factor (TGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), immunosuppressive drugs (eg cyclosporine), anti-inflammatory agents, etc. be able to.
  • pluripotent stem cells can be en
  • Stem cells can be used as they are for transplantation treatment, but various tissue engineering techniques can be used to guide them to the desired cells and design them into a certain organ shape.
  • the pluripotent stem cells of the present invention can be administered to a subject together with a scaffold material (acting as an extracellular matrix). Therefore, the regenerative medical material or drug of the present invention may further contain a scaffold material together with pluripotent stem cells.
  • the use of scaffold materials can facilitate the growth and differentiation of stem cells into certain forms in vitro or in vivo.
  • Scaffold materials that can be used in the present invention include, for example, proteins such as collagen, albumin, and fibrin, polylactic acid, polydaricholic acid, a copolymer of lactic acid and glycolic acid, poly-one ⁇ -force prolatathon, ⁇ —Copolymers of prolataton and lactic acid or darlicolic acid, polycenoic acid, polymalic acid, polymono-cyanate, poly-is-hydroxybutyric acid, polytrimethylene oxalate, polytetramethylene oxalate, poly Ortho Este Synthetic molecules such as Nore, Polyonolecarbonate, Polyethylene carbonate, Polypropylene carbonate, Poly ⁇ -Benzylu L glutamate, Poly ⁇ -Methyl-L glutamate, Poly L-alanine, Starch, Alginic acid, Hyanorenoic acid, Chitin, Pectinic acid And polysaccharides such as their derivatives.
  • proteins such as collagen, albumin
  • the form of the scaffold material is not particularly limited, and can be, for example, a sponge form, a mesh form, a non-woven form, a disk form, a film form, a rod form, a particle form, a paste form, or the like.
  • the scaffold material may be made of a biodegradable material, and it is a material that can release stem cells, cell growth factors, etc. for a certain period of time (eg:! ⁇ 3 weeks) in vivo. It ’s okay.
  • stem cells of the present invention various differentiated cells and / or progenitor cells can be obtained. A specific procedure is described below.
  • the present invention provides a method for preparing differentiated cells from stem cells.
  • Differentiated cells obtained from stem cells include not only finally differentiated cells but also progenitor cells.
  • Methods for preparing differentiated cells from lipoaspirate-derived stem cells include (1) a method of adding a differentiation inducer (eg, dexamethasone) to the culture medium, and (2) differentiation corresponding to a desired site.
  • a differentiation inducer eg, dexamethasone
  • the cell mixture of the present invention further comprises a factor that promotes differentiation into a differentiated cell corresponding to a desired site. May be included. This factor can be any factor known or confirmed to promote differentiation into differentiated cells corresponding to the desired site. Examples of preferred differentiation-inducing agents include corticosteroids such as dexamethasone, insulin, gnolecose, indomethacin, isobutyl monomethylxanthine ( ⁇ ), ascorbate 2_phosphate, ascorbic acid and its derivatives.
  • corticosteroids such as dexamethasone, insulin, gnolecose, indomethacin, isobutyl monomethylxanthine ( ⁇ ), ascorbate 2_phosphate, ascorbic acid and its derivatives.
  • any culture solution can be used as long as the cells to be mixed and the differentiation into differentiated cells corresponding to the desired site are maintained.
  • examples of such cultures include DMEM, Ml 99, MEM, HBSS (Hanks' Balanced Salt Solution), Ham's F12, BME, RPMI1640, MCDB104, MCDB153 (KGM), etc. Nare ,.
  • Such cultures include corticosteroids such as dexamethasone, insulin, glucose, indomethacin, isobutyl-methylxanthine (IBMX), ascorbate 2-phosphate, ascorbine Growth factors including acids and their derivatives, glyceguchi phosphate, estrogens and their derivatives, progesterone and its derivatives, androgens and their derivatives, a FGF, ⁇ FGF, EGF, IGF, TGF ⁇ , ECGF, BMP, PDGF Pituitary extract, pineal gland extract, retinoic acid, vitamin D, thyroid hormone, feline serum, horse serum, human serum, heparin, sodium bicarbonate, HEP ES, anolebumin, transferrin, selenate ( For example, sodium selenite), linolenic acid, 3_isobutyl_1-methyl Demethylating agents such as xanthine and 5-hazancytidine, histone deacetylating agents such as trichostat
  • a condition for inducing differentiation of a lipoaspirate-derived stem cell into an adipocyte for example, culturing the stem cell in a DMEM medium supplemented with isoptirumethylxanthine, dexamethasone, insulin, and indomethacin.
  • the power S that can be mentioned, and not limited to this.
  • the conditions for induction of differentiation of lipoaspirate-derived stem cells into chondrocytes include, for example, the stem cells in DMEM medium supplemented with insulin, ascorbate 2-phosphate, and TGF- ⁇ 1. Although culture
  • the stem cell in a DM EM medium supplemented with dexamethasone, ascorbate 1_2 phosphate, ⁇ -glyce mouth phosphate for example, the stem cell in a DM EM medium supplemented with dexamethasone, ascorbate 1_2 phosphate, ⁇ -glyce mouth phosphate
  • dexamethasone ascorbate 1_2 phosphate
  • ⁇ -glyce mouth phosphate a condition for inducing differentiation of a lipoaspirate-derived stem cell into a bone cell.
  • the conditions for inducing differentiation of lipoaspirate-derived stem cells into myocytes include, for example, a DMEM medium containing 10% feline serum (FBS) and 5% horse serum (HS).
  • FBS feline serum
  • HS horse serum
  • Examples include culturing the stem cells in a medium supplemented with dexamethasone and hyodecortisone 1S, but is not limited thereto.
  • differentiated cells containing the desired properties By co-culturing the stem cells and differentiated cells corresponding to the desired site, differentiated cells containing the desired properties, preferably homogeneously, can be provided in a certain amount or more.
  • This method comprises: A) a) mixing adipose-derived progenitor cells with b) differentiated cells corresponding to the desired site to obtain a mixture; and B) sufficient to cause differentiation of the adipose-derived progenitor cells And culturing the mixture under various conditions.
  • Differentiated cells corresponding to the desired site can also be prepared using techniques well known in the art.
  • commercially available cell lines for example, cell lines distributed from ATCC and the like
  • Differentiated cells may be cells cultured primarily from a subject intended for transplantation (eg, hepatocytes, kidney cells, adipocytes, bone cells, chondrocytes, etc.).
  • any cell may be used as a differentiated cell as long as it corresponds to a desired site.
  • a mesenchymal cell is used, and such a mesenchymal cell is used.
  • the systemic cells include adipocytes, bone marrow cells, osteoblasts, chondrocytes, fibroblasts, myofibroblasts, neurons, skeletal muscle cells, cardiomyocytes, vascular endothelial cells, vascular smooth muscle cells, liver Examples include, but are not limited to, cells and knee cells.
  • Such a differentiated cell may be an identified cell, but even if the nature of the cell is unknown, for example, a cell corresponding to a desired site using a sorting technique such as FACS. Can be used to prepare
  • the cell mixture of the present invention may further contain a factor that promotes differentiation into differentiated cells corresponding to a desired site.
  • factors can be forces known to promote differentiation into the desired differentiated cells, or any factor identified.
  • Preferred differentiation promoting factor Examples of such children include corticosteroids such as dexamethasone, insulin, dalcos, indomethacin, isobutyl-methylxanthine (IBMX), ascorbate 2-phosphate, ascorbic acid and its derivatives. Glycate oral phosphate, estrogen and its derivatives, progesterone and its derivatives, androgen and its derivatives, FGF.
  • FGF-EGF-IGF-TGF- ⁇ -ECGF-BM P 'PDGF and other growth factors pituitary gland Extract, pineal extract, retinoic acid, vitamin D, thyroid hormone, baby serum, horse serum, human serum, heparin, sodium bicarbonate, HEPES, anolebumin, transferrin, selenate (selenite) Sodium), linolenic acid, 3_isobutyl_1-methylxanthine, 5-azane Demethylating agents such as thymidine, histone deacetylating compounds such as trichostatin, cytosines such as activin, LIF, IL_2, and IL-6, hexamethylenebisacetamide (HMBA), dimethylacetate Amide (DMA), Dibutyl cAMP (dbcAMP), Dimethyl sulfoxide (DMSO), Rhododeoxyuridine (IdU), Hydroxyurea (HU), Cytosine arabinoside (Ara
  • any culture solution can be used as long as the cells to be mixed and the differentiation into differentiated cells corresponding to the desired site are maintained.
  • examples of such cultures include DMEM, Ml 99, MEM, HBSS (Hanks' Balanced Salt Solution), Ham's F12, BME, RPMI1640, MCDB104, MCDB153 (KGM), etc. Nare ,.
  • Such cultures include corticosteroids such as dexamethasone, insulin, glucose, indomethacin, isobutyl monomethylxanthine (IBMX), ascorbate 2_phosphate, Ascorbic acid and its derivatives, glyceguchi phosphate, estrogen and its derivatives, progesterone and its derivatives, androgen and its derivatives, FGF, ⁇ FGF, EGF, IGF, TGF— ⁇ , ECGF, BMP, PD GF and others Growth factor, pituitary extract, pineal extract, retinoic acid, vitamin D, thyroid hormone, feline serum, horse serum, human serum, heparin, sodium bicarbonate, HEPES, albumin, transferrin, selenate ( Sodium selenite), linolene Demethylating agents such as acid, 3-isobutyl-1-methylxanthine and 5-azazancydine, histone deacetylated chiral compounds such as trichostatin IJ, activin,
  • the present invention provides a cell mixture comprising stem cells derived from lipoaspirate and differentiated cells corresponding to a desired site.
  • a cell mixture comprising stem cells derived from lipoaspirate and differentiated cells corresponding to a desired site.
  • Such a mixture is useful for cell transplantation, and has an advantage that each component is less than the conventional transplantation using cells alone.
  • the cell mixture of the present invention is preferably after being subjected to conditions sufficient for the differentiation of adipose stem cells, but the present invention is not limited thereto.
  • the cell mixture after being subjected to differentiation conditions may be used for transplantation as it is, or may be differentiated into a tissue or organ.
  • the present invention provides compositions for cell transplantation (eg, tissue pieces and grafts, and compositions comprising them).
  • This transplantation may be performed by any eye as long as it treats or prevents a disease, disorder or abnormal condition associated with the loss or deterioration of differentiated cells corresponding to a desired site, or treats or improves a cosmetic condition.
  • the transplantation is transplanted to a desired site, but is not limited thereto. If the desired site can be finally treated or prevented, the cell-containing composition of the present invention can be used. It can be administered or transplanted at any site.
  • the mixing ratio of the lipoaspirate-derived stem cells and differentiated cells may be any ratio as long as desired differentiation occurs, but is usually about 1:10 to about 10: 1, preferably About 1: 5 to about 5: 1, more preferably about 1: 2 to about 2: 1 and most preferably about equal amounts.
  • the differentiated cells used for transplantation and the stem cells derived from lipoaspirates are in any form or embodiment as described in “Method for preparing differentiated cells” in the present specification. Can take.
  • These differentiated cells and adipose-derived progenitor cells are different from each other, allogeneic or syngeneic with respect to the host to be transplanted.
  • they are allogeneic or syngeneic, more preferably syngeneic, but the present invention is not limited thereto.
  • the present invention may further include a step of avoiding rejection. Procedures to avoid rejection are well known in the art (eg, New Surgery System, Section 12, Organ Transplantation (from Heart Transplantation / Lung Transplantation, Technical / Ethical Development to Implementation [New Whole Surgery , Vol.
  • Immunosuppressants to prevent rejection are currently “cyclosporine” (Sandy Miyun / Neo-Iral); “Tacrolimus” (Prograf) "Azathioprine” (Imran); “steroid hormones” (predonin, methylpredonin); “T cell antibodies” (OKT3, ATG, etc.) Prophylactic immunosuppressive therapy
  • the method used in the world is a combination of cyclosporine, azathioprine, and steroid hormones, and it is desirable that the immunosuppressant be administered at the same time as the drug of the present invention.
  • the immunosuppressive agent can be administered before or after the regenerative method / therapeutic method of the present invention as long as an immunosuppressive effect is achieved.
  • the differentiated cells and adipose-derived progenitor cells are each different, allogeneic or syngeneic, preferably allogeneic or syngeneic, and more preferably syngeneic.
  • allogeneic or syngeneic are more differentiated cells. This is because stem cells derived from lipoaspirates tend to form a uniform cell population.
  • the cell mixture or composition described above may be provided as a medicament.
  • Such medicaments can be used to treat or prevent a disease, disorder or abnormal condition associated with the loss or deterioration of differentiated cells corresponding to the desired site, or to treat or ameliorate a cosmetic condition.
  • the medicament of the present invention may contain a pharmaceutically acceptable carrier in addition to the cell mixture or the composition containing it.
  • a pharmaceutically acceptable carrier any carrier described in the present specification can be selected, and can be used by those skilled in the art depending on its use.
  • One skilled in the art can modify the ingredients to be included in the present invention.
  • the present invention provides a method for treating or preventing a disease, disorder or abnormal condition resulting from a defect in differentiated cells, and a method for treating or ameliorating a cosmetic condition.
  • These methods comprise the steps of: A) providing a composition comprising a) adipose-derived progenitor cells; and b) differentiated cells corresponding to a desired site, and B) administering the composition to a subject, Is included.
  • the stem cells derived from differentiated cells and lipoaspirates used for transplantation can take any form and embodiment as described in “Methods for preparing differentiated cells” herein. .
  • any method of administration can be used, including those known in the art of the invention.
  • the above composition is not limited to the force injected using a syringe, catheter, tube, or the like.
  • administration routes include, but are not limited to, local injection (subcutaneous injection, intramuscular or intramuscular injection), intravenous injection, intraarterial injection, tissue administration, and the like.
  • This method of treatment or prevention by transplantation of the present invention has advantages over, for example, the following conventional techniques, but is not limited to these: (1) Necessary to create regenerative tissue outside the tissue (ex vivo production) (2) Larger tissue regeneration is possible more reliably; (3) Can be achieved by simple and short processing; (4) Surgery for incising an organ such as the skin It is possible to administer (transplant) cells and tissues by puncturing a needle without the need for a device.
  • the present invention corresponds to a) adipose-derived progenitor cells, b) desired sites Provided is the use of a mixture with differentiated cells for the preparation of a medicament for treating or preventing a disease, disorder or abnormal condition resulting from a defect in differentiated cells, or for treating or ameliorating a cosmetic condition.
  • Differentiated cells and adipose-derived progenitor cells used in the cell mixture for transplantation can take any form as described herein in “Methods for Preparing Differentiated Cells”.
  • liposuction aspirates The lipoaspirate was divided into two parts, a floating fat part (also called lipoaspirates) and the remaining liquid part (see figure la). Both parts were used as cell sources for PLA cells and LA F cells, respectively.
  • PLA cells were isolated from the fat portion of the lipoaspirate using a modified procedure of the method of Zuk and coworkers (Zuk et al. Tissu Eng 7: 211-28; 2001). Briefly, aspirated fat was digested for 30 minutes at 37 ° C in PBS containing 0.075% collagenase on a shaker. Mature adipocytes and connective tissue were separated from the pellets by centrifugation (800 g, 10 minutes). Resuspend pellet in erythrocyte lysis buffer (155 mM NH 4 Cl, 10 mM KHCO, 0. ImM EDTA) for 5 minutes at room temperature
  • the pellet was resuspended and filtered through a lOO x m mesh (Millipore, MA, USA).
  • LAF cells were taken from the liquid portion of the lipoaspirate. The aspirate was centrifuged (400 g, 10 minutes) and the resulting pellet was resuspended in erythrocyte lysis buffer for 5 minutes at room temperature and then filtered through a 100 ⁇ m mesh.
  • the pellets were then treated with Ficoll (Amersham Biosciences, NJ, USA) for density gradient centrifugation. After centrifugation (800 g, 20 minutes), cells at the gradient interface were collected, washed in PBS, and filtered through 100 ⁇ mesh. Density gradient centrifugation was not performed for flow cytometry of freshly isolated LAF cells. Nucleated cysts were counted using the NucleoCounter (Chemometec, Denmark).
  • Freshly isolated PLA or LAF cells are plated on medium at a density of 5 x 10 6 nucleated cells / 100 mm gelatin-coated dish and are at 37 ° C in a humidified atmosphere with 5% CO
  • the culture medium was M-199 containing 10% ushi fetal serum (FBS), 100 IU penicillin, 1 OO mg / ml streptomycin, 5 ⁇ g / ml heparin, and 2 ng / ml acidic FGF.
  • FBS ushi fetal serum
  • the same medium with different serum concentrations containing 15% FBS was also used only for doubling time measurements.
  • Primary cells were cultured for 7 days until they reached confluence and defined as “passage 0”. The medium was changed every 2 days and the cells were passaged weekly. After 7 days of primary culture, the attached cells were passaged by trypsinization and plated at a density of 2,000 cells Zcm 2 in the same medium. The doubling time was calculated using the number of adherent cells 3 and 5 days after seeding.
  • colony forming unit analysis also uses intracellular lipids. This was done to quantify the colonies.
  • 150 Itoda vesicles were plated on 100 mm culture dishes and cultured for 10-14 days, followed by incubation with chondrogenic medium for 2 weeks and stained with Oil-Red O as described above. The number of positive or negative colonies for staining was counted under a microscope.
  • Freshly isolated PLA or LAF cells were tested for expression of surface or intracellular molecules using flow cytometry. Cultured PLA or LAF cells were also tested at 1, 2, 4, 6, 8, 10, or 20 weeks.
  • the following monoclonal antibodies (MAbs) conjugated with fluorescent dyes were used: anti-CD4_FITC, CD10_PE, CD1 3—PE, CD16—PE, CD29—PE, CD31—PE, CD34—PE, CD34—FITC, CD36 — PE, CD44—PE, CD45—PE, CD45—FITC, CD49d—PE, CD49e—PE, CD54_PE, CD56_PE, CD57_FITC, CD62E_PE, CD62P_PE, C D69—FITC, CD73—PE, CD90—PE, CD106—FITC , CD117—PE, CD13 5—PE, CD144—PE, CD—151—PE, HLA—A, B, C—PE, Tie—2—PE, (BD Biosciences, San Diego, CA, USA), CD59
  • Analysis was performed using a flow cytometer (Becton Dickinson, San Jose, CA, USA). Data collection and analysis were then performed (Cell Quest software, Beet on Dickinson). A gate was set based on staining with a combination of related and irrelevant MAbs so that no more than 0.1% of cells were positive using an irrelevant antibody. Perform cell sorting and subsequent analysis using a FACSAria cell sorter (Becton Dickinson).
  • the new PLA cells contain a higher percentage of cells positive for CD29, CD34, and CD90 expression, and few CD45 + cells of hematopoietic origin.
  • the new LAF cells have a higher proportion of CD31 + and CD45 + cells, which means that the new LAF cell power is higher than peripheral PLA-derived vascular endothelial cells and cells. Suggests containing a large number of populations.
  • FSC and SSC forward and square scatter properties of new LAF cells
  • CD34 + and CD45_ cells are located on clusters corresponding to monocytes ( Figure 3a).
  • FIG. 4 shows representative FACS data for freshly isolated or cultured PLA and LAF cells. Sampu Nore from 5 patients was used. As shown, newly isolated PLA and LAF cells showed a well-defined profile of cell surface markers. In contrast, PLA and LAF cells cultured for 2 weeks showed very similar expression profiles. These differences are thought to arise from the fact that freshly isolated LAF cells contain a large percentage of peripheral blood-derived cells.
  • FIG. 5 shows a continuous change in the expression of cell surface markers.
  • CD34 + cells increased in proportion in PLA cells and uniformly expressed mesenchymal markers such as CD13, CD29 (j31 integrin), CD44, CD73, and CD90 (Yhy-1).
  • CD10, CD49e, CD59, and CD151 were also uniformly expressed in PLA cells cultured for longer than 1 week.
  • CD105 Endoglin
  • CD105 one of the mesenchymal stem cell-related markers
  • CD45, Flk—l (VEGFR—2), Tie—2, CD31 (PECAM—1), CD117 (c—kit), and CD13 3 / AC133 (one of the stem cell-related markers) are found in PLA and LAF cells. The rate decreased significantly.
  • CD4 All cell populations were tested for CD4, CD45, CD6 2E (E-selectin), CD62P (P-selectin), CD69, CD135, and CD144 (VE cadherin) in the first week after incubation. The results were negative for CD16, CD31, CD57, CD106 (VC AM_1), CD133, Flk_l and Tie_2.
  • CD34 + cells were sorted, about half of the force cells cultured under the same culture protocol Power cultures were found to be negative for CD34 after 2 weeks ( Figure 3b). This suggests that some CD34 + cells have lost CD34 expression in culture and others have grown without losing CD34 expression.
  • CD34 expression in adherent PLA cells and adherent LAF cells was confirmed up to 20 weeks (passage 19) and 10 weeks (passage 9), respectively.
  • FIGs 6 and 7 show graphs showing the ratio of CD34 positive cells and CD105 positive cells up to 20 weeks, respectively. As shown, it was unexpectedly confirmed that CD34 positive cells were maintained even after 20 weeks, about 10-20%. It was found that about 50% of CD105 positive cells were maintained.
  • CD34 is generally cultured in DMEM, and it has been reported that CD34 becomes completely negative in 2 weeks (Mizuno, J Nippon Med Sch 2003; 70 (5) 428-431)) 0 There is also a paper that has been reported as CD34 (_) from the beginning (Mizuno J Nippon Med Sch 2 003; 70 (5) 428-431)). Therefore, it was concluded that the cells remained undifferentiated even after long-term culture.
  • the collected cells are cultured in DMEM medium at a concentration of 1.8 ⁇ 10 7 cells Z dish on a 60 mm dish. After 10 days of culture, the cells are close to confluence, at which point their current medium and differentiation induction medium (composition: an example of osteogenic medium is DMEM containing 10% FBS and 5% horse serum). , 1 ⁇ dexamethasone, 50 ⁇ ascorbate _ 2_phosphate, 10 mM / 3-glyceose phosphate, and 1% ABAM), and incubate for 22 days. The cells are observed using von Kossa staining.
  • phosphate buffered saline PBS
  • This sample is contacted with 2.5% silver nitrate for 20 minutes at a certain place.
  • the sample is washed with milliQ water. Leave this sample under fluorescent light for 15 minutes.
  • the sample is incubated for 2 minutes with 0.5% hydroquinone followed by 2 minutes with 5% sodium thiosulfate.
  • the sample is then washed with milliQ water. This sample is stained for 2 minutes with Nuclear First Red.
  • the sample is then washed with milliQ water. Mount this sample with Mount Quick (Daido Sangyo, Japan) and observe with a microscope.
  • the collected cells are cultured in DMEM medium at a concentration of 1.8 ⁇ 10 7 cells Z dish on a 60 mm dish. After 10 days of culture, the cells are close to confluence, at which point their current medium and differentiation induction medium (composition: an example of chondrogenic medium is 6.25 zg in DMEM containing 1% FBS. / ml insulin, 6.25 ⁇ g Zml transferrin, 10 ng / ml TGF j3 1, 50 nM ascorbate 2_phosphate, and 1% ABAM The medium to be added may be replaced) and cultured for 22 days. The cells are observed using Alcian blue staining.
  • Discard the medium from the desired sample The sample is fixed with 4% paraformaldehyde for 10 minutes. The sample is washed with milliQ water. Wash the sample with 3% acetate for 3 minutes. This sample was added to an Alcian Blue staining solution (composition: example of adipogenic medium, DMEM containing 10% FBS, 0.5 mM IBMX, 1 ⁇ M dexamethasone, 10 ⁇ insulin, 200 ⁇ indomethacin, and Immerse in medium supplemented with 1% koji for 30 minutes. The sample is washed with 3% acetate for 3 minutes. Wash the sample with milliQ water. The sample is dyed with Nuclea Fast Red for 2 minutes. The sample is then washed with milliQ water. The sample is then mounted with MountQuick (Daido Sangyo, Japan) and observed with a microscope.
  • MountQuick Daido Sangyo, Japan
  • the collected cells were cultured in DMEM medium at a concentration of 1.8 ⁇ 10 7 cells / dish on a 60 mm dish. After 10 days of culture, the cells are close to confluence, at which point their current medium and differentiation induction medium (composition: an example of adipogenic medium is DMEM containing 10% FBS, 0.5 mM IBMX, Medium) supplemented with 1 ⁇ dexamethasone, 10 ⁇ insulin, 200 / i M indomethacin, and 1% AMAM) and cultured for 12 days. The cells are observed using oil red O staining.
  • the culture medium was discarded, and the target sample was washed with phosphate buffered saline (PBS). Fix the washed sampnore with 4% paraformaldehyde (PFA) for 10 minutes. Wash this sample with milliQ water. Immerse this sample in 60% isopropyl alcohol for 1 minute. The sample is washed with milliQ water. Immerse this sample in hematoxylin for 10 minutes. The sample is washed with milliQ water. The sample is then immersed in lithium carbonate for a few seconds and then washed with milliQ water. Mount this sample with Mount Quick (Daido Sangyo, Japan) and observe with a microscope.
  • PBS phosphate buffered saline
  • PFA paraformaldehyde
  • composition example of osteogenic medium is DMEM containing 10% FBS and 5% horse serum, 1 ⁇ dexamethasone, 50 ⁇ ascorbate 2 phosphate In a medium supplemented with 10 mM ⁇ -glycose mouth phosphate, and 1%)) 1. Seed on a 60 mm dish at a concentration of 8 ⁇ 10 7 cells / dish.
  • composition adipogenic medium is an example of DMEM containing 10% FBS, 0.5 mM IBMX, 1 ⁇ dexamethasone, 10 ⁇ insulin, 200 ⁇ indomethacin, and 1 Seed on a 60 mm dish at a concentration of 8 X 10 7 cells / dish.
  • composition is an example of DMEM containing 10% FBS, 0.5 mM IBMX, 1 ⁇ dexamethasone, 10 ⁇ insulin, 200 ⁇ indomethacin, and 1 Seed on a 60 mm dish at a concentration of 8 X 10 7 cells / dish.
  • Oil Red Staining Oil red O staining is performed as described in Example 17 above.
  • the stem cells prepared in Example 1 or Example 2 exhibit high-quality pluripotency using a differentiation-inducing medium without culturing to a state close to confluence. It is understood.
  • the present invention can supply a large amount of ASC maintaining clinically useful properties, the medical field (particularly, fat regeneration, bone regeneration, myocardial regeneration, angiogenesis, refractory ulcer, etc.), etc. Useful for application.
  • the present invention proved that lipoaspirate-derived stem cells that can be obtained by a simple method can be applied to regenerative medicine. Accordingly, those skilled in the art easily find the industrial applicability of the present invention in the pharmaceutical industry and the like.

Abstract

Disclosed are: a fat-derived stem cell which is isolated from a fat aspirate; and a detailed characterization of the fat-derived stem cell in culture. A culture method for an undifferentiated pluripotent stem cell and an isolated or cultured pluripotent stem cell, particularly a novel long-term culture proliferation method for a fat-derived stem cell and a fat-derived stem cell produced by using the method; and a method for preparation of a fat-derived CD34+ and/or CD105+ stem cell comprising the step of cultivating an isolated human fat-derived stem cell. The cultivation is preferably performed at least for two weeks.

Description

明 細 書  Specification
脂肪由来幹細胞の長期培養増殖法  Long-term growth method for adipose-derived stem cells
技術分野  Technical field
[0001] 本発明は、未分化多能性幹細胞の培養方法、および単離または培養された多能 性幹細胞に関する。特に、本発明は、脂肪由来幹細胞の新規な長期培養増殖法、 およびそれを用いて得られる脂肪由来幹細胞に関する。  [0001] The present invention relates to a method for culturing undifferentiated pluripotent stem cells, and an isolated or cultured pluripotent stem cell. In particular, the present invention relates to a novel long-term culture growth method for adipose-derived stem cells, and adipose-derived stem cells obtained using the same.
背景技術  Background art
[0002] 現在、再生医学にぉレ、ては、幹細胞移植により目的とする組織及び臓器を修復し、 再生する技術の開発が進められている。多能性幹細胞は、増殖し、継代を繰り返す 自己複製能、および多種の成熟細胞（例えば、骨細胞、心筋細胞、血液細胞、皮膚 細胞、脂肪細胞、骨髄細胞など）に分化する分化能 (多能性)とを有している。生体か ら取り出された多能性幹細胞を培養し、更に分化誘導させた後、再度、生体内に戻 すことにより、組織及び臓器を修復し、再生することができると考えられている。多能 性幹細胞としては、一般的に、胚性幹細胞 (ES細胞）、間葉系幹細胞などが知られ ている。  [0002] Currently, in the field of regenerative medicine, development of a technique for repairing and regenerating target tissues and organs by transplanting stem cells is in progress. Pluripotent stem cells can proliferate and repeat passages, and have the ability to differentiate into various types of mature cells (eg, bone cells, cardiomyocytes, blood cells, skin cells, adipocytes, bone marrow cells, etc. Pluripotency). It is considered that tissue and organs can be repaired and regenerated by culturing pluripotent stem cells removed from a living body, further inducing differentiation, and then returning to the living body again. As pluripotent stem cells, embryonic stem cells (ES cells), mesenchymal stem cells, etc. are generally known.
[0003] 脂肪組織から単離された間質一血管画分細胞は、線維芽細胞様の形態を有する 脂肪生成前駆体であることが知られており（Van et al. J Clin Invest 1976 ; 58 : 699— 704 :非特許文献 1)、脂肪前駆細胞、血管間質系細胞などと呼ばれてき た。本明細書中以下では、脂肪組織から単離された接着間質細胞を脂肪由来間質（ または幹）細胞（adipose— derived stromal (or stem) cells) (ASC)と呼ぶ。 モノクローナノレ（Zuk et al. Mol Biol Cell 2002 ; 13 : 4279— 4295 :非特許 文献 2)またはポリクローナノレ（Zuk et al. Tissue Eng 2001 ; 7 : 211 - 228 : 非特許文献 3)の細胞培養研究によって、ヒト ASCが脂肪吸引物から得られ、中胚葉 起源または外胚葉起源の多数の系統に分化することが明らかにされている。ヒト ASC は、月旨肪生成、車欠' 形成 (Erickson et al. Biochem Biophys Res Commun 2002 ; 290 : 763— 769 :非特許文献4)、骨形成（^½1 0 611 et al. Int J Ob es Relat Metab Disord 2000 ; 24 Suppl 4 : S41— 44：非特許文献 5)、お よび筋原性（Mizuno et al. Plast Reconstr Surg 2002 ; 109 : 199— 209 : 非特許文献 6)の系統のような間葉系統のみでなぐ神経原性（Safford et al. Bi ochem Biophys Res Commun 2002 ; 294 : 371— 379：非特許文献 7)、血 管形成（Planat— Benard et al. Circulation 2004； 109： 656— 663：非特 許文献 8)、および造血'性 (Cousin et al. Biochem Biophys Res Commun 2003 ; 301 : 1016 - 1022 :非特許文献 9)の系統へも分化することが、多数のイン ビトロおよびインビボの研究によって示されている。 [0003] Stromal vascular fraction cells isolated from adipose tissue are known to be adipogenic precursors with a fibroblast-like morphology (Van et al. J Clin Invest 1976; 58 : 699-704: Non-patent document 1), which has been called adipose precursor cells, vascular stromal cells, and the like. Hereinafter, adherent stromal cells isolated from adipose tissue are referred to as adipose—derived stromal (or stem) cells (ASC). Monoclonal nanole (Zuk et al. Mol Biol Cell 2002; 13: 4279-4295: Non-patent literature 2) or Polyclonal nanole (Zuk et al. Tissue Eng 2001; 7: 211-228: non-patent literature 3) Culture studies have shown that human ASCs are obtained from lipoaspirates and differentiate into a number of strains of mesodermal or ectoderm origin. Human ASCs are known to produce luteum, occupancy (Erickson et al. Biochem Biophys Res Commun 2002; 290: 763—769: Non-patent document 4), bone formation (^ ½1 0 611 et al. Int J Ob es Relat Metab Disord 2000; 24 Suppl 4: S41—44: Non-patent literature 5), And neurogenicity (Safford et al. Biochem Biophys Res Commun 2002) only in the mesenchymal line such as Mizuno et al. Plast Reconstr Surg 2002; 109: 199—209: Non-patent document 6) 294: 371-379: Non-patent document 7), angiogenesis (Planat- Benard et al. Circulation 2004; 109: 656-663: non-patent document 8), and hematopoietic (Cousin et al. Biochem Biophys Res) Commun 2003; 301: 1016-1022: Non-patent literature 9) It has also been shown by numerous in vitro and in vivo studies.
[0004] 脂肪由来幹細胞 (ASC)は、大容量 (例えば、 500ml< )の脂肪吸引物を処理する ことによって、十分な数の ASCが得られるため、細胞培養を行うことなく臨床的に使 用され得る。新たに単離した細胞（freshly isolated cells)を使用することにより、 実際の処置においてより高い安全性および有効性が導かれ得る。実際、新たに単離 した ASCは、骨損傷（Lendeckel et al. J Craniomaxilofac Surg 2004 ; 3 2 : 370— 373 :非特許文献 10)、直腸膣瘻孔（Garcia— Olmo et al. Int J Co lorectal Dis 2003 ; 18 : 451— 454 :非特許文献 11)を処置するため、または軟 部組織増大術ようなレ、くつかの臨床試験におレ、て既に使用されてレ、る。  [0004] Adipose-derived stem cells (ASC) can be used clinically without cell culture because a sufficient number of ASCs can be obtained by treating a large volume (eg, 500 ml <) of lipoaspirate. Can be done. The use of freshly isolated cells can lead to higher safety and effectiveness in actual treatment. In fact, newly isolated ASCs are found in bone lesions (Lendeckel et al. J Craniomaxilofac Surg 2004; 3 2: 370—373: Non-patent literature 10) and rectal vaginal fistulas (Garcia— Olmo et al. Int J Colorectal Dis 2003; 18: 451-454: Non-patent literature11), or used in some clinical trials, such as soft tissue augmentation.
[0005] 脂肪吸引術は、最も人気のある美容形成術の 1つであり、毎年 80万を超える施術 が世界中で行われていると推定される。脂肪吸引術は、臨床的に大容量の脂肪組織 を取得可能であり、 ASCを回収するための典型的な方法であると考えられている。し 力 ながら、現時点において、脂肪吸引物は、臨床的な状況で使用されるものとして 十分に研究されていない。  [0005] Liposuction is one of the most popular cosmetic surgery, and it is estimated that over 800,000 procedures are performed every year worldwide. Liposuction is clinically capable of obtaining large volumes of adipose tissue and is considered a typical method for recovering ASC. However, at present, lipoaspirates have not been well studied for use in clinical situations.
[0006] 脂肪吸引物は、脂肪部分および液体部分から構成される。脂肪部分は、力ニュー レと減圧との相互動作によって「寸断されて」いる吸引された脂肪組織を意味する。 一方、液体部分は、脂肪部分とともに吸引された液体を意味する。液体は、主に、 (1 [0006] A lipoaspirate is composed of a fat portion and a liquid portion. By fat part is meant aspirated adipose tissue that has been “chopped” by the interaction of force neutrons and reduced pressure. On the other hand, the liquid part means a liquid sucked together with the fat part. Liquid is mainly (1
)神経および血管を損傷せずに脂肪吸引を促進するために、前もってその部位に注 入された生理食塩水、（2)末梢血、および（3)脂肪組織に由来する細胞または組織 断片から構成されている。 ) Consists of saline previously injected into the site to promote liposuction without damaging nerves and blood vessels, (2) peripheral blood, and (3) cells or tissue fragments derived from adipose tissue Has been.
[0007] これまで、脂肪吸引物のうちの脂肪部分は調べられてきた力 液体部分はほとんど 研究対象とされてこなかった。 特許文献 1：国際公開第 WO2005/035738パンフレット [0007] So far, the fat portion of the lipoaspirate has been examined. The liquid portion has hardly been studied. Patent Document 1: International Publication No. WO2005 / 035738 Pamphlet
特許文献 2：国際公開第 WO2005/042730パンフレット  Patent Document 2: International Publication No. WO2005 / 042730 Pamphlet
非特許文献 1: Van et al. J Clin Invest 1976 ; 58 : 699— 704  Non-Patent Document 1: Van et al. J Clin Invest 1976; 58: 699—704
非特許文献 2:Zuk et al. Mol Biol Cell 2002;13:4279-4295 非特許文献 3:Zuk et al. Tissue Eng 2001;7:211-228  Non-patent literature 2: Zuk et al. Mol Biol Cell 2002; 13: 4279-4295 Non-patent literature 3: Zuk et al. Tissue Eng 2001; 7: 211-228
非特許文献 4 : Erickson et al. Biochem Biophys Res Commun 2002 ;29 Non-Patent Document 4: Erickson et al. Biochem Biophys Res Commun 2002; 29
0:763-769 0: 763-769
特許文献 5 : Halvorsen et al. Int J Obes Relat Metab Disord 2000; 24 Suppl 4:S41-44  Patent Document 5: Halvorsen et al. Int J Obes Relat Metab Disord 2000; 24 Suppl 4: S41-44
非特許文献 6 : Mizuno et al. Plast Reconstr Surg 2002;109:199-20 9  Non-Patent Document 6: Mizuno et al. Plast Reconstr Surg 2002; 109: 199-20 9
非特許文献 7 : Saff ord et al. Biochem Biophys Res Commun 2002 ;29 4:371-379  Non-Patent Document 7: Saff ord et al. Biochem Biophys Res Commun 2002; 29 4: 371-379
非特許文献 8:Planat— Benard et al. Circulation 2004 ; 109 : 656— 663 非特許文献 9 : Cousin et al. Biochem Biophys Res Commun 2003 ;30 1:1016-1022  Non-patent literature 8: Planat— Benard et al. Circulation 2004; 109: 656-663 Non-patent literature 9: Cousin et al. Biochem Biophys Res Commun 2003; 30 1: 1016-1022
非特許文献 10:Lendeckel et al. J Craniomaxilofac Surg 2004 ;32 :370 -373  Non-Patent Document 10: Lendeckel et al. J Craniomaxilofac Surg 2004; 32: 370 -373
非特許文献 11: Garcia— Olmo et al. Int J Colorectal Dis 2003; 18:45 1-454  Non-Patent Document 11: Garcia— Olmo et al. Int J Colorectal Dis 2003; 18:45 1-454
発明の開示  Disclosure of the invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0008] 上述のような状況下で、臨床的な状況で使用されるものとしての脂肪吸引物につい て、さらに詳細な研究が行われることが求められている。 [0008] Under the circumstances described above, further detailed research is required to be conducted on lipoaspirates used in clinical situations.
課題を解決するための手段  Means for solving the problem
[0009] そこで、本発明者らは、脂肪吸引物の脂肪部分のみならず、液体部分についても 細胞の供給源としての詳細な特徴付けを行った。その結果、脂肪吸引物の液体部分 力、らも相当数の脂肪由来幹細胞 (ASC)が単離され得ることを見出した。 [0010] さらに、本発明者らは、新たに単離された ASCと培養された ASCとの比較実験を 行った。その結果、予想外にも、本発明者らのインビトロ培養条件を用いることにより 、 ASCの長期培養（例えば、 20週）後においてもなお、 CD34および CD105の発現 能を維持した ASCが、脂肪吸引物の脂肪部分および液体部分の両方から得られる ことを見出した。また、脂肪部分および液体部分から得られた ASCを、分化誘導培 地で培養し、これらが脂肪生成、軟骨形成、および骨形成の系統に沿って類似の分 化能を有することを確認した。 [0009] Therefore, the present inventors have performed detailed characterization as a cell source not only for the fat portion of the lipoaspirate but also for the liquid portion. As a result, it was found that the liquid partial force of lipoaspirate, and a considerable number of fat-derived stem cells (ASC) can be isolated. [0010] Furthermore, the present inventors conducted a comparative experiment between newly isolated ASC and cultured ASC. As a result, unexpectedly, by using the in vitro culture conditions of the present inventors, ASC that maintained the expression ability of CD34 and CD105 even after long-term culture of ASC (for example, 20 weeks) was liposuctioned. It was found that it can be obtained from both the fat and liquid parts of the product. In addition, ASCs obtained from the fat portion and the liquid portion were cultured in a differentiation-inducing medium, and it was confirmed that they had similar differentiation ability along the adipogenic, cartilage-forming, and osteogenic strains.
[0011] 本発明はこれらの知見に基づいて完成したものであり、以下のような特徴を有する 単離された多能性幹細胞、培養された多能性幹細胞、培養された脂肪由来幹細胞、 脂肪由来幹細胞の調製方法、および上記幹細胞の再生医療等における使用を提供 する。  The present invention has been completed based on these findings, and has the following characteristics: isolated pluripotent stem cell, cultured pluripotent stem cell, cultured adipose-derived stem cell, fat Provided are a method for preparing a stem cell, and use of the stem cell in regenerative medicine.
(1)単離された多能性幹細胞であって、 CD34、 CD105、またはこれらの両方の細 胞表面マーカーを発現し得る、細胞。  (1) An isolated pluripotent stem cell that can express a cell surface marker of CD34, CD105, or both.
(2)培養された多能性幹細胞であって、 CD34、 CD105、またはこれらの両方の細 胞表面マーカーを発現し得る、細胞。  (2) A cultured pluripotent stem cell that is capable of expressing CD34, CD105, or both cell surface markers.
(3)培養された脂肪由来幹細胞であって、 CD34、 CD105、またはこれらの両方の 細胞表面マーカーを発現し得る、細胞。  (3) A cultured adipose-derived stem cell that can express cell surface markers of CD34, CD105, or both.
(4)少なくとも 2週間培養された、上記（2)または（3)に記載の細胞。  (4) The cell according to (2) or (3), which has been cultured for at least 2 weeks.
(5)少なくとも 10週間培養された、上記（2)または（3)に記載の細胞。  (5) The cell according to (2) or (3), which has been cultured for at least 10 weeks.
(6)少なくとも 20週間培養された、上記（2)または（3)に記載の細胞。  (6) The cell according to (2) or (3), which has been cultured for at least 20 weeks.
(7)新たに単離された脂肪由来幹細胞を培養する工程を包含する、 CD34+および /または CD105+脂肪由来幹細胞の調製方法。 (7) A method for preparing CD34 + and / or CD105 + adipose-derived stem cells, comprising a step of culturing newly isolated adipose-derived stem cells.
(8)上記脂肪由来幹細胞を少なくとも 2週間培養する、上記（7)に記載の方法。 (8) The method according to (7) above, wherein the adipose-derived stem cells are cultured for at least 2 weeks.
(9)上記脂肪由来幹細胞を少なくとも 10週間培養する、上記（7)に記載の方法。(9) The method according to (7) above, wherein the adipose-derived stem cells are cultured for at least 10 weeks.
(10)上記脂肪由来幹細胞を少なくとも 20週間培養する、上記（7)に記載の方法。(10) The method according to (7) above, wherein the adipose-derived stem cells are cultured for at least 20 weeks.
(11)上記培養の基本培地が、 M _ 199培地である、上記（ 7)〜（： 10)のレ、ずれかに 記載の方法。 (11) The method according to any one of (7) to (: 10) above, wherein the basic medium for the culture is M_199 medium.
(12)接着培養を行う、上記（7)〜（： 11)のいずれかに記載の方法。 (13)上記培養が、 (12) The method according to any one of (7) to (: 11) above, wherein adhesion culture is performed. (13) The culture is
5〜20% ゥシ胎仔血清  5-20% urine fetal serum
50〜400IUまたは 50〜400mg/ml 抗生物質、  50-400 IU or 50-400 mg / ml antibiotic,
1〜： !O g/ml へパリン、および  1 to:! O g / ml heparin, and
0. 5〜： 10ng/ml 酸性線維芽細胞増殖因子  0.5 ~: 10ng / ml acidic fibroblast growth factor
を含有する培地にぉレ、て行われる、上記（7)〜（： 12)のレ、ずれかに記載の方法,The method according to any one of (7) to (: 12) above, which is performed on a medium containing
(14)上記培地が、 (14) The medium is
5〜20% ゥシ胎仔血清  5-20% urine fetal serum
50〜200IU ペニシリン、  50-200 IU penicillin,
50〜200mg/ml ス卜レプ卜マイシン、および  50-200 mg / ml sulepomycin, and
l ^lO g/ml へノ ジン、  l ^ lO g / ml
0. 5〜： 10ng/ml 酸性線維芽細胞増殖因子、  0.5 to 10 ng / ml acidic fibroblast growth factor,
を含有する、上記（13)に記載の方法。 The method according to (13) above, comprising
(15)上記培養のための培地が、  (15) The culture medium for the culture is
7〜： 15% ゥシ胎仔血清  7 ~: 15% urine fetal serum
70〜： 150IU ペニシリン  70 ~: 150IU penicillin
70〜： 150mgZml ストレプ卜マイシン、  70 ~: 150mgZml streptomycin,
2〜7 /i g/ml へパリン、および  2-7 / i g / ml heparin, and
l〜5ng/ml 酸性線維芽細胞増殖因子  l-5ng / ml acidic fibroblast growth factor
を含有する、上記（14)に記載の方法。 The method as described in said (14) containing.
(16)上記培養が、  (16) The culture is
10% ゥシ胎仔血清  10% urine fetal serum
100IU ペニシリン  100IU penicillin
lOOmg/ml ストレプトマイシン、  lOOmg / ml streptomycin,
5 μ g/ml へパリン、および  5 μg / ml heparin, and
2ng/ml 酸性線維芽細胞増殖因子  2ng / ml acidic fibroblast growth factor
を含有する培地において行われる、上記（15)に記載の方法。 The method according to (15) above, which is carried out in a medium containing
(17)脂肪由来幹細胞を調製する方法であって、 ヒトドナー由来の脂肪吸引物を得る工程、および (17) A method for preparing adipose-derived stem cells, Obtaining a lipoaspirate from a human donor, and
上記脂肪吸引物から、脂肪由来幹細胞を単離する工程、  Isolating fat-derived stem cells from the lipoaspirate,
上記単離した脂肪由来幹細胞を培養する工程  The step of culturing the isolated adipose-derived stem cells
を包含し、 Including
上記培養が、  The culture is
5〜20% ゥシ胎仔血清  5-20% urine fetal serum
50〜400IUまたは 50〜400mg/ml 抗生物質、  50-400 IU or 50-400 mg / ml antibiotic,
l^lO z g/ml へパリン、および  l ^ lO z g / ml heparin, and
0. 5〜： 10ng/ml 酸性線維芽細胞増殖因子  0.5 ~: 10ng / ml acidic fibroblast growth factor
を含有する培地にぉレ、て行われる、 Performed in a medium containing
方法。 Method.
(18)上記脂肪由来幹細胞を単離する工程が、上記脂肪吸引物の脂肪部分から、脂 肪由来幹細胞を単離することを含む、上記（17)に記載の方法。  (18) The method according to (17) above, wherein the step of isolating the fat-derived stem cells comprises isolating fat-derived stem cells from the fat portion of the lipoaspirate.
(19)上記脂肪由来幹細胞を単離する工程が、上記脂肪吸引物の液体部分から、脂 肪由来幹細胞を単離することを含む、上記（17)に記載の方法。  (19) The method according to (17), wherein the step of isolating the fat-derived stem cells comprises isolating the fat-derived stem cells from the liquid portion of the lipoaspirate.
(20)上記培養のための基本培地力 M— 199培地である、上記（17)〜（： 19)のい ずれかに記載の方法。  (20) The method according to any one of (17) to (: 19), wherein the basic medium force for the culture is M-199 medium.
(21)上記培養のための培地が、  (21) The culture medium for the culture is
5〜20% ゥシ胎仔血清  5-20% urine fetal serum
50〜200IU ペニシリン、  50-200 IU penicillin,
50〜200mg/ml ス卜レプ卜マイシン、および  50-200 mg / ml sulepomycin, and
l^lO z g/ml へノ ジン、  l ^ lO z g / ml
0. 5〜： 10ng/ml 酸性線維芽細胞増殖因子、  0.5 to 10 ng / ml acidic fibroblast growth factor,
を含有する、上記（17)〜（20)のレ、ずれかに記載の方法。 The method according to any one of (17) to (20) above, which contains
(22)上記培養のための培地が、  (22) The medium for the culture is
7〜： 15% ゥシ胎仔血清  7 ~: 15% urine fetal serum
70〜： 150IU ペニシリン  70 ~: 150IU penicillin
70~150mg/ml ストレプトマイシン、 2〜7 μ g/ml へパリン、および 70-150 mg / ml streptomycin, 2-7 μg / ml heparin, and
l〜5ng/ml 酸性線維芽細胞増殖因子  l-5ng / ml acidic fibroblast growth factor
を含有する、上記（21)に記載の方法。 The method according to (21) above, comprising
(23)上記培養のための培地が、  (23) The medium for the culture is
10% ゥシ胎仔血清  10% urine fetal serum
100IU ペニシリン  100IU penicillin
lOOmg/ml ストレプトマイシン、  lOOmg / ml streptomycin,
5 μ g/ml へパリン、および  5 μg / ml heparin, and
2ng/ml 酸性線維芽細胞増殖因子  2ng / ml acidic fibroblast growth factor
を含有する、上記（22)に記載の方法。 The method according to (22) above, comprising
(24)脂肪吸引物の液体部分を用いて脂肪由来幹細胞を調製する方法であって、 ヒトドナー由来の脂肪吸引物を得る工程  (24) A method for preparing adipose-derived stem cells using a liquid portion of a lipoaspirate, and a step of obtaining a lipoaspirate derived from a human donor
上記脂肪吸引物の液体部分から、脂肪由来幹細胞を単離する工程、および 上記単離した脂肪由来幹細胞を培養する工程  A step of isolating adipose-derived stem cells from the liquid portion of the lipoaspirate, and a step of culturing the isolated adipose-derived stem cells
を包含する、方法。 Including the method.
(25)上記脂肪由来幹細胞を単離する工程が、  (25) The step of isolating the fat-derived stem cells comprises
上記液体部分を遠心分離してペレットを回収すること、  Centrifuging the liquid part to recover the pellet;
上記ペレットから赤血球を溶血除去すること、および  Lysing red blood cells from the pellet; and
密度勾配遠心分離法により、上記赤血球を除去したペレットから脂肪由来幹細胞を 単離することを含み、  Isolating the adipose-derived stem cells from the pellet from which the red blood cells have been removed by density gradient centrifugation,
上記液体部分を酵素処理することを含まない、上記（24)に記載の方法。  The method according to (24) above, which does not include enzymatic treatment of the liquid portion.
(26)上記培養のための基本培地が、 M—199である、上記（24)または（25)に記載 の方法。  (26) The method according to (24) or (25) above, wherein the basic medium for the culture is M-199.
(27)上記培養のための培地が、  (27) The culture medium for the culture is
5〜20% ゥシ胎仔血清  5-20% urine fetal serum
50〜400IUまたは 50〜400mg/ml 抗生物質、  50-400 IU or 50-400 mg / ml antibiotic,
：!〜 10 μ g/ml へパリン、および  :! ~ 10 μg / ml heparin, and
0. 5〜： 10ng/ml 酸性線維芽細胞増殖因子 を含有する、上記（24)〜（26)のレ、ずれかに記載の方法。 0.5 ~: 10ng / ml acidic fibroblast growth factor The method according to any one of (24) to (26) above, which contains
(28)上記培養のための培地が、  (28) The medium for the culture is
5〜20% ゥシ胎仔血清  5-20% urine fetal serum
50〜200IU ペニシリン、  50-200 IU penicillin,
50〜200mg/ml ス卜レプ卜マイシン、および  50-200 mg / ml sulepomycin, and
l^lO z g/ml へノ ジン、  l ^ lO z g / ml
0. 5〜： 10ng/ml 酸性線維芽細胞増殖因子、  0.5 to 10 ng / ml acidic fibroblast growth factor,
を含有する、上記（27)に記載の方法。 The method according to (27) above, comprising
(29)上記培養のための培地が、  (29) The medium for the culture is
7〜： 15% ゥシ胎仔血清  7 ~: 15% urine fetal serum
70〜： 150IU ペニシリン  70 ~: 150IU penicillin
70~150mg/ml ストレプトマイシン、  70-150 mg / ml streptomycin,
2〜7 μ g/ml へパリン、および  2-7 μg / ml heparin, and
l〜5ng/ml 酸性線維芽細胞増殖因子  l-5ng / ml acidic fibroblast growth factor
を含有する、上記（28)に記載の方法。 The method according to (28) above, comprising
(30)上記培養のための培地が、  (30) The culture medium for the culture is
10% ゥシ胎仔血清  10% urine fetal serum
100IU ペニシリン  100IU penicillin
lOOmg/ml ストレプトマイシン、  lOOmg / ml streptomycin,
5 μ g/ml へパリン、および  5 μg / ml heparin, and
2ng/ml 酸性線維芽細胞増殖因子  2ng / ml acidic fibroblast growth factor
を含有する、上記（29)に記載の方法。 The method according to (29) above, comprising
(31)ゼラチンで被覆した基板上で接着培養を行う、上記（17)〜（30)のレ、ずれかに 記載の方法。  (31) The method according to any one of (17) to (30) above, wherein adhesion culture is performed on a substrate coated with gelatin.
(32)上記培養を少なくとも 2週間行う、上記（17)〜（31)のいずれかに記載の方法。  (32) The method according to any one of (17) to (31) above, wherein the culture is performed for at least 2 weeks.
(33)上記培養を少なくとも 10週間行う、上記（ 17)〜（31 )のレ、ずれかに記載の方法  (33) The method according to any one of (17) to (31) above, wherein the culture is performed for at least 10 weeks.
(34)上記培養を少なくとも 20週間行う、上記（ 17)〜（31 )のレ、ずれかに記載の方法 (35)培地交換を少なくとも週 2回、細胞継代を少なくとも週 1回行う、上記（32)〜（3 4)のレ、ずれかに記載の方法。 (34) The method according to any one of (17) to (31) above, wherein the culture is performed for at least 20 weeks. (35) The method according to any one of (32) to (34) above, wherein the medium is exchanged at least twice a week and cell passage is performed at least once a week.
(36)上記（17)〜（35)のいずれかに記載の方法によって調製された脂肪由来幹細 胞であって、 CD34、 CD105、またはこれらの両方の細胞表面マーカーを発現する 、細胞。  (36) A fat-derived stem cell prepared by the method according to any one of (17) to (35) above, which expresses a cell surface marker of CD34, CD105, or both of them.
(37)上記（1)〜（6)のいずれか、または上記（36)に記載の細胞を含有する、再生 医療用材料。  (37) A regenerative medical material comprising the cell according to any one of (1) to (6) above or (36) above.
(38)上記（1)〜（6)のレ、ずれか、または上記（36)に記載の細胞を使用する工程を 包含する、再生医療用材料の製造方法。  (38) A method for producing a material for regenerative medicine, which comprises the step of using the cells according to (1) to (6) above or any one of (1) to (6) above or (36).
(39)上記（1)〜（6)のいずれか、または上記（36)に記載の細胞を含有する、ヒトもし くは動物の疾患の治療、またはヒトもしくは動物の細胞、組織、もしくは臓器の再生に 使用するための薬剤。  (39) Treatment of a disease of a human or animal or a cell, tissue, or organ of a human or animal containing the cell according to any of (1) to (6) above or (36) above Drug for use in regeneration.
(40)上記（1)〜（6)のいずれか、または上記（36)に記載の細胞を含有する、ヒトもし くは動物の疾患を治療するため、またはヒトもしくは動物の細胞、組織、もしくは臓器 を再生するための方法。  (40) In order to treat a human or animal disease containing the cell according to any one of (1) to (6) above or (36) above, or a human or animal cell, tissue, or A method for regenerating organs.
(41)多能性幹細胞を含む組成物であって、該組成物は、 CD34、 CD105、または これらの両方の細胞表面マーカーを発現し得る多能性幹細胞を少なくとも 10%含む 、組成物。  (41) A composition comprising pluripotent stem cells, the composition comprising at least 10% pluripotent stem cells capable of expressing CD34, CD105, or both cell surface markers.
(42)上記組成物は、 CD34、 CD105、またはこれらの両方の細胞表面マーカーを 発現し得る多能性幹細胞を少なくとも 50%含む、（41)に記載の組成物。  (42) The composition according to (41), wherein the composition comprises at least 50% of pluripotent stem cells capable of expressing a cell surface marker of CD34, CD105, or both.
(43)上記組成物は、 CD105の細胞表面マーカーを発現し得る多能性幹細胞を少 なくとも 50%含む、（41)に記載の組成物。  (43) The composition according to (41), wherein the composition contains at least 50% of pluripotent stem cells capable of expressing a cell surface marker of CD105.
(44)上記組成物は、 CD34の細胞表面マーカーを発現し得る多能性幹細胞を少な くとも 10%含む、（41)に記載の組成物。  (44) The composition according to (41), wherein the composition comprises at least 10% of pluripotent stem cells capable of expressing a cell surface marker of CD34.
(45) CD34+および/または CD105+脂肪由来幹細胞を含む組成物を調製する方 法であって、 A)新たに単離された脂肪由来幹細胞を培養する工程を包含する、方 法。 (46)上記脂肪由来幹細胞を少なくとも 2週間培養する、 （45)に記載の方法。 (45) A method for preparing a composition comprising CD34 + and / or CD105 + adipose-derived stem cells, comprising the step of A) culturing newly isolated adipose-derived stem cells. (46) The method according to (45), wherein the adipose-derived stem cells are cultured for at least 2 weeks.
(47)上記脂肪由来幹細胞を 1〜2週間培養する、 (45)に記載の方法。  (47) The method according to (45), wherein the adipose-derived stem cells are cultured for 1 to 2 weeks.
(48)上記脂肪由来幹細胞を少なくとも 10週間培養する、 （45)に記載の方法。 (48) The method according to (45), wherein the adipose-derived stem cells are cultured for at least 10 weeks.
(49)上記脂肪由来幹細胞を少なくとも 20週間培養する、 （45)に記載の方法。(49) The method according to (45), wherein the adipose-derived stem cells are cultured for at least 20 weeks.
(50)上記培養が、 (50) The culture is
5〜20% ゥシ胎仔血清  5-20% urine fetal serum
50〜400IUまたは 50〜400mg/ml 抗生物質、  50-400 IU or 50-400 mg / ml antibiotic,
l^lO z g/ml へパリン、および  l ^ lO z g / ml heparin, and
0. 5〜： 10ng/ml 酸性線維芽細胞増殖因子  0.5 ~: 10ng / ml acidic fibroblast growth factor
を含有する培地を用いて行われる、（45)〜（49)のいずれ力、 1項に記載の方法。 The method according to any one of (45) to (49), wherein the method is performed using a medium containing
(51)上記脂肪由来幹細胞は、 (51) The adipose-derived stem cells are
ヒトドナー由来の脂肪吸引物を得る工程、および  Obtaining a lipoaspirate from a human donor, and
上記脂肪吸引物から、脂肪由来幹細胞を単離する工程  Isolating fat-derived stem cells from the lipoaspirate
によって、単離される、（45)に記載の方法。  The method according to (45), which is isolated by
(52)上記脂肪吸引物は、液体部分を含む、 (51)に記載の方法。  (52) The method according to (51), wherein the lipoaspirate includes a liquid portion.
(53)上記脂肪吸引物を得る工程は、液体部分を分離する工程をさらに包含する、 ( 51)に記載の方法。  (53) The method according to (51), wherein the step of obtaining the lipoaspirate further includes a step of separating the liquid portion.
発明の効果  The invention's effect
[0012] 本発明の単離または培養された多能性幹細胞は、 CD34または CD105のような臨 床的に重要な表面マーカーを発現することができるため、脂肪再生、骨再生、心筋 再生、血管再生、難治性潰瘍などの再生医療に使用するのに有利である。 CD34は 広く幹細胞の未分化状態を代表するマーカーと考えられており、間葉系マーカーで ある CD105との両陽性細胞は、間葉系細胞 *組織の再生に有効であると期待されて いるため。  [0012] Since the isolated or cultured pluripotent stem cell of the present invention can express a clinically important surface marker such as CD34 or CD105, fat regeneration, bone regeneration, myocardial regeneration, blood vessel It is advantageous for use in regenerative medicine such as regenerative and refractory ulcers. CD34 is widely considered to be a marker that represents the undifferentiated state of stem cells, and both positive cells with CD105, a mesenchymal marker, are expected to be effective for mesenchymal cell * tissue regeneration. .
[0013] 本発明の ASCの調製方法により得られるヒト由来の ASCは、長期培養増殖された 後も CD34および CD105のような臨床的に重要な表面マーカーを発現することがで きる。このような性質を有する ASCは、脂肪再生、骨再生、心筋再生、血管新生、難 治性潰瘍などの再生医療への応用におレ、て、有利な材料を提供し得る。 [0014] 本発明の多能性幹細胞の調製方法により、 CD34、 CD105のような臨床的に重要 な表面マーカーを発現し得る多能性幹細胞が大量に調製することができるため、再 生医療等に使用し得る材料が有利に提供され得る。 [0013] The human-derived ASC obtained by the method for preparing ASC of the present invention can express clinically important surface markers such as CD34 and CD105 even after long-term culture growth. ASC having such properties can provide an advantageous material for application to regenerative medicine such as fat regeneration, bone regeneration, myocardial regeneration, angiogenesis, and refractory ulcer. [0014] The method for preparing pluripotent stem cells of the present invention can prepare a large number of pluripotent stem cells capable of expressing clinically important surface markers such as CD34 and CD105. Materials that can be used for the present invention can be advantageously provided.
[0015] 通常行われる脂肪吸引では吸引脂肪は日常的に廃棄されている組織であるが、本 発明により有効利用が可能である。さらに、これまで廃棄されていた液体部分の有効 禾 U用という利点も提供する。 [0015] In normal liposuction, suctioned fat is a tissue that is routinely discarded, but can be effectively used according to the present invention. In addition, it provides the advantage of effective use of previously discarded liquid parts.
図面の簡単な説明  Brief Description of Drawings
[0016] [図 1]脂肪吸引物の脂肪部分または液体部分力 単離された脂肪由来間質細胞 (A SC)を示す写真およびグラフである。  [0016] FIG. 1 is a photograph and a graph showing isolated fat-derived stromal cells (ASC) of a lipoaspirate.
[図 2]継代 3〜 5の接着 PLA細胞または接着 LAF細胞を用いた細胞分化分析の結 果を示す写真およびグラフである。  FIG. 2 is a photograph and graph showing the results of cell differentiation analysis using adherent PLA cells or adherent LAF cells at passage 3-5.
[図 3-1]図 3は、新たに単離された LAF細胞の CD34および CD45発現を示すグラフ および写真（aおよび b)である。 [FIG. 3-1] FIG. 3 is a graph and photographs ( a and b) showing CD34 and CD45 expression of newly isolated LAF cells.
[図 3-2]図 3— 2は、図 3—1の続きである。  [Fig. 3-2] Fig. 3-2 is a continuation of Fig. 3-1.
[図 4-1]図 4は、新たに単離したか、または 2週間培養した PLA細胞および LAF細胞 のフローサイトメトリーのデータの比較を示す図（a〜n)である。 [FIG. 4-1] FIG. 4 is a diagram ( a to n) showing a comparison of flow cytometry data of PLA cells and LAF cells newly isolated or cultured for 2 weeks.
園 4- -2]図 4一 .2は、図 4— ■1の続きである。  (Section 4--2) Figure 4.1.2 is a continuation of Figure 4— ■ 1.
園 4- -3]図 4一 .3は、図 4— ■2の続きである。  4-3 is a continuation of Figure 4— ■ 2.
園 4- -4]図 4— .4は、図 4— ■3の続きである。  4-4) Figure 4-4 is a continuation of Figure 4-3.
園 4- -5]図 4一 .5は、図 4— ■4の続きである。  (Section 4--5) Figure 4 1.5 is a continuation of Figure 4—4.
園 4- -6]図 4— .6は、図 4— ■5の続きである。  (Section 4--6) Figure 4-6 is a continuation of Figure 4-5.
園 4- -7]図 4一 .7は、図 4— ■6の続きである。  (Section 4--7) Figure 4 1.7 is a continuation of Figure 4—6.
園 4- -8]図 4— .8は、図 4— ■7の続きである。  (Section 4--8) Figure 4-8 is a continuation of Figure 4-8.
園 4- -9]図 4— .9は、図 4— ■8の続きである。  4-9 of Figure 4-9 is a continuation of Figure 4-8.
[図 4-10]図 4— 10は、図 4— 9の続きである。  [Figure 4-10] Figure 4-10 is a continuation of Figure 4-9.
[図 4-11]図 4— 11は、図 4— 10の続きである。  [Figure 4-11] Figure 4-11 is a continuation of Figure 4-10.
[図 4-12]図 4—12は、図 4—11の続きである。  [Figure 4-12] Figure 4-12 is a continuation of Figure 4-11.
[図 4-13]図 4— 13は、図 4— 12の続きである。 [図 4-14]図 4—14は、図 4—13の続きである。 [Figure 4-13] Figure 4-13 is a continuation of Figure 4-12. [Figure 4-14] Figure 4-14 is a continuation of Figure 4-13.
[図 5-1]図 5は、新たに単離した、または培養した PLA細胞および LAF細胞の代表 的な細胞表面マーカーの発現の連続的な変化を示す図（a〜t)である。  [FIG. 5-1] FIG. 5 is a diagram (at) showing a continuous change in the expression of representative cell surface markers of newly isolated or cultured PLA cells and LAF cells.
：図 5- -2]図 5— •2は、図 5—1の続きである。  : Figure 5--2] Figure 5— • 2 is a continuation of Figure 5-1.
：図 5- -3]図 5— •3は、図 5— 2の続きである。  : Figure 5--3] Figure 5— • 3 is a continuation of Figure 5-2.
：図 5- -4]図 5— •4は、図 5— 3の続きである。  : Figure 5--4] Figure 5— • 4 is a continuation of Figure 5-3.
：図 5- -5]図 5— •5は、図 5— 4の続きである。  : Figure 5--5] Figure 5— • 5 is a continuation of Figure 5-4.
：図 5- -6]図 5— •6は、図 5— 5の続きである。  Figure 5--6] Figure 5— • 6 is a continuation of Figure 5-5.
：図 5- -7]図 5— •7は、図 5— 6の続きである。  : Fig. 5--7] Fig. 5— • 7 is a continuation of Fig. 5-6.
：図 5- -8]図 5— •8は、図 5— 7の続きである。  : Figure 5--8] Figure 5— • 8 is a continuation of Figure 5-7.
：図 5- -9]図 5— •9は、図 5— 8の続きである。  : Figure 5--9] Figure 5— • 9 is a continuation of Figure 5-8.
：図 5- -10]図 5- —10は、図 5 _ 9の続きである。  : Fig. 5--10] Fig. 5--10 is a continuation of Fig. 5_9.
：図 5- - 11]図 5- —11は、図 5— 10の続きである。  : Figure 5--11] Figure 5--11 is a continuation of Figure 5-10.
：図 5- - 12]図 5- —12は、図 5— 11の続きである。  : Figure 5--12] Figure 5--12 is a continuation of Figure 5-11.
：図 5- - 13]図 5- —13は、図 5— 12の続きである。  Figure 5--13] Figure 5--13 is a continuation of Figure 5-12.
：図 5- - 14]図 5- —14は、図 5— 13の続きである。  : Figure 5--14] Figure 5--14 is a continuation of Figure 5-13.
：図 5- - 15]図 5- —15は、図 5— 14の続きである。  : Figure 5--15] Figure 5--15 is a continuation of Figure 5-14.
：図 5- - 16]図 5- —16は、図 5— 15の続きである。  : Fig. 5--16] Fig. 5--16 is a continuation of Fig. 5-15.
：図 5- - 17]図 5- —17は、図 5— 16の続きである。  Figure 5--17] Figure 5--17 is a continuation of Figure 5-16.
：図 5- - 18]図 5- —18は、図 5— 17の続きである。  : Fig. 5--18] Fig. 5--18 is a continuation of Fig. 5-17.
：図 5- - 19]図 5- —19は、図 5— 18の続きである。  : Figure 5--19] Figure 5--19 is a continuation of Figure 5-18.
：図 5- -20]図 5- —20は、図 5—19の続きである。  : Fig. 5--20] Fig. 5--20 is a continuation of Fig. 5-19.
[図 6]新たに単離された PLA細胞および LAF細胞の CD34陽性細胞の比率の変動 を 20週間まで示した図である。  [FIG. 6] A graph showing changes in the ratio of CD34 positive cells in newly isolated PLA cells and LAF cells up to 20 weeks.
[図 7]新たに単離された PLA細胞および LAF細胞の CD105陽性細胞の比率の変 動を 20週間まで示した図である。  FIG. 7 is a graph showing changes in the ratio of CD105 positive cells in newly isolated PLA cells and LAF cells up to 20 weeks.
発明を実施するための最良の形態 BEST MODE FOR CARRYING OUT THE INVENTION
以下、本発明に関して、発明の実施の形態を説明する。本明細書の全体にわたり、 単数形の表現 (例えば、英語の場合は「a」、「an」、「the」など、独語の場合の「ein」、 「der」、「das」、「die」などおよびその格変化形、仏語の場合の「un」、「une」、「le」、 「la」など、スペイン語における「un」、「una」、「el」、「la」など、他の言語における対 応する冠詞、形容詞など）は、特に言及しない限り、その複数形の概念をも含むこと が理解されるべきである。また、本明細書において使用される用語は、特に言及しな い限り、当該分野で通常用いられる意味で用いられることが理解されるべきである。し たがって、他に定義されない限り、本明細書中で使用される全ての専門用語および 科学技術用語は、本発明の属する分野の当業者によって一般的に理解されるのと同 じ意味を有する。矛盾する場合、本明細書 (定義を含めて）が優先する。 Hereinafter, embodiments of the present invention will be described with respect to the present invention. Throughout this specification, Singular expressions (e.g. "a", "an", "the" for English, "ein", "der", "das", "die" for German, etc. Corresponding articles in other languages such as “un”, “una”, “el”, “la” in Spanish, such as “un”, “une”, “le”, “la” in French, It is to be understood that adjectives, etc. also include the plural concept, unless otherwise stated. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. . In case of conflict, the present specification, including definitions, will control.
[0018] (用語の定義）  [0018] (Definition of terms)
以下に本明細書において特に使用される用語の定義を列挙する。  Listed below are definitions of terms particularly used in the present specification.
[0019] 用語「細胞」は、当該分野におけるその最も広義の意味で本明細書において使用 され、内部に自己再生能を備え、遺伝情報およびその発現機構を有し、そして細胞 のような生物体を外界力も隔離する膜構造に包まれる、多細胞生物の組織の構成単 位をいう。本発明の方法において、任意の細胞が対象として使用され得る。本発明で 使用される細胞の数は、光学顕微鏡により計数することができる。光学顕微鏡を使用 して計数する場合は、核の数が計数される。組織を組織切片スライスとし、次いでへ マトキシリン一ェォシン (HE)染色し、細胞外マトリクス（例えば、エラスチンまたはコラ 一ゲン)および細胞に由来する核を識別する。この組織切片を光学顕微鏡にて検鏡 し、特定の面積（例えば、 200 μ ΐη Χ 200 μ ΐη)あたりの核の数を細胞数と見積って 計数することができる。本明細書において使用される細胞は、天然に存在する細胞 であっても、人工的に改変された細胞（例えば、融合細胞、遺伝子改変細胞など）で あってもよレ、。細胞の供給源の例としては、単一の細胞培養物；正常に成長したトラ ンスジヱニック動物の胚、血液、または体組織；あるいは正常に成長した細胞株由来 の細胞のような細胞混合物が挙げられるが、それらに限定されない。このような供給 源自体が、細胞として使用され得る。  [0019] The term "cell" is used herein in its broadest sense in the art, and has an internal self-regenerative capability, has genetic information and its expression mechanism, and is an organism such as a cell. Is a structural unit of tissue of multicellular organisms that is enveloped in a membrane structure that also isolates external forces. In the method of the present invention, any cell can be used as a subject. The number of cells used in the present invention can be counted with an optical microscope. When counting using an optical microscope, the number of nuclei is counted. Tissue is made into tissue section slices and then hematoxylin-eosin (HE) stained to identify extracellular matrix (eg, elastin or collagen) and cell-derived nuclei. This tissue section can be examined with an optical microscope, and the number of nuclei per specific area (for example, 200 μΐηΧ200 μΐη) can be estimated and counted. The cells used herein may be naturally occurring cells or artificially modified cells (eg, fusion cells, genetically modified cells, etc.). Examples of cell sources include a single cell culture; a normally grown embryo of a transgenic animal, blood, or body tissue; or a cell mixture such as cells from a normally grown cell line. However, it is not limited to them. Such sources themselves can be used as cells.
[0020] 本明細書において使用される脂肪細胞（fat cell, adipocyte)およびそれに対応 する物質は、生物が脂肪細胞またはそれに対応する細胞を有しさえすれば、任意の 生物 (例えば、メタラウナギ類、ャッメゥナギ類、軟骨魚類、硬骨魚類、両生類、爬虫 類、鳥類、哺乳動物など)、より好ましくは、哺乳動物 (例えば、単孔類、有袋類、貧歯 類、皮翼類、翼手類、食肉類、食虫類、長鼻類、奇蹄類、偶蹄類、管歯類、有鱗類、 海牛類、クジラ目、霊長類、齧歯類、ゥサギ目など）に由来し得る。 1つの実施形態に おいて、霊長類 (例えば、チンパンジー、二ホンザル、ヒト）由来の細胞が使用される。 最も好ましくは、ヒト由来の細胞が使用されるが、本発明はそれに限定されない。 [0020] As used herein, fat cells and corresponding substances may be any substance as long as the organism has fat cells or cells corresponding thereto. Organisms (e.g. metallunagi, shark eels, cartilaginous fish, teleosts, amphibians, reptiles, birds, mammals, etc.), more preferably mammals (e.g. single pores, marsupials, rodents, skins) Wings, wings, carnivores, carnivores, long-nosed animals, odd-hoofed animals, even-hoofed animals, rodents, scales, sea cattle, cetaceans, primates, rodents, maggots) Can be derived from In one embodiment, cells from a primate (eg, chimpanzee, dihonkey, human) are used. Most preferably, human-derived cells are used, but the invention is not so limited.
[0021] 本明細書において使用される場合、「幹細胞」とは、単分化能性、多能性、または 全能性を有する、分化細胞の前駆体 (または前駆細胞）をいう。幹細胞は、特定の刺 激に応答して分化され得る。代表的に、幹細胞は、損傷された組織を再生することが できる。本明細書で使用される幹細胞は、胚性幹 (ES)細胞、組織幹細胞 (組織性幹 細胞、組織特異的幹細胞または体性幹細胞ともいう）、あるいは他の前駆細胞であり 得る力 これらに限定されない。上述の能力を有している限り、幹細胞は、人工的に 作製した細胞（例えば、本明細書において記載される融合細胞、再プログラム化され た細胞など）であってもよい。胚性幹細胞は、初期胚に由来する多能性幹細胞である 。胚性幹細胞は、 1981年に初めて樹立され、 1989年以降ノックアウトマウス作製に も応用されている。 1998年にはヒト胚性幹細胞が樹立され、最近は再生医学にも利 用されつつある。組織幹細胞は、胚性幹細胞とは異なり、相対的に限定されたレべ ルの分化能を有する。組織幹細胞は、組織中に存在し、未分化な細胞内構造を有 する。組織幹細胞は、より高い核/細胞質比を有し、そしてわずかな細胞内オルガネ ラを有する。ほとんどの組織幹細胞は、多能性を有し、細胞周期が遅ぐ個体の一生 以上に増殖能を維持する。本明細書において使用される場合、幹細胞は、好ましく は組織幹細胞であり得るが、状況に応じて胚性幹細胞も使用され得る。  [0021] As used herein, "stem cell" refers to a precursor (or progenitor cell) of a differentiated cell having unipotency, pluripotency, or totipotency. Stem cells can be differentiated in response to specific stimuli. Typically, stem cells can regenerate damaged tissue. Stem cells used herein can be embryonic stem (ES) cells, tissue stem cells (also referred to as tissue stem cells, tissue-specific stem cells or somatic stem cells), or other progenitor cells. Not. As long as it has the above-mentioned ability, the stem cell may be an artificially produced cell (for example, a fusion cell described herein, a reprogrammed cell, etc.). Embryonic stem cells are pluripotent stem cells derived from early embryos. Embryonic stem cells were first established in 1981 and have been applied since 1989 to the production of knockout mice. In 1998, human embryonic stem cells were established and recently used in regenerative medicine. Tissue stem cells, unlike embryonic stem cells, have a relatively limited level of differentiation potential. Tissue stem cells are present in tissues and have an undifferentiated intracellular structure. Tissue stem cells have a higher nucleus / cytoplasm ratio and a few intracellular organelles. Most tissue stem cells are pluripotent and maintain proliferative capacity over the life of an individual with a slow cell cycle. As used herein, stem cells are preferably tissue stem cells, although embryonic stem cells can also be used depending on the situation.
[0022] 由来する部位により分類すると、組織幹細胞は、例えば、皮膚系、消化器系、骨髄 系、神経系などに分けられる。皮膚系の組織幹細胞としては、表皮幹細胞、毛嚢幹 細胞などが挙げられる。消化器系の組織幹細胞としては、膝 (共通）幹細胞、肝幹細 胞などが挙げられる。骨髄系の組織幹細胞としては、造血幹細胞、間葉系幹細胞な どが挙げられる。神経系の組織幹細胞としては、神経幹細胞、網膜幹細胞などが挙 げられる。 [0023] 本明細書において使用される場合、「前駆細胞」とは、子孫にあたる細胞が特定の 分化形質を発現することが明らかな場合、分化形質を発現していない未分化な親細 胞をいい、多能性未分化細胞だけでなく単分化能性未分化細胞も含む。例えば、子 孫にあたる細胞が、血管内皮細胞である場合、その前駆細胞を血管内皮前駆細胞と いう。本明細書において使用される場合、用語「幹細胞」は、前駆細胞を含む。しかし 、幹細胞の分化により得られる前駆細胞は、その幹細胞から見ると「分化細胞」に対 応するとレ、うこと力できる。用語「PLA (吸引脂肪由来細胞)」は、脂肪吸引物の脂肪 部分 (吸引脂肪)から得られる前駆細胞をいう。脂肪吸引物の液体部分に由来する 前駆細胞は、「吸引物細胞」または「LAF」ということができる。脂肪由来前駆細胞は 、 PLA (吸引脂肪由来細胞）および吸引物細胞を含む。 [0022] When classified according to the site of origin, tissue stem cells are classified into, for example, the skin system, digestive system, myeloid system, nervous system and the like. Examples of skin tissue stem cells include epidermal stem cells and hair follicle stem cells. Examples of digestive tissue stem cells include knee (common) stem cells and liver stem cells. Examples of myeloid tissue stem cells include hematopoietic stem cells and mesenchymal stem cells. Neural tissue stem cells include neural stem cells and retinal stem cells. [0023] As used herein, the term "progenitor cell" refers to an undifferentiated parent cell that does not express a differentiation trait when it is clear that a cell that is a progeny expresses a specific differentiation trait. It includes not only pluripotent undifferentiated cells but also unipotent undifferentiated cells. For example, when a cell corresponding to a progeny is a vascular endothelial cell, the progenitor cell is called a vascular endothelial progenitor cell. As used herein, the term “stem cell” includes progenitor cells. However, progenitor cells obtained by stem cell differentiation can respond to “differentiated cells” when viewed from the stem cells. The term “PLA (aspirated fat-derived cells)” refers to progenitor cells obtained from the fat portion (aspirated fat) of lipoaspirate. Progenitor cells derived from the liquid portion of lipoaspirate can be referred to as “aspirate cells” or “LAF”. Adipose-derived progenitor cells include PLA (aspirated fat-derived cells) and aspirate cells.
[0024] いわゆる吸引脂肪 (lipoaspirates) (脂肪吸引物の脂肪部分)から単離された細胞 は、回収した起源にちなんで PLA (processed lipoaspirate)細胞と呼ばれている（ ί列えば、 Zuk et al. Mol Biol Cell 2002 ; 13 : 4279— 4295)。そこで、本明 細書中でも同様に、脂肪吸引物の脂肪部分から単離した細胞を、 PLA細胞という。 また、本明細書中、脂肪吸引物の液体部分から単離された細胞を、 LAF Qiposucti on aspirate fluid :脂肪吸引物液）細胞という。  [0024] Cells isolated from so-called lipoaspirates (the fat part of the lipoaspirate) are called PLA (processed lipoaspirate) cells after their recovered origin (for example, Zuk et al Mol Biol Cell 2002; 13: 4279-4295). Therefore, similarly in this specification, cells isolated from the fat portion of the lipoaspirate are referred to as PLA cells. Moreover, in this specification, the cell isolated from the liquid part of the lipoaspirate is called LAF Qiposucti on aspirate fluid (cell).
[0025] 本明細書において使用される場合、用語「脂肪由来前駆細胞」は、脂肪吸引より得 られた、幹細胞および他の前駆細胞をレ、い、他の前駆細胞は、この脂肪吸引により 得られた、末梢血由来の幹細胞または血管間質細胞（脂肪前駆細胞（preadipocyt e) )のようなものである。脂肪由来前駆細胞は、脂肪組織または脂肪吸引法により得 られた任意の多能性前駆細胞または単分化能性前駆細胞の集団を意味する。この 細胞としては、脂肪由来血管間質細胞（=脂肪前駆細胞 (preadipocyte)、脂肪由 来間質細胞）、脂肪由来幹細胞、脂肪幹細胞、内皮前駆細胞、造血性幹細胞などが 挙げられる。このような幹細胞を単離するためのレ、くつかの技術力、例えば、 Nakats ujiら「幹細胞研究プロトコール [Stem Cell/Clone Research Protocol]」、 Yo dosha (2001)； WO00Z53795 ;W〇03Z022988 ;W〇0l/62901に記載され るように知られてレ、る。これらの文献は、その関連部分において、参考として本明細 書中に援用される。本明細書中において使用される場合、用語「脂肪由来前駆細胞 」とは、これらの公知な単離方法により得られた脂肪組織由来幹細胞を含む、全ての 脂肪組織由来幹細胞をいう。本明細書中で使用される場合、「前駆細胞」は、多能性 未分化細胞だけでなぐ単分化能性未分化細胞も含む。本明細書において使用さ れる場合、用語「幹細胞」は、前駆細胞を含む。用語「PLA (吸引脂肪由来細胞）」は 、脂肪吸引物の脂肪部分 (吸引脂肪)から得られる前駆細胞をいう。脂肪吸引物の液 体部分に由来する前駆細胞は、「吸引物細胞」ということができる。脂肪由来前駆細 胞は、 PLA (吸引脂肪由来細胞）および吸引物細胞を含む。 [0025] As used herein, the term "adipose-derived progenitor cell" refers to stem cells and other progenitor cells obtained by liposuction, and other progenitor cells are obtained by this liposuction. Such as peripheral blood-derived stem cells or vascular stromal cells (preadipocytes). An adipose-derived progenitor cell means any pluripotent progenitor cell or a population of unipotent progenitor cells obtained by adipose tissue or liposuction. Examples of such cells include adipose-derived vascular stromal cells (= preadipocytes, adipose-derived stromal cells), adipose-derived stem cells, adipose stem cells, endothelial progenitor cells, hematopoietic stem cells, and the like. A number of technical capabilities for isolating such stem cells, such as Nakats uji et al., “Stem Cell / Clone Research Protocol”, Yo dosha (2001); WO00Z53795; W003Z022988; W ○ Known as described in 0l / 62901. These documents are incorporated herein by reference in their relevant parts. As used herein, the term “adipose-derived progenitor cells” "Means all adipose tissue-derived stem cells including adipose tissue-derived stem cells obtained by these known isolation methods. As used herein, “progenitor cells” include unipotent undifferentiated cells as well as pluripotent undifferentiated cells. As used herein, the term “stem cell” includes progenitor cells. The term “PLA (aspirated fat-derived cells)” refers to progenitor cells obtained from the fat portion (aspirated fat) of lipoaspirate. Progenitor cells derived from the liquid part of the lipoaspirate are called “aspirate cells”. Adipose-derived precursor cells include PLA (aspirated fat-derived cells) and aspirate cells.
[0026] これまで、脂肪吸引物のうちの脂肪部分のみが調べられてきたが、本発明者らは、 予備的な研究を通して、脂肪吸引物の液体部分からも有意な量の前駆体細胞が単 離され得ることを認識した。さらに、新たに単離した脂肪由来幹細胞 (ASC)は、培養 された ASCよりも、細胞ベースの治療のためのツールとして好ましレ、（特に、臨床試 験の初期段階で)ことから、脂肪組織力 新たに単離された細胞の特徴付けも詳細 になされるべきである。 [0026] So far, only the fat portion of the lipoaspirate has been investigated, but through preliminary studies, the inventors have found that a significant amount of precursor cells are also present from the liquid portion of the lipoaspirate. Recognized that it can be isolated. In addition, freshly isolated adipose-derived stem cells (ASCs) are preferred as a tool for cell-based therapy over cultured ASCs (especially in the early stages of clinical trials). Organizational power The characterization of newly isolated cells should also be detailed.
[0027] 細胞の起源は、外胚葉、内胚葉および中胚葉に分類される。外胚葉起源の幹細胞 は、主に脳に存在し、これには神経幹細胞が含まれる。内胚葉由来の細胞は、主に 骨髄に存在し、これには血管幹細胞およびその分化細胞、造血幹細胞およびその 分化細胞、間葉系幹細胞およびその分化細胞などが含まれる。中胚葉由来の細胞 は主に臓器に存在し、これには肝幹細胞およびその分化細胞、膝幹細胞およびその 分化細胞などが含まれる。本明細書では、体細胞はどの胚葉由来であってもよい。 好ましくは、間葉系幹細胞が使用され得る。  [0027] The origin of cells is classified into ectoderm, endoderm and mesoderm. Stem cells of ectoderm origin are mainly present in the brain, including neural stem cells. Endoderm-derived cells are mainly present in the bone marrow, and include vascular stem cells and their differentiated cells, hematopoietic stem cells and their differentiated cells, mesenchymal stem cells and their differentiated cells, and the like. Cells derived from mesoderm are mainly present in organs, and include hepatic stem cells and differentiated cells thereof, knee stem cells and differentiated cells thereof. As used herein, somatic cells may be derived from any germ layer. Preferably, mesenchymal stem cells can be used.
[0028] 本明細書において使用される場合、用語「間葉系幹細胞」とは、間葉に見出される 幹細胞をいう。本明細書では、用語「間葉系幹細胞」は、「MSC」と略され得る。間葉 とは、多細胞動物の発生各期に認められる、星状または不規則な突起をもち、上皮 組織間の間隙をうめる遊離細胞の集団をいう。間葉はまた、細胞と結合する細胞内 接着因子とともに形成された組織をいう。間葉系幹細胞は、増殖能と、骨細胞、軟骨 細胞、筋細胞、ストローマ細胞、腱細胞、および脂肪細胞への分化能とを有する。間 葉系幹細胞は、患者力 採取した骨髄細胞等を培養または増殖、あるいは軟骨細胞 または骨芽細胞に分化させるために使用される。間葉系幹細胞はまた、歯槽骨；関 節症等の骨、軟骨、または関節などの再建材料として使用されておいる。間葉系幹 細胞に対する大きな需要が存在している。また、間葉系幹細胞は、血球、リンパ系細 胞へも分化し得る。従って、間葉系幹細胞に対する需要がますます高まっている。 [0028] As used herein, the term "mesenchymal stem cell" refers to a stem cell found in mesenchyme. As used herein, the term “mesenchymal stem cell” may be abbreviated as “MSC”. Mesenchyme refers to a population of free cells with stellate or irregular protrusions found in each stage of multicellular animal development that fill the gaps between epithelial tissues. Mesenchyme also refers to tissue formed with intracellular adhesion factors that bind to cells. Mesenchymal stem cells have the ability to proliferate and differentiate into bone cells, chondrocytes, muscle cells, stromal cells, tendon cells, and adipocytes. Mesenchymal stem cells are used for culturing or proliferating bone marrow cells and the like collected from patient force or differentiating them into chondrocytes or osteoblasts. Mesenchymal stem cells also have alveolar bone; It is used as a reconstruction material for bone, cartilage, or joints with arthropathy. There is a great demand for mesenchymal stem cells. Mesenchymal stem cells can also differentiate into blood cells and lymphoid cells. Therefore, there is an increasing demand for mesenchymal stem cells.
[0029] 語句「脂肪吸引由来物質」とは、脂肪吸引が行われた際に生成する任意の物質を いう。代表的に、このような脂肪吸引由来物質は、脂肪組織および脂肪吸引物を含 む。  [0029] The phrase "substance derived from liposuction" refers to any substance that is produced when liposuction is performed. Typically, such liposuction-derived materials include adipose tissue and lipoaspirate.
[0030] 語句「脂肪吸引物」とは、脂肪吸引が行われる際に生成する液体をいう。このような 脂肪吸引物は、以下：（1)脂肪吸引時に一緒に吸引された液体 (例えば、トウメセント 液および血液を含む）または、（2)吸引脂肪を溶液 (例えば、生理食塩水）で洗浄す る際に生じた廃液、をいう。  [0030] The phrase "lipoaspirate" refers to a liquid that is generated when liposuction is performed. Such lipoaspirates are: (1) liquid aspirated together at the time of liposuction (for example, containing tomesent fluid and blood) or (2) washing the aspirated fat with a solution (for example, physiological saline) This refers to the waste liquid that is generated during the process.
[0031] 本明細書において使用される場合、用語「脂肪細胞（adipocyte)」とは、組織間に 位置されるか、あるいは、疎性結合組織または毛細血管周辺の一群として脂肪組織 を形成し、そして大量の脂質を含む細胞をいう。脂肪細胞（Fat Cell)としては、黄 色脂肪細胞（yellow adipocyte)または褐色脂肪細胞（brown adipocyte)が挙 げられる。これらの細胞は、本明細書中において等価に使用され得る。細胞内の脂 肪は、スダン III (Sudan III)または四酸化オスミウムを用いて容易に検出され得る。  [0031] As used herein, the term "adipocyte" is located between tissues or forms adipose tissue as a group of loose connective tissue or capillaries, It refers to cells that contain large amounts of lipids. Examples of fat cells include yellow adipocytes and brown adipocytes. These cells can be used equivalently herein. Intracellular fat can be easily detected using Sudan III or osmium tetroxide.
[0032] 本明細書において使用される場合、用語「組織」とは、多細胞生物において、実質 的に同一の機能および/または形態をもつ細胞の集合体をいう。通常「組織」は、同 じ起源の細胞の集合体であるが、その細胞が同一の機能および/または形態を有 する限り、異なる起源の細胞の集合体であり得る。従って、本発明の幹細胞が組織再 生に使用される場合、 2以上の異なる起源の細胞の集合体力 構成され得る。通常、 組織は、臓器の一部を構成する。動物の組織は、形態的、機能的または発生的根拠 に基づき、上皮組織、結合組織、筋肉組織、神経組織などに区別される。植物では、 構成細胞の発達段階によって分裂組織と永久組織とに大別される。あるいは、組織 は、構成細胞の種類によって単一組織と複合組織とに区別され得る。このように組織 は、種々の分類に区別される。任意の組織が、本明細書中において処置される標的 として意図され得る。  [0032] As used herein, the term "tissue" refers to a collection of cells having substantially the same function and / or morphology in a multicellular organism. A “tissue” is usually a collection of cells of the same origin, but can be a collection of cells of different origins as long as the cells have the same function and / or morphology. Thus, when the stem cells of the present invention are used for tissue regeneration, an aggregate strength of cells of two or more different origins can be constructed. Usually, the tissue forms part of an organ. Animal tissues are classified into epithelial tissues, connective tissues, muscle tissues, nerve tissues, etc. based on morphological, functional or developmental basis. Plants are roughly divided into meristems and permanent tissues depending on the stage of development of the constituent cells. Alternatively, the tissue can be distinguished into a single tissue and a composite tissue depending on the type of constituent cells. In this way, organizations are divided into various categories. Any tissue can be contemplated as a target to be treated herein.
[0033] 任意の臓器が、本発明の (処置等の)標的となり得る。本発明により標的とされる組 織または細胞は、任意の臓器由来であり得る。本明細書において、用語「臓器」とは 、特定の機能が行われる個体生物の特定の部分に位置する独立的な構造体をいう。 多細胞生物（例えば、動物、植物）において、臓器は、特定の様式において空間的 に配列された複数の組織からなり、各組織は多数の細胞から構成される。そのような 臓器の例としては、血管系に関連する臓器が挙げられる。 1つの実施形態において、 本発明により対象とされる臓器としては、皮膚、血管、角膜、腎臓、心臓、肝臓、臍帯 、腸、神経、肺、胎盤、膝臓、脳、四肢末梢、網膜などが挙げられるが、これらに限定 されなレ、。本明細書において、任意の臓器が標的として使用され得る。好ましくは、 間葉系の組織 (例えば、脂肪、骨、靭帯など）が標的とされ得るが、これらに限定され ない。 [0033] Any organ can be a target (such as a treatment) of the present invention. Pairs targeted by the present invention The tissue or cell can be from any organ. As used herein, the term “organ” refers to an independent structure located in a specific part of an individual organism that performs a specific function. In multicellular organisms (eg, animals, plants), an organ is composed of a plurality of tissues spatially arranged in a specific manner, and each tissue is composed of a large number of cells. An example of such an organ is an organ related to the vascular system. In one embodiment, organs targeted by the present invention include skin, blood vessels, cornea, kidney, heart, liver, umbilical cord, intestine, nerve, lung, placenta, knee, brain, extremity, retina, etc. But not limited to these. Any organ can be used as a target herein. Preferably, mesenchymal tissues (eg, fat, bone, ligaments, etc.) can be targeted, but are not limited to these.
[0034] 本明細書において使用される場合、細胞、組織、 S蔵器などに関連する用語「所望の 部位に対応する」とは、本発明における移植または再生などを目的とするときの、 目 的とする部位から取得した力 (例えば、心臓であれば、心臓由来の細胞）または目的 とする部位に存在する細胞などと実質的に同じ性質を有する細胞など (例えば、心臓 細胞へと分化させた細胞）を示す。従って、所望の部位に対応する細胞は、実質的 に同一の特徴 (例えば、細胞表面マーカーなど）を有することによって確認することが できる。  [0034] As used herein, the term "corresponding to a desired site" related to cells, tissues, S-ware, etc. means that when the purpose is transplantation or regeneration in the present invention, Forces obtained from the target site (for example, heart-derived cells in the case of the heart) or cells having substantially the same properties as cells existing in the target site (for example, differentiated into heart cells) Cells). Therefore, cells corresponding to a desired site can be confirmed by having substantially the same characteristics (for example, cell surface markers).
[0035] そのような所望の部位に対応する細胞を判別するために有用なマーカーの例とし ては、（1)脂肪：細胞質内におけるトリグリセリドの存在、 OilRed—〇染色、グリセロホ スフェートデヒドロゲナーゼ (Glycerophosphate dehydrogenase = GPDH)活十生 、細胞質内の GLUT4 、 Ap2 (脂肪酸結合タンパク質）、 LPL (リポタンパク質リパー ゼ）、 PPAR y 1 , 2 (ペルォキシソーム増殖活性化レセプター γ 1 , 2)、およびレプチ ン (Leptin)の発現；（2)骨細胞、骨組織：アルカリホスファターゼの存在、ある程度の 骨石灰化（カルシウムの沈着）の確認；およびォステオカルシン（Osteocalcin)、ォス テオポンチン（〇steopontin)、またはォステオネクチン（Osteonectin)の発現；（3) 軟骨細胞、軟骨組織:ムコ多糖の存在、 II型コラーゲン、コンドロイチン _4 _サルフ エート（chondroitin— 4— sulfate)の発現/存在；（4)骨格筋細胞：細胞質内のミオ シンの豊富な存在；などが挙げられるが、これらに限定されなレ、。 [0036] 本明細書において使用される場合、用語「移植（implantation)」および「移植 (tra nsplantation)」とは、本明細書中で互換的に使用され、本発明の細胞、組成物、医 薬などを、単独で、または他の治療剤と組み合わせて、体内に移入することを意味す る。本明細書において「移植片」とは、体内に移入される本発明の組織片、細胞、組 成物、医薬などを意味する。本発明において、治療部位 (例えば、骨など）への導入 のための以下の方法、形態および量が使用され得:本発明の医薬は、損傷部位へ直 接注入、貼付後および縫合、挿入などされる。本発明の脂肪由来前駆細胞と分化細 胞との組み合わせは、同時に (例えば、混合物として同時に、別々であるが同時にも しくは並行して）；または逐次的にかのいずれかで投与され得る。これは、組み合わさ れた薬剤が、治療混合物として一緒に投与される提示を含み、そして組み合わせた 薬剤が、別々であるが同時に (例えば、分化促進因子)投与される手順もまた含む。 「 組み合わせ」投与は、第 1に与えられ、続いて第 2に与えられる化合物または薬剤の うちの 1つを別々に投与することをさらに含む。 [0035] Examples of markers useful for discriminating cells corresponding to such desired sites include (1) fat: presence of triglycerides in the cytoplasm, OilRed-O staining, glycerophosphate dehydrogenase (Glycerophosphate dehydrogenase = GPDH), GLUT4 in the cytoplasm, Ap2 (fatty acid binding protein), LPL (lipoprotein lipase), PPAR y 1 and 2 (peroxisome proliferator activated receptor γ 1 and 2), and leptin (Leptin) (2) Bone cells, bone tissue: presence of alkaline phosphatase, confirmation of some bone mineralization (calcium deposition); and osteocalcin, osteopontin, or osteonectin (3) chondrocytes, cartilage tissue: presence of mucopolysaccharide, type II collagen, chondroitin _4 _sulfate (chondr oitin—4-sulfate) expression / presence; (4) skeletal muscle cells: abundant presence of myosin in the cytoplasm; [0036] As used herein, the terms "implantation" and "transplantation" are used interchangeably herein and refer to the cells, compositions, medical devices of the invention. Means that a drug or the like is transferred into the body alone or in combination with other therapeutic agents. As used herein, “graft” means a tissue piece, cell, composition, medicine, etc. of the present invention that is transferred into the body. In the present invention, the following methods, forms and amounts for introduction into a treatment site (for example, bone, etc.) can be used: the medicament of the present invention can be directly injected into an injured site, applied and sutured, inserted, etc. Is done. The combination of adipose-derived progenitor cells and differentiated cells of the invention can be administered either simultaneously (eg, simultaneously as a mixture, separately but simultaneously or concurrently); or sequentially. This includes presentation where the combined agents are administered together as a therapeutic mixture, and also includes a procedure where the combined agents are administered separately but simultaneously (eg, differentiation promoting factors). “Combination” administration further includes the separate administration of one of the compounds or agents given first, followed by the second.
[0037] 本明細書において使用される場合、用語「レシピエント」（受容者）とは、移植される 細胞などを受け取る個体といい、「宿主」とも呼ばれる。これに対し、移植される細胞 などを提供する個体は、「ドナー」（供与者）と呼ばれる。レシピエントとドナーとは同じ であっても異なってレ、てもよレ、。  [0037] As used herein, the term "recipient" (recipient) refers to an individual that receives cells to be transplanted and the like, and is also referred to as "host". In contrast, an individual that provides cells to be transplanted is called a “donor”. Recipients and donors can be the same or different.
[0038] 本発明において使用される細胞は、 自系由来（自己由来）でも、同種異系由来 (非 自己由来）でも、異種由来でもよい。拒絶反応の観点から、自己由来の細胞が好まし レ、。拒絶反応が問題でない場合、同種異系細胞が使用され得る。  [0038] The cells used in the present invention may be autologous (autologous), allogeneic (non-autologous), or heterologous. From the viewpoint of rejection, self-derived cells are preferred. If rejection is not an issue, allogeneic cells can be used.
[0039] 本明細書において使用される場合、用語「分化細胞の欠損に起因する疾患、障害 または異常状態」とは、分化細胞が関与する任意の疾患、障害または異常状態をい う。このような分化細胞としては、好ましくは、間葉系細胞であることが好ましいが、こ れに限定されない。  [0039] As used herein, the term "disease, disorder or abnormal condition resulting from the loss of differentiated cells" refers to any disease, disorder or abnormal condition involving differentiated cells. Such differentiated cells are preferably mesenchymal cells, but are not limited thereto.
[0040] 1つの実施形態において、本発明が対象とする疾患および障害は、循環器系（血 液細胞など）のものであり得る。そのような疾患または障害の例としては、貧血 (例え ば、再生不良性貧血 (特に重症再生不良性貧血)、腎性貧血、癌性貧血、二次性貧 血、不応性貧血など）、癌または腫瘍 (例えば、白血病）およびそれらの化学療法処 置後の造血不全、血小板減少症、急性骨髄性白血病（特に、第 1寛解期（High— ri sk群）、第 2寛解期およびそれ以降）、急性リンパ性白血病（特に、第 1寛解期、第 2 寛解期およびそれ以降)、慢性骨髄性白血病（特に、慢性期、移行期）、悪性リンパ 腫 (特に、第 1寛解期 (High—risk群）、第 2寛解期およびそれ以降)、多発性骨髄腫 (特に、発症後早期)など；心不全、狭心症、心筋梗塞、不整脈、弁膜症、心筋 Z心 膜疾患、先天性心疾患 (例えば、心房中隔欠損、動脈管開存、ファロー四徴など）、 動脈疾患 (例えば、動脈硬化、動脈瘤）、静脈疾患 (例えば、静脈瘤）、リンパ管疾患 (例えば、リンパ浮腫)などが挙げられるが、これらに限定されない。 [0040] In one embodiment, diseases and disorders targeted by the present invention may be those of the circulatory system (such as blood cells). Examples of such diseases or disorders include anemia (eg, aplastic anemia (particularly severe aplastic anemia), renal anemia, cancerous anemia, secondary anemia, refractory anemia, etc.), cancer Or tumors (eg, leukemia) and their chemotherapy Post-placement hematopoietic failure, thrombocytopenia, acute myeloid leukemia (especially the first remission phase (High-risk group), second remission phase and beyond), acute lymphoblastic leukemia (especially the first remission phase, 2nd remission phase and later), chronic myelogenous leukemia (especially chronic phase, transitional phase), malignant lymphoma (especially 1st remission phase (High-risk group), 2nd remission phase and later), frequent occurrence Myeloma (especially early after onset); heart failure, angina pectoris, myocardial infarction, arrhythmia, valvular disease, myocardial Z pericardial disease, congenital heart disease (eg, atrial septal defect, patent ductus arteriosus, fallow) 4), arterial disease (eg, arteriosclerosis, aneurysm), venous disease (eg, varicose vein), lymphatic vessel disease (eg, lymphedema), and the like.
[0041] 別の実施形態において、本発明が対象とする疾患および障害は、神経系のもので あり得る。そのような疾患または障害の例としては、痴呆症、脳卒中およびその後遺 症、脳腫瘍、脊髄損傷が挙げられるが、これらに限定されない。  [0041] In another embodiment, the diseases and disorders targeted by the present invention may be those of the nervous system. Examples of such diseases or disorders include, but are not limited to, dementia, stroke and its sequelae, brain tumors, spinal cord injury.
[0042] 別の実施形態において、本発明が対象とする疾患および障害は、免疫系のもので あり得る。そのような疾患または障害の例としては、 T細胞欠損症、白血病などが挙げ られる力 S、これらに限定されない。  [0042] In another embodiment, the diseases and disorders targeted by the present invention may be those of the immune system. Examples of such diseases or disorders include, but are not limited to, force S, including T cell deficiency, leukemia and the like.
[0043] 別の実施形態において、本発明が対象とする疾患および障害は、運動器および骨 格系のものであり得る。そのような疾患または障害の例としては、骨折、骨粗鬆症、関 節の脱臼、亜脱臼、捻挫、靱帯損傷、変形性関節症、骨肉腫、ユーイング肉腫、筋 ジストロフィー、骨形成不全症、骨軟骨異形成症が挙げられるが、これらに限定され ない。  [0043] In another embodiment, diseases and disorders targeted by the present invention may be those of the musculoskeletal and skeletal systems. Examples of such diseases or disorders include fractures, osteoporosis, joint dislocation, subluxation, sprains, ligament damage, osteoarthritis, osteosarcoma, Ewing sarcoma, muscular dystrophy, osteogenesis imperfecta, osteochondral dysfunction Examples include, but are not limited to, dysplasia.
[0044] 別の実施形態において、本発明が対象とする疾患および障害は、皮膚系のもので あり得る。そのような疾患または障害の例としては、無毛症、黒色腫、皮膚悪性リンパ 腫、血管肉腫、組織球症、水疱症、膿疱症、皮膚炎、湿疹などが挙げられるが、これ らに限定されない。  [0044] In another embodiment, the diseases and disorders targeted by the present invention may be of the skin system. Examples of such diseases or disorders include, but are not limited to, alopecia, melanoma, cutaneous malignant lymphoma, angiosarcoma, histiocytosis, blistering, pustulosis, dermatitis, eczema. Not.
[0045] 別の実施形態において、本発明が対象とする疾患および障害は、内分泌系のもの であり得る。そのような疾患または障害の例としては、視床下部 Z下垂体疾患、甲状 腺疾患、副甲状腺 (上皮小体)疾患、副腎皮質/髄質疾患、糖代謝異常、脂質代謝 異常、タンパク質代謝異常、核酸代謝異常、先天性代謝異常 (フェニールケトン尿症 、ガラクトース血症、ホモシスチン尿症、メープルシロップ尿症）、無アルブミン血症、 ァスコルビン酸合成能欠如、高ビリルビン血症、高ピリルビン尿症、カリクレイン欠損、 肥満細胞欠損、尿崩症、ノくソプレツシン分泌異常、侏儒症、ウォルマン病（酸リパー ゼ (Acid lipase)欠損症）、ムコ多糖症 VI型などが挙げられる力 これらに限定され ない。 [0045] In another embodiment, the diseases and disorders targeted by the present invention may be of the endocrine system. Examples of such diseases or disorders include hypothalamic Z pituitary disease, thyroid disease, parathyroid (parathyroid) disease, adrenal cortex / medullary disease, abnormal glucose metabolism, abnormal lipid metabolism, abnormal protein metabolism, nucleic acid Metabolic disorders, inborn errors of metabolism (phenylketonuria, galactosemia, homocystinuria, maple syrup urine), abuminemia, Ascorbic acid synthesis deficiency, hyperbilirubinemia, hyperpyrurubinuria, kallikrein deficiency, mast cell deficiency, diabetes insipidus, abnormal sonopressin secretion, gonorrhea, Wolman's disease (acid lipase deficiency), Powers including mucopolysaccharidosis type VI, but not limited to.
[0046] 別の実施形態において、本発明が対象とする疾患および障害は、呼吸器系のもの であり得る。そのような疾患または障害の例としては、肺疾患（例えば、肺炎、肺癌な ど）、気管支疾患などが挙げられるが、これらに限定されない。  [0046] In another embodiment, diseases and disorders targeted by the present invention may be of the respiratory system. Examples of such diseases or disorders include, but are not limited to, pulmonary diseases (eg, pneumonia, lung cancer, etc.), bronchial diseases and the like.
[0047] 別の実施形態において、本発明が対象とする疾患および障害は、消化器系のもの であり得る。そのような疾患または障害の例としては、食道疾患（例えば、食道癌など )、胃/十二指腸疾患 (例えば、胃癌、十二指腸癌など）、小腸疾患/大腸疾患 (例 えば、結腸ポリープ、結腸癌、直腸癌など）、胆道疾患、肝臓疾患 (例えば、肝硬変、 肝炎 (A型、 B型、 C型、 D型、 E型など）、劇症肝炎、慢性肝炎、原発性肝癌、アルコ 一ル性肝障害、薬物性肝障害など)、勝臓疾患 (急性膝炎、慢性膝炎、膝臓癌、嚢 胞性膝疾患など）、腹膜/腹壁/横隔膜疾患 (ヘルニアなど）、ヒルシュスプラング病 などが挙げられる力 これらに限定されない。  [0047] In another embodiment, diseases and disorders targeted by the present invention may be of the digestive system. Examples of such diseases or disorders include esophageal diseases (eg, esophageal cancer), gastric / duodenal diseases (eg, gastric cancer, duodenal cancer), small intestine / colon diseases (eg, colon polyps, colon cancer, Rectal cancer, etc.), biliary tract disease, liver disease (eg, cirrhosis, hepatitis (A, B, C, D, E, etc.), fulminant hepatitis, chronic hepatitis, primary liver cancer, alcoholic liver Disorders, drug-induced liver disorders, etc.), spleen diseases (acute knee inflammation, chronic knee inflammation, knee cancer, cystic knee disease, etc.), peritoneal / abdominal wall / diaphragm diseases (hernia, etc.), Hirschsprung disease, etc. Power is not limited to these.
[0048] 別の実施形態において、本発明が対象とする疾患および障害は、泌尿器系のもの であり得る。そのような疾患または障害の例としては、腎疾患 (例えば、腎不全、原発 性糸球体疾患、腎血管障害、尿細管機能異常、間質性腎疾患、全身性疾患による 腎障害、腎臓癌など)、膀胱疾患 (例えば、膀胱炎、膀胱癌など)などが挙げられるが 、これらに限定されない。  [0048] In another embodiment, the diseases and disorders targeted by the present invention may be of the urinary system. Examples of such diseases or disorders include renal diseases (e.g., renal failure, primary glomerular disease, renal vascular disorder, tubular dysfunction, interstitial renal disease, renal disorder due to systemic disease, renal cancer, etc. ), Bladder diseases (eg, cystitis, bladder cancer, etc.), but are not limited thereto.
[0049] 別の実施形態において、本発明が対象とする疾患および障害は、生殖器系のもの であり得る。そのような疾患または障害の例としては、男性生殖器疾患（例えば、男性 不妊、前立腺肥大症、前立腺癌、精巣癌など)、女性生殖器疾患 (例えば、女性不 妊、卵巣機能障害、子宮筋腫、子宮腺筋症、子宮癌、子宮内膜症、卵巣癌、絨毛性 疾患など)などが挙げられるが、これらに限定されない。  [0049] In another embodiment, diseases and disorders targeted by the present invention may be of the reproductive system. Examples of such diseases or disorders include male genital diseases (eg, male infertility, benign prostatic hyperplasia, prostate cancer, testicular cancer), female genital diseases (eg, female infertility, ovarian dysfunction, uterine fibroids, uterus) Adenomyosis, uterine cancer, endometriosis, ovarian cancer, chorionic disease, etc.).
[0050] 本明細書において使用される場合、「診断、予防、処置または予後のために有効な 量」とは、それぞれ、診断、予防、処置 (または治療）または予後において、医療上有 効であると認められる程度の量をいう。このような量は、当該分野において周知の技 術を用いて種々のパラメータを参酌することにより、当業者により決定され得る。 [0050] As used herein, "amount effective for diagnosis, prevention, treatment or prognosis" refers to a medically effective in diagnosis, prevention, treatment (or therapy) or prognosis, respectively. An amount that is recognized as being. Such amounts are well known in the art. It can be determined by one skilled in the art by taking into account various parameters using surgery.
[0051] 別の実施形態において、本発明は、美容目的の治療、処置または改善において使 用され得る。そのような美容目的は、純粋に健常状態への美容目的と同様に、手術 後または外傷後の変形および先天性の変形に対する美容治療も含まれる。本発明 は例えば、乳房の組織増大術 (豊胸術）、頰ゃ上下眼瞼の陥凹に対する組織増大術 、および顔面半側萎縮症、顔面神経麻痺後の組織萎縮、漏斗胸などへの組織増大 術に適用され得るが、これらに限定されない。さらに本発明は、鼻形成術、整鼻術、 オトガイ形成術 (組織増大術)、前額形成術 (組織増大術)、小耳症など耳介変形/ 奇形に対する耳介軟骨形成術へ適用され得るが、これらに限定されない。  [0051] In another embodiment, the present invention may be used in cosmetic treatment, treatment or improvement. Such cosmetic purposes include cosmetic treatment for post-surgical or post-traumatic and congenital deformities as well as purely healthy cosmetic purposes. The present invention includes, for example, breast tissue augmentation (breast augmentation), tissue augmentation for upper and lower eyelid depression, and tissue enlargement to facial atrophy, tissue atrophy after facial paralysis, funnel chest, etc. It can be applied to surgery, but is not limited thereto. Furthermore, the present invention can be applied to auricular chondrogenesis for auricular deformation / deformation such as nasal plastic surgery, rhinoplasty, genioplasty (tissue augmentation), forehead plasticity (tissue augmentation), and microtia. However, it is not limited to these.
[0052] 本発明が医薬として使用される場合、その医薬は、薬学的に受容可能なキャリアを さらに含み得る。当該分野において公知な任意の薬学的に受容可能なキャリアが、 本発明の医薬において使用され得る。  [0052] When the present invention is used as a medicament, the medicament may further comprise a pharmaceutically acceptable carrier. Any pharmaceutically acceptable carrier known in the art can be used in the medicament of the present invention.
[0053] 薬学的に受容可能なキャリアまたは適切な処方材料としては、抗酸化剤、保存剤、 着色料、風味料、希釈剤、乳化剤、懸濁化剤、溶媒、フィラー、増量剤、緩衝剤、送 達ビヒクル、および/または薬学的アジュバントが挙げられる力 これらに限定されな レ、。代表的には、本発明の医薬は、本発明の細胞および他の活性成分を、 1つ以上 の生理的に受容可能なキャリア、賦形剤または希釈剤とともに含む組成物の形態で 投与される。例えば、適切なビヒクルは、注射用水、生理的溶液、または人工脳脊髄 液であり得、これらには、非経口送達のための組成物に一般に使用される他の物質 を補充することが可能である。  [0053] Pharmaceutically acceptable carriers or suitable formulation materials include antioxidants, preservatives, colorants, flavors, diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffering agents. Power, including, but not limited to, delivery vehicles, and / or pharmaceutical adjuvants. Typically, the medicament of the present invention is administered in the form of a composition comprising the cells of the present invention and other active ingredients together with one or more physiologically acceptable carriers, excipients or diluents. . For example, a suitable vehicle can be water for injection, physiological solution, or artificial cerebrospinal fluid, which can be supplemented with other substances commonly used in compositions for parenteral delivery. is there.
[0054] 本明細書で使用される受容可能なキャリア、賦形剤または安定化剤は、好ましくは レシピエントに対して非毒性であり、そして好ましくは、使用される投薬量および濃度 において不活性であり、そして好ましくは、リン酸塩、クェン酸塩、または他の有機酸； ァスコルビン酸、 ひ—トコフエロール；低分子量ポリペプチド；タンパク質（例えば、血 清アルブミン、ゼラチンまたは免疫グロブリン）；親水性ポリマー（例えば、ポリビュルピ 口リドン）；アミノ酸 (例えば、グリシン、グノレタミン、ァスパラギン、アルギニンまたはリジ ン）；モノサッカリド、ジサッカリドおよび他の炭水化物（グルコース、マンノース、または デキストリンを含む）；キレート剤（例えば、 EDTA)；糖アルコール (例えば、マンニト ールまたはソルビトール）；塩形成対イオン (例えば、ナトリウム）；ならびに/あるいは 非イオン性表面活性化剤（例えば、 Tween、プル口ニック（pluronic)またはポリェチ レングリコール（PEG) )などが挙げられる力 これらに限定されない。 [0054] Acceptable carriers, excipients or stabilizers used herein are preferably non-toxic to the recipient and are preferably inert at the dosages and concentrations used. And preferably, phosphate, kenate, or other organic acids; ascorbic acid, hytocopherol; low molecular weight polypeptide; protein (eg, serum albumin, gelatin or immunoglobulin); hydrophilic polymer Amino acids (eg glycine, gnoretamine, asparagine, arginine or lysine); monosaccharides, disaccharides and other carbohydrates (including glucose, mannose or dextrin); chelating agents (eg EDTA) Sugar alcohols (eg mannito Or salt-forming counterions (eg, sodium); and / or non-ionic surfactants (eg, Tween, pluronic, or polyethylene glycol (PEG)) It is not limited to these.
[0055] 適切なキャリアの例としては、中性緩衝化生理食塩水、または血清アルブミンと混 合された生理食塩水が挙げられる。好ましくは、その生成物は、適切な賦形剤（例え ば、スクロース）を用いて凍結乾燥剤として処方される。他の標準的なキャリア、希釈 剤および賦形剤が、所望に応じて含まれ得る。他の例示的な組成物は、 pH7. 0〜8 . 5の Tris緩衝剤または pH4. 0〜5. 5の酢酸緩衝剤を含み、これらはさらに、ソルビ トールまたはその適切な代替物を含み得る。  [0055] Examples of suitable carriers include neutral buffered saline or saline mixed with serum albumin. Preferably, the product is formulated as a lyophilizer using a suitable excipient (eg, sucrose). Other standard carriers, diluents and excipients may be included as desired. Other exemplary compositions include Tris buffer at pH 7.0-8.5 or acetate buffer at pH 4.0-5.5, which may further include sorbitol or a suitable replacement thereof. .
[0056] 本発明の医薬組成物の一般的な調製法を以下に記載する。動物薬組成物、医薬 部外品、水産薬組成物、食品組成物および化粧品組成物なども、公知の技術により 製造すること力 Sできる。  [0056] A general method for preparing the pharmaceutical composition of the present invention is described below. Veterinary drug compositions, quasi-drugs, marine drug compositions, food compositions and cosmetic compositions can also be produced by known techniques.
[0057] 本発明の細胞などは、必要に応じて薬学的に受容可能なキャリアと配合され、注射 剤、懸濁剤、溶液剤、スプレー剤等の液状製剤として非経口的に投与され得る。薬 学的に受容可能なキャリアの例としては、賦形剤、潤滑剤、結合剤、崩壊剤、崩壊ィ ンヒビター、吸収促進剤、吸収剤、保湿剤、溶剤、溶解補助剤、懸濁化剤、等張化剤 、緩衝剤、無痛化剤などが挙げられる。防腐剤、抗酸化剤、着色剤、甘味剤などの製 剤添加物も、必要に応じて使用され得る。本発明の組成物は、本発明のポリヌクレオ チド、ポリペプチドなど以外の物質を配合され得る。非経口の投与経路の例としては 、静脈内、筋肉内、皮下投与、皮内投与、粘膜投与、直腸内投与、膣内投与、局所 投与、経皮投与などが挙げられるが、これらに限定されない。全身投与される場合、 本発明において使用される医薬は、発熱物質を含まない、薬学的に受容可能な水 溶液の形態であり得る。そのような薬学的に受容可能な組成物の調製は、 pH、等張 性、安定性などを考慮することにより、当該技術の範囲内である。  [0057] The cells of the present invention can be combined with a pharmaceutically acceptable carrier as necessary and administered parenterally as liquid preparations such as injections, suspensions, solutions, sprays and the like. Examples of pharmaceutically acceptable carriers include excipients, lubricants, binders, disintegrants, disintegrators, absorption enhancers, absorbents, humectants, solvents, solubilizers, suspending agents. , Tonicity agents, buffering agents, soothing agents and the like. Product additives such as preservatives, antioxidants, colorants, sweeteners and the like may be used as needed. The composition of the present invention can be blended with substances other than the polynucleotide, polypeptide and the like of the present invention. Examples of parenteral routes of administration include, but are not limited to, intravenous, intramuscular, subcutaneous, intradermal, mucosal, rectal, vaginal, topical, and transdermal. . When administered systemically, the medicament used in the present invention may be in the form of a pharmaceutically acceptable aqueous solution free of pyrogens. The preparation of such pharmaceutically acceptable compositions is within the skill of the art by considering pH, isotonicity, stability, and the like.
[0058] 本発明の医薬組成物は、着色料、保存剤、香料、矯味矯臭剤、甘味料等、ならび に他の薬剤をさらに含み得る。  [0058] The pharmaceutical composition of the present invention may further contain a colorant, a preservative, a fragrance, a flavoring agent, a sweetener and the like, and other agents.
[0059] 本発明の処置方法において使用される組成物の量は、使用目的、対象疾患（種類 、重篤度など）、患者の年齢、体重、性別、既往歴、細胞の形態または種類などを考 慮して、当業者により容易に決定され得る。本発明の処置方法を被験体 (または患者 )に対して施す頻度もまた、使用目的、対象疾患 (種類、重篤度など）、患者の年齢、 体重、性別、既往歴、および治療経過などを考慮して、当業者により容易に決定され 得る。頻度の例としては、毎日〜数ケ月に 1回（例えば、 1週間に 1回〜 1ヶ月に 1回） が挙げられる。好ましくは、投与は、経過を見ながら 1週間〜 1ヶ月に 1回施される。投 薬量は、処置される部位が必要とする量を見積もることによって、確定され得る。 [0059] The amount of the composition used in the treatment method of the present invention depends on the purpose of use, target disease (type, severity, etc.), patient age, weight, sex, medical history, cell morphology or type, and the like. Thought Therefore, it can be easily determined by those skilled in the art. The frequency with which the treatment method of the present invention is applied to a subject (or patient) also depends on the purpose of use, target disease (type, severity, etc.), patient age, weight, gender, medical history, treatment history, etc. In view of this, it can be easily determined by one skilled in the art. Examples of frequencies include daily to once every few months (eg once a week to once a month). Preferably, the administration is performed once a week to once a month while observing the course. The dosage can be determined by estimating the amount required by the area to be treated.
[0060] 本明細書において使用される場合、用語「指示書」は、医薬を投与する方法または 診断のための方法などを、医師、患者など投与を行う人または投与される人、診断す る人 (例えば、医師、患者など）または診断される人に対して記載したものである。こ の指示書は、本発明の診断薬、医薬などを投与するための適切な方法を指示する文 言が記載されている。この指示書は、本発明が実施される国の監督官庁（例えば、 日 本であれば厚生労働省、米国であれば食品医薬品局（FDA)など）が規定した様式 に従って作成され、その監督官庁により承認を受けた旨が明記される。指示書は、い わゆる添付文書 (package insert)であり、通常は紙媒体で提供される。この指示書 は、これに限定されず、例えば、電子媒体 (例えば、インターネットで提供される電子 媒体 (例えば、ウェブサイト）、電子メール)のような形態でも提供され得る。  [0060] As used herein, the term "instruction" diagnoses a method of administering a medicine or a method for diagnosis, etc., such as a doctor, a patient, etc. Described for a person (eg, a doctor, patient, etc.) or a person being diagnosed. This instruction describes a word indicating an appropriate method for administering the diagnostic agent, medicine and the like of the present invention. This instruction is prepared according to the format prescribed by the national supervisory authority (for example, the Ministry of Health, Labor and Welfare in Japan and the Food and Drug Administration (FDA) in the United States, etc. in the US). It will be clearly stated that it has been approved. Instructions are so-called package inserts, usually provided on paper. This instruction is not limited to this, and may be provided in the form of, for example, an electronic medium (for example, an electronic medium provided on the Internet (for example, a website), an electronic mail).
[0061] 本発明の方法による治療の終了の判断は、商業的に利用可能なアツセィもしくは 機器を使用する標準的な臨床検査の結果または意図される処置に関連する疾患（ 例えば、骨疾患、心臓疾患、神経疾患など）に特徴的な臨床症状の消滅あるいは美 容状態の回復 (例えば、外見の回復など）によって支持され得る。治療は、分化細胞 の欠損などに関連する疾患 (例えば、神経疾患）の再発または美容状態の損傷によ り再開され得る。 [0061] The end of treatment according to the methods of the present invention may be determined by the results of standard laboratory tests using commercially available accessories or equipment or diseases associated with the intended treatment (eg, bone disease, heart It can be supported by the disappearance of the clinical symptoms characteristic of the disease, neurological disease, etc. or the restoration of the cosmetic state (eg, recovery of appearance). Treatment can be resumed by a recurrence of a disease (eg, neurological disease) associated with a deficiency of differentiated cells or a cosmetic condition injury.
[0062] 本発明はまた、 1つ以上の医薬組成物を満たした 1つ以上の容器を備える医薬品 パッケージまたはキットを提供する。医薬品または生物学的製品の製造、使用または 販売を規制する政府機関が定めた形式の通知が、このような容器に任意に付属され 得、この通知は、ヒトへの投与に対する製造、使用または販売に関する政府機関によ る承認を表す。このキットは、注射用デバイスを備え得る。  [0062] The present invention also provides a pharmaceutical package or kit comprising one or more containers filled with one or more pharmaceutical compositions. A notice in the form of government agencies regulating the manufacture, use or sale of pharmaceuticals or biological products may optionally be attached to such containers, which may be produced, used or sold for human administration. Represents approval by the government agency. The kit can include an injectable device.
[0063] 毒性研究が、種々の動物モデル (例えば、マウス、ゥサギ、または非げつ歯類）を用 いて行われ得る。これらの動物モデルにおいて、一般状態、体重、摂餌量、飲水量 などの観察、ならびに、血液検査、血液化学的検査、尿検査、および眼科学的検査 などの検査が行われる。各種検査は、当該分野において周知であり、例えば、以下 のものが挙げられる力 これらに限定されない。 [0063] Toxicity studies have used various animal models (eg, mouse, rabbit, or non-rodent) Can be done. In these animal models, general conditions, body weight, food consumption, water consumption, etc., and tests such as blood tests, blood chemistry tests, urine tests, and ophthalmological tests are performed. Various tests are well known in the art, and include, but are not limited to, for example, the following forces.
[0064] 血液学的検查：赤血球数、白血球数、血小板数、血色素量、へマトクリット、血液像  [0064] Hematological examination: red blood cell count, white blood cell count, platelet count, hemoglobin content, hematocrit, blood image
(白血球型別百分率）、その他網赤血球数、プロトロンビン時間、活性化部分トロンボ プラスチン時間、などの測定が行われる。例えば、血液細胞組成物は、以下のように 測定される：（1)薬剤がマウスに投与される (未処置のコントロールマウスもまた、測定 されるべきである）； (2)各々の処置群中の 1匹のマウスから尾静脈を介して血液サン プノレを周期的に得る；そして（3)上記サンプノレを、赤血球数および白血球数、血液細 胞組成物ならびにリンパ球と多形核細胞との割合について分析する。各々の投薬レ ジメンおよびコントロールは、毒性が存在するか否かを示す。  (Percentage of leukocytes), other reticulocyte count, prothrombin time, activated partial thromboplastin time, etc. are measured. For example, blood cell composition is measured as follows: (1) Drug is administered to mice (untreated control mice should also be measured); (2) Each treatment group A blood sample is obtained periodically from one mouse through the tail vein; and (3) the sample is obtained from the red blood cell and white blood cell counts, the blood cell composition and the lymphocytes and polymorphonuclear cells. Analyze percentages. Each dosing regimen and control indicates whether toxicity is present.
[0065] 血液化学的検査：血清（血漿)タンパク、アルブミン、 A/C比、タンパク分画、ブドウ 糖、コレステロール、トリグリセリド、ピリルビン、尿素窒素、クレアチニン、トランスァミナ ーゼ（ASAT (GOT)、 ALAT (GPT) )、アルカリホスファターゼ、電解質（ナトリウム、 カリウム、塩素、カルシウム、無機リンなど）の測定。  [0065] Blood chemistry tests: serum (plasma) protein, albumin, A / C ratio, protein fraction, glucose, cholesterol, triglyceride, pyrilbin, urea nitrogen, creatinine, transaminase (ASAT (GOT), ALAT ( GPT)), alkaline phosphatase, electrolytes (sodium, potassium, chlorine, calcium, inorganic phosphorus, etc.).
[0066] 尿検査：尿量、 pH、タンパク、糖、ケトン体、ビリルビン、潜血、沈渣、比重又は浸透 圧、電解質 (ナトリウム，カリウムなど）の測定。  [0066] Urinalysis: measurement of urine volume, pH, protein, sugar, ketone body, bilirubin, occult blood, sediment, specific gravity or osmotic pressure, electrolyte (sodium, potassium, etc.).
[0067] 眼科学的検査：肉眼および検眼鏡を使用し、前眼部、中間透光体、眼底を分析す る。  [0067] Ophthalmological examination: Using the naked eye and an ophthalmoscope, the anterior segment, intermediate translucent body, and fundus are analyzed.
[0068] 各々の毒性研究の終了の際に、動物を屠殺することによって、さらなる研究が行わ れ得る (好ましくは、 American Veterinary Medical Association guidelines Report of the American Veterinary Medical Assoc. Panel on Euth anasia, (1993)J. Am. Vet. Med. Assoc. 202 : 229 _ 249 (こ従う）。次レヽで、各 処置群からの代表的な動物が、転移、異常疾患または毒性の直接的な証拠のため に、全体的な検屍によって試験され得る。組織における全体の異常が記載され、そ の組織が組織学的に試験される。体重の減少または血液成分の減少を引き起こす 医薬は、主要な器官に対する有害作用を有する医薬と同様に好ましくない。一般的 に、有害作用が大きいほど、その医薬は好ましくない。 [0068] Further studies can be performed by sacrificing animals at the end of each toxicity study (preferably American Veterinary Medical Association guidelines Report of the American Veterinary Medical Assoc. Panel on Euth anasia, (1993 ) J. Am. Vet. Med. Assoc. 202: 229-249 (follow this), at the next level, representative animals from each treatment group will receive direct evidence of metastasis, abnormal disease or toxicity. Can be tested by holistic examination, describes the overall abnormalities in the tissue, and the tissue is examined histologically, drugs that cause weight loss or loss of blood components are harmful to major organs As well as pharmaceuticals with In addition, the greater the adverse effect, the less preferred the medicament.
[0069] (好ましい実施形態の説明）  [0069] (Description of Preferred Embodiment)
以下に本発明の好ましい実施形態が説明される。以下の実施形態は、本発明のよ りよい理解のために提供されるものであり、本発明の範囲は以下の記載に限定される べきでない。本明細書中を参酌して、本発明の範囲を逸脱することなぐ変更および 修正を行い得ることが、当業者に明らかに理解される。  In the following, preferred embodiments of the present invention will be described. The following embodiments are provided for a better understanding of the present invention, and the scope of the present invention should not be limited to the following description. It will be apparent to those skilled in the art that changes and modifications may be made without departing from the scope of the invention in light of the present specification.
[0070] 脂肪吸引物の脂肪部分 (吸引脂肪)から単離され得る。具体的には、例えば、（1) 吸引された脂肪を、 1リットルの分液漏斗を使用して生理食塩水でよく洗浄する；（2) 下層の生理食塩水から上層の吸引脂肪が十分に分離されたことを確認し、その下層 を捨てる。この手順は、その生理食塩水が肉眼で見たときに実質的に透明になるま で繰り返される；（3) 0. 075%のコラゲナーゼ /PBSを、吸引された脂肪の量と等量 添加し、続いてよく攪拌しながら 37°Cにて 30分間インキュベートする；（4)等量の 10 %血清補充 DMEMを、上記のサンプルに添加する；（5)このサンプルを、 1200 X g にて 10分間遠心分離する；（6)生じるペレットを、 0. 16Mの NH C1/PBSに懸濁し  [0070] It can be isolated from the fat portion (aspiration fat) of the lipoaspirate. Specifically, for example, (1) The aspirated fat is thoroughly washed with physiological saline using a 1 liter separatory funnel; (2) The upper aspirated fat is sufficiently removed from the lower physiological saline. Check that it has been separated, and discard the lower layer. This procedure is repeated until the saline is substantially clear when viewed with the naked eye; (3) Add 0.075% collagenase / PBS equal to the amount of aspirated fat. Incubate for 30 minutes at 37 ° C with good agitation; (4) Add an equal volume of 10% serum-supplemented DMEM to the above sample; (5) Add this sample at 1200 X g (6) The resulting pellet is suspended in 0.16M NH 3 C1 / PBS.
4  Four
、続いて室温にて 10分間インキュベートする；（7)このサンプルを、 100 μ ΐη直径メッ シュを使用して吸引濾過する；そして（8)生じる濾液を、 1200 X gにて 5分間遠心分 離する。上記プロトコールは、製剤の量に依存して、当業者によりスケールアップまた はスケールダウンされ得る。  Then incubate at room temperature for 10 minutes; (7) Suction-filter the sample using a 100 μΐη diameter mesh; and (8) Centrifuge the resulting filtrate at 1200 X g for 5 minutes. To do. The protocol can be scaled up or down by those skilled in the art depending on the amount of the formulation.
[0071] 他方、脂肪由来前駆細胞は、脂肪吸引物の液体部分 (脂肪吸引液)から、例えば 以下のように単離され得る：（1)脂肪吸引物の液体部分を調製する；（2)その液体部 分を遠心分離し、細胞画分を得る；（3)その細胞画分を密度勾配遠心分離し、比重 に基づレ、て細胞の分離を行う；そして（4)赤血球よりも比重の軽レ、細胞層から細胞を 回収する。吸引液の液体部分は、生理食塩水またはリンゲル注射液を使用して調製 され得る。この遠心分離は、約 800 X g以下、あるいは約 400 X g以上の速度にて行 われ得る。その密度勾配遠心分離は、約 370 X g〜： 1， 100 X gの速度にて行われる 。この密度勾配遠心分離は、約 1. 076g/ml〜： 1. 078gZmlの比重（20°C)を有す る媒体を使用して行われる。この密度勾配遠心分離法において使用される媒体は、 Ficoll™, Percoll™またはスクロースであり得る。回収される細胞層の比重は、約 1. 050-1. 075の範囲であり得る。この細胞層は、ピペットを使用して収集され得る。 [0071] On the other hand, adipose-derived progenitor cells can be isolated from the liquid portion of lipoaspirate (lipoaspirate), for example as follows: (1) preparing the liquid portion of lipoaspirate; (2) Centrifuge the liquid portion to obtain the cell fraction; (3) density gradient centrifuge the cell fraction to separate the cells based on specific gravity; and (4) specific gravity over red blood cells. Collect cells from the cell layer. The liquid part of the aspirate can be prepared using saline or Ringer's injection. This centrifugation can be performed at a speed of about 800 X g or less, or about 400 X g or more. The density gradient centrifugation is performed at a speed of about 370 Xg to: 1,100 X g. This density gradient centrifugation is performed using a medium having a specific gravity (20 ° C.) of about 1.076 g / ml to: 1.078 gZml. The medium used in this density gradient centrifugation can be Ficoll ™, Percoll ™ or sucrose. The specific gravity of the recovered cell layer is approximately 1. It can be in the range of 050-1. This cell layer can be collected using a pipette.
[0072] 脂肪吸引方法は、当業者に周知である技術を用いて、実施することができる。脂肪 吸引方法に用いられる装置としては、ケイセィ脱脂吸引機サルポンプ SAL 76 -A ( ケイセィ医科工業 (株）、東京都文京区本郷 3— 19— 6)が挙げられるが、これらに限 定されない。例えばヒトの場合、 0. 0001 %アドレナリン入り生理食塩水などの液体を 、予定吸引脂肪量の等量から 2倍量、脂肪組織内に注入し、その後、内径 2〜3mm の力ニューレ（金属製吸引管）を用いて、— 250〜900mmHg程度の陰圧で吸引す ることによって行う。 [0072] The liposuction method can be performed using techniques well known to those skilled in the art. Examples of devices used for the fat suction method include, but are not limited to, KSEI degreasing and suction machine monkey pump SAL 76-A (KASEY Medical Co., Ltd., Hongo 3-19-6, Bunkyo-ku, Tokyo). For example, in the case of a human, a liquid such as a saline solution containing 0.0001% adrenaline is injected into the adipose tissue by an amount equal to twice the expected amount of aspirated fat, and then a force nucleus (made of metal) having an inner diameter of 2 to 3 mm. Using a suction tube), suction is performed at a negative pressure of about 250 to 900 mmHg.
[0073] 吸引した脂肪細胞を洗浄するために使用される液体として通常は、生理食塩水が 用いられる。しかし、生理食塩水以外にも、単離すべき幹細胞に悪影響を与えない 液体であれば、任意の液体が使用可能である。具体的には、例えば、リンゲル液、お よび哺乳動物培養培地（例えば、 DMEM、 MEM、 Ml 99、および MCDB153など )のような、任意の等張液が、本発明において使用可能である。  [0073] As the liquid used for washing the aspirated fat cells, physiological saline is usually used. However, any liquid other than physiological saline can be used as long as it does not adversely affect the stem cells to be isolated. Specifically, any isotonic solution can be used in the present invention, such as, for example, Ringer's solution and mammalian culture media (eg, DMEM, MEM, Ml 99, MCDB153, etc.).
[0074] 脂肪吸引物から幹細胞を調製する場合、必要に応じて、脂肪吸引物にコラゲナー ゼなどの酵素処理を行なうことが可能である。しかし、脂肪吸引物中に含まれるコラ 一ゲンの量は、脂肪組織と比較して相対的に少ないため、幹細胞調製の原料として 脂肪吸引物を用いる場合、コラゲナーゼ処理に必要とされる時間、および/または 酵素処理に必要とされる酵素量が、脂肪組織の場合よりも、相対的に低い。  [0074] When preparing stem cells from a lipoaspirate, it is possible to perform an enzyme treatment such as collagenase on the lipoaspirate when necessary. However, since the amount of collagen contained in the lipoaspirate is relatively small compared to that of adipose tissue, when using lipoaspirate as a raw material for stem cell preparation, the time required for collagenase treatment, and The amount of enzyme required for the enzyme treatment is relatively lower than in the case of adipose tissue.
[0075] 脂肪吸引物を遠心分離して細胞画分を得る工程においては、細胞画分と、その他 の成分 (例えば、血漿、手術時に注入した生理食塩水、麻酔薬、止血薬、破壊された 脂肪細胞力 漏出した脂質成分)とを分離する任意の条件を用いることができる。例 えば、 400〜800 X gでの 5〜： 15分程度の遠心分離を用いることが可能である。  [0075] In the step of obtaining the cell fraction by centrifuging the lipoaspirate, the cell fraction and other components (for example, plasma, physiological saline injected at the time of surgery, anesthetic, hemostatic, destroyed Any conditions for separating the fat cell force (leaked lipid component) can be used. For example, centrifugation at 400 to 800 X g for 5 to 15 minutes can be used.
[0076] 比重による遠心分離により細胞を単離する工程においては、例えば単離された細 胞画分を、密度勾配遠心分離にかけることができる。任意の密度勾配遠心分離のた めに使用可能な媒体が、この媒体として使用され得る。好ましい媒体は、 1. 076〜1 . 078g/mlの比重を有し得る。さらに、好ましい媒体の pHは、 4. 5と 7. 5との間で ある。媒体中の好ましいエンドトキシンレベルは、 0. 12EUZml以下である。代表的 な媒体としては、スクロース、フイコール（スクロースとェピクロロヒドリンとの共重合物） 、およびパーコール (ポリビニルピロリドンの被膜を有するコロイド状シリカ製品）が挙 げられる。フイコーノレは、例えば、 Ficoll-Paque PLUS (フアルマシアバイオテク 株式会社、東京）、 Histopaque— 1077 (シグマ アルドリッチ ジャパン株式会社、 東京）などの名称で市販されている。パーコールは、 PercolKシグマ アルドリッチ ジャパン株式会社、東京）という名称で市販されている。 [0076] In the step of isolating cells by centrifugation by specific gravity, for example, the isolated cell fraction can be subjected to density gradient centrifugation. Any medium that can be used for density gradient centrifugation can be used as this medium. A preferred medium may have a specific gravity of 1.076 to 1.078 g / ml. Furthermore, the preferred medium pH is between 4.5 and 7.5. The preferred endotoxin level in the medium is 0.12 EUZml or less. Typical media include sucrose and ficoll (copolymer of sucrose and epichlorohydrin). , And Percoll (a colloidal silica product with a polyvinylpyrrolidone coating). For example, Ficollore is commercially available under the names Ficoll-Paque PLUS (Falmacia Biotech Co., Ltd., Tokyo), Histopaque-1077 (Sigma Aldrich Japan Co., Ltd., Tokyo). Percoll is marketed under the name PercolK Sigma Aldrich Japan, Tokyo).
[0077] 比重による遠心分離の条件は、 Ficoll— Paque PLUSを媒体として用いる場合、 400 X g、 30〜40分力 S用レヽられ、 Histopaque— 1077を媒体として用レヽる場合、 37 Ocg、約 30分が用いられる。リンフォプレップを媒体として用いる場合、条件としては 8 00 X g、約 20分または 1 100 X g、約 10分が挙げられる力 これらに限定されない。  [0077] Centrifugation by specific gravity is performed when Ficoll—Paque PLUS is used as a medium, 400 X g, 30-40 component force S, and when Histopaque—1077 is used as a medium, 37 Ocg, approximately 30 minutes is used. When using lymphoprep as a medium, conditions include, but are not limited to, 800 xg, about 20 minutes or 1 100 xg, about 10 minutes.
[0078] 比重による遠心分離が用いられるときに、最も多い細胞は、通常赤血球細胞であり 、赤色の細胞層として確認することができる。幹細胞は、赤血球よりも比重が軽いため 、幹細胞を分離する場合には、赤血球よりも軽い細胞層を収集する。好ましくは、比 重 1 · 050〜： 1. 075の範囲の細胞層を収集する。  [0078] When centrifugation by specific gravity is used, the most abundant cells are usually red blood cells, which can be confirmed as a red cell layer. Stem cells are lighter in specific gravity than red blood cells, and therefore, when separating stem cells, a cell layer lighter than red blood cells is collected. Preferably, a cell layer with a specific gravity in the range of 1 · 050 to 1.075 is collected.
[0079] おおまかな細胞の比重は、パーコール、レディグラッドのような密度勾配遠心分離 媒体を塩ィヒナトリウム溶液やスクロース溶液で調製し、収集した細胞と共にデンシティ 一マーカービーズ（density marker beads)を加えて遠心し、ビーズによって分け られた 5〜： 10層のうち、どの層に細胞があるかを確認することで、試験され得る。  [0079] The specific gravity of the cells is roughly determined by preparing density gradient centrifugation media such as Percoll and Readygrad in sodium chloride solution or sucrose solution, adding density marker beads together with the collected cells, and centrifuging. However, it can be tested by checking which of the 5 to 10 layers separated by the beads is in which layer.
[0080] さらに、層に分離した細胞の回収は、例えば、ピペットを用いて行なうことができる。  [0080] Furthermore, the cells separated into layers can be collected using, for example, a pipette.
[0081] 比重による遠心分離においては、セルセパレーター（例えば、血液成分分離装置 A STEC204、（株）アムコ）を使用することが可能である。  [0081] A cell separator (for example, blood component separation apparatus A STEC204, Amco Co., Ltd.) can be used for centrifugation by specific gravity.
[0082] 脂肪組織が細胞から分離される条件は、脂肪組織が細胞から分離され、かつ細胞 の単離を促進する条件であり得る。このような条件としては、例えば、 Wakoより入手 可能なコラゲナーゼ（細胞分散のためのコラゲナーゼ、 032— 10534)を添カロするよ うな、細胞外マトリクスの分解が挙げられる力 これに限定されない。このようなコラゲ ナーゼは、濃度 0. 075%にて PBS中に溶解し、この溶液を 37°Cまで温めて脂肪と 等量を添加する。そのサンプノレを 37°Cにて 80rpmで 30分間攪拌するために、アジ テーターが使用される。  [0082] The conditions under which adipose tissue is separated from cells can be those under which adipose tissue is separated from cells and facilitates cell isolation. Such conditions include, but are not limited to, for example, the degradation of the extracellular matrix, such as collagenase available from Wako (collagenase for cell dispersion, 032-10534). Such collagenase is dissolved in PBS at a concentration of 0.075%, and the solution is warmed to 37 ° C and added in an amount equivalent to fat. An agitator is used to stir the sampnore at 37 ° C and 80 rpm for 30 minutes.
[0083] 好ましくは、本発明の方法は、高度の幹細胞純度を達成するために、血球細胞を 除去する工程をさらに包含する。しかし、このような血球細胞は、続いてディッシュ上 で培養し、この細胞集団を継代し、 80%を超える（好ましくは、 90%を超える）幹細胞 を生じさせることにより除去され得る。これもまた、先行技術からは予想されていなか つた驚くべき効果である。このような血球細胞は、血球細胞を分解する成分を添加す る工程を包含する血球除去工程により、除去され得る。 [0083] Preferably, the method of the present invention is used to treat blood cells in order to achieve a high degree of stem cell purity. The step of removing is further included. However, such blood cells can be removed by subsequent culturing on a dish and passage of this cell population to generate more than 80% (preferably more than 90%) stem cells. This is also a surprising effect that was not expected from the prior art. Such blood cells can be removed by a blood cell removal step including a step of adding a component that degrades blood cells.
[0084] (幹細胞マーカーの長期維持）  [0084] (Long-term maintenance of stem cell markers)
本発明者らは、将来の臨床試験のために、脂肪吸引物の液体部分力 新たに単離 された細胞および培養された細胞の特徴付けを行レ、、吸引脂肪由来のものと比較し た。さらに、細胞表面マーカーの発現プロフィールにおける長期的な変化を、両方の 細胞集団について決定した。  We have characterized the liquid partial force of lipoaspirates for future clinical trials and characterized newly isolated and cultured cells with those derived from aspirated fat. . In addition, long-term changes in the expression profile of cell surface markers were determined for both cell populations.
[0085] その結果、予想外にも、本発明者らは、本発明者らの使用した培養条件下で、長 期培養（例えば、 20週）後においてもなお、 CD34および CD105の発現能を維持し た ASC (以下、特に断らない限り、 PLA細胞および LAF細胞の両方を含むものとす る）が、脂肪吸引物の脂肪部分および液体部分の両方から得られることを見出した。  [0085] As a result, unexpectedly, the present inventors showed the ability to express CD34 and CD105 even after long-term culture (eg, 20 weeks) under the culture conditions used by the present inventors. It was found that the maintained ASC (hereinafter including both PLA and LAF cells unless otherwise specified) is obtained from both the fat and liquid portions of the lipoaspirate.
CD34は、最も確立されている幹細胞マーカーであり、 CD34+細胞は、臨床的な応 用が期待できるものである。 CD105は、間葉系幹細胞関連マーカーとして知られて おり、 ASCの脂肪、軟骨、および骨の様な間葉系起源の系統への分化に寄与してい ると考えられる。 CD34 is the most established stem cell marker, and CD34 + cells are expected to be clinically applicable. CD105 is known as a mesenchymal stem cell-related marker, and is thought to contribute to the differentiation of ASC into strains of mesenchymal origin such as fat, cartilage, and bone.
(脂肪由来幹細胞 (ASC)の調製方法）  (Preparation method of adipose-derived stem cells (ASC))
したがって、本発明は、臨床的に有用なヒト由来の培養された ASC、およびそのよ うな ASCを調製するための新規な方法を提供する。この方法は、典型的には、以下 の工程を包含する：  Accordingly, the present invention provides a clinically useful human-derived cultured ASC and a novel method for preparing such ASC. This method typically includes the following steps:
ヒトドナー由来の脂肪吸引物を得る工程、および  Obtaining a lipoaspirate from a human donor, and
前記脂肪吸引物から、脂肪由来幹細胞を単離する工程、  Isolating adipose-derived stem cells from the lipoaspirate,
前記単離した脂肪由来幹細胞を培養する工程。  Culturing the isolated adipose-derived stem cells.
[0086] 本明細書中、「ヒトドナー」とは、脂肪を提供するヒトを意味する。典型的には、脂肪 吸引術を受けているヒト（男性および女性を含む）であり、より典型的には、体型改善 などの目的で脂肪吸引術を受けている健常女性である。脂肪吸引術を受けるドナー には、十分なインフォームドコンセントが行われていることが好ましい。 [0086] As used herein, "human donor" means a human who provides fat. Typically, humans (including men and women) undergoing liposuction, and more typically healthy women undergoing liposuction for purposes such as improving body shape. Donor undergoing liposuction It is preferable that sufficient informed consent is provided.
[0087] 本明細書中、「脂肪吸引物」とは、脂肪吸引術により得られた吸引物のことを意味す る。脂肪吸引術は、通常、体型改善等の目的で、力ニューレと呼ばれる吸引管を、身 体の皮下脂肪の厚い部分 (例えば、腹部、腰部、大腿など）に挿入し、脂肪を吸引除 去するものである。脂肪吸引物は、既に記載したように、「脂肪部分」と「液体部分」と 力 構成される。 「脂肪部分」は、典型的には、力ニューレと減圧との相互動作によつ て「寸断されて」いる吸引された脂肪組織を意味する。一方、「液体部分」は、脂肪部 分とともに吸引された液体を意味する。液体は、主に、（1)神経および血管を損傷せ ずに脂肪吸引を促進するために、前もってその部位に注入された生理食塩水、（2) 末梢血、および（3)脂肪組織に由来する細胞または組織断片などから構成されてい る。本発明の ASCの調製方法では、脂肪吸引物のうちの脂肪部分および液体部分 のいずれも使用し得る。  [0087] In this specification, "lipoaspirate" means an aspirant obtained by liposuction. Liposuction usually involves the insertion of a suction tube called force neulet into the thick part of the body's subcutaneous fat (eg, abdomen, waist, thigh, etc.) for the purpose of improving body shape, etc. Is. As already described, the lipoaspirate is composed of a “fat part” and a “liquid part”. “Fat portion” typically refers to aspirated adipose tissue that has been “chopped” by the interaction of force neuules and reduced pressure. On the other hand, the “liquid part” means the liquid sucked together with the fat part. The fluid is primarily derived from (1) saline previously injected into the site to promote liposuction without damaging nerves and blood vessels, (2) peripheral blood, and (3) adipose tissue Cell or tissue fragment. In the method for preparing ASC of the present invention, either the fat portion or the liquid portion of the lipoaspirate can be used.
[0088] 得られた脂肪吸引物からの脂肪由来幹細胞の単離は、典型的には、 Zukら（Zuk et al. Tissu Eng 2001 ; 7 : 211—228)の方法またはその改変方法を用いて、 以下のように行い得る。  [0088] Isolation of adipose-derived stem cells from the resulting lipoaspirate is typically performed using the method of Zuk et al. (Zuk et al. Tissu Eng 2001; 7: 211-228) or a modification thereof. It can be done as follows.
[0089] 脂肪部分を使用する場合、脂肪部分からの材料は、まず、細胞外マトリックスから細 胞を解離するために、コラゲナーゼのような酵素で処理される。次いで、遠心分離に よって成熟脂肪細胞、結合組織などをペレットから分離し、回収したペレットを赤血球 溶解緩衝液で処理した後フィルター（例えば、孔径 100 μ m)を通して赤血球および 非細胞成分を除去する。次いで、フィルターを通過したろ過液から密度勾配遠心分 離法によって目的の ASC (すなわち、 PLA細胞）を濃縮単離する。得られた ASCは さらにフィルターを通して不純物を除いても良い。  [0089] When using the fat portion, the material from the fat portion is first treated with an enzyme such as collagenase to dissociate the cells from the extracellular matrix. Subsequently, mature adipocytes, connective tissue, and the like are separated from the pellet by centrifugation, and the collected pellet is treated with an erythrocyte lysis buffer, and then erythrocytes and non-cellular components are removed through a filter (for example, 100 μm in pore diameter). Next, the target ASC (ie, PLA cells) is concentrated and isolated from the filtrate that has passed through the filter by density gradient centrifugation. The resulting ASC may be further filtered to remove impurities.
[0090] 液体部分を使用する場合、液体部分からの液体は、直接、遠心分離にかけられて ペレットが回収される。そのペレットを赤血球溶解緩衝液で処理した後フィルターに 通して赤血球および非細胞成分を除去する。次いで、フィルターを通過したろ過液か ら密度勾配遠心分離法によって目的の ASC (すなわち、 LAF細胞）を濃縮単離する 。液体部分を使用する場合には、脂肪部分を使用する場合のような酵素処理が要ら ないという利点がある。 [0091] 本発明の ASCの調製方法において、脂肪吸引物から単離された ASCは、さらに 培養され、増殖される。培養のための手順は、常法に従う（例えば、新生化学実験講 座（18)「細胞培養技術」、 日本生化学会編、第 1版、第 1刷、 1990、東京化学同人 等を参照）。細胞は、懸濁液中、または固定培養基上で培養することができる。好まし くは、細胞は、基板（例えば、ガラス製またはプラスチック製のシャーレ、フラスコ、プレ ート等）上に接着されてインビトロで培養される。プラスチック製品は細胞が接着しや すいように表面処理された製品を使用してもよい。また、必要に応じて、接着を十分 なものとし、効率の良い培養を実現するために、任意の細胞外マトリックス (例えば、 コラーゲン、ヒアルロン酸、ゼラチン、ポリ一L—リジン、ポリグリコール酸、ポリ一 ε _ 力プロラタトン等）で代表される細胞接着因子を使用してもよい。その際、一般的には 、培養容器の接着面に細胞外マトリックスをコートする。細胞外マトリックスは、当業者 にとつて公知の方法で使用前に培養容器の接着面にコートされてもよいし、または細 胞外マトリックスが予めコートされている製品を、当業者に周知の業者力も商業的に 入手しても良い。 [0090] When using a liquid portion, the liquid from the liquid portion is directly centrifuged to recover the pellet. The pellet is treated with erythrocyte lysis buffer and then passed through a filter to remove erythrocytes and non-cellular components. Next, the target ASC (ie, LAF cells) is concentrated and isolated from the filtrate that has passed through the filter by density gradient centrifugation. When using a liquid part, there is an advantage that an enzyme treatment is not required as in the case of using a fat part. [0091] In the ASC preparation method of the present invention, ASC isolated from lipoaspirate is further cultured and expanded. The procedure for culturing is in accordance with conventional methods (see, for example, the New Chemistry Laboratory (18) “Cell Culture Technology”, edited by the Japanese Biochemical Society, 1st edition, 1st edition, 1990, Tokyo Kagaku Dojin). Cells can be cultured in suspension or on a fixed culture medium. Preferably, the cells are attached to a substrate (eg, glass or plastic petri dish, flask, plate, etc.) and cultured in vitro. As the plastic product, a product which has been surface-treated so that cells can be easily adhered may be used. If necessary, any extracellular matrix (eg, collagen, hyaluronic acid, gelatin, poly-L-lysine, polyglycolic acid, (1) __force prolatatatone etc.) and the like may be used. At that time, generally, an extracellular matrix is coated on the adhesion surface of the culture vessel. The extracellular matrix may be coated on the adhesive surface of the culture vessel prior to use in a manner known to those skilled in the art, or a product pre-coated with the extracellular matrix can be obtained by a manufacturer known to those skilled in the art. Power may also be obtained commercially.
[0092] 本発明の ASCの調製方法において、細胞培養に使用する培地は、基本培地と添 加物とから構成される。基本培地は、 L—アルギニン、 L—グルタミン、 L—ヒスチジン 、 L—イソロイシン等のようなアミノ酸、 CaCl · 2Η 0、 KC1、 NaCl等のような無機塩、  [0092] In the method for preparing ASC of the present invention, a medium used for cell culture is composed of a basic medium and an additive. The basic medium consists of amino acids such as L-arginine, L-glutamine, L-histidine, L-isoleucine, inorganic salts such as CaCl · 20, KC1, NaCl,
2 2  twenty two
ニコチンアミドのようなビタミン、緩衝液、その他の成分を含む。本発明の ASCの調製 方法において、基本培地として典型的には、 M— 199培地（Morgan et al. Proc • Soc. Exp. Biol. 1950 ; 73 : 1)が使用される。 M— 199培地は、主に哺乳動物の 正常細胞を培養するための基本培地として慣用されており、当業者に周知の供給業 者（例えば、 Invitrogen Corporationなど）力、ら粉末または液体で市販されてレ、る 。他に本発明の ASCの調製方法において使用し得る基本培地としては、イーグルの 最小必須培地（MEM)、イーグルの基本培地（BME)、ダルベッコ変法イーグル培 地（DMEM)、 Ham' s F12培地、 MCDB104培地、 MCDB153倍地などが挙げ られる。これらの培地は、当業者に周知の供給業者（例えば、 Invitrogen Corpora tionなど）から、粉末または液体として商業的に入手可能である。  Contains vitamins such as nicotinamide, buffers and other ingredients. In the ASC preparation method of the present invention, M-199 medium (Morgan et al. Proc • Soc. Exp. Biol. 1950; 73: 1) is typically used as the basic medium. M-199 medium is commonly used as a basic medium mainly for culturing mammalian normal cells, and is commercially available from suppliers (eg, Invitrogen Corporation) force, powder or liquid well known to those skilled in the art. It's terrible. Other basic media that can be used in the ASC preparation method of the present invention include Eagle's minimum essential medium (MEM), Eagle's basic medium (BME), Dulbecco's modified Eagle medium (DMEM), and Ham's F12 medium. MCDB104 medium, MCDB153 medium and the like. These media are commercially available as powders or liquids from suppliers well known to those skilled in the art (eg, Invitrogen Corporation).
[0093] 本発明の ASCの調製方法において、好ましくは、細胞培養に必要とされる栄養素 や因子などの供給源となる添加物が、細胞の増殖能および/または生存性の刺激ま たは維持の目的で基本培地に添加されて使用される。そのような添加物の好適な例 としては、血清、細胞成長因子、抗生物質などが挙げられる。 [0093] In the method for preparing ASC of the present invention, preferably, nutrients required for cell culture Additives that serve as sources such as or are added to the basal medium for the purpose of stimulating or maintaining cell growth and / or viability. Preferable examples of such additives include serum, cell growth factor, antibiotics and the like.
[0094] 本発明において使用され得る「血清」の種類としては、ゥシ胎仔血清、ゥシ新生仔 血清、仔ゥシ血清、ゥマ血清、ヒト血清などが挙げられる力 とりわけ、ゥシ胎仔血清 が好ましい。好ましくは、非動化された血清が使用される。血清は、 2〜25%程度の 濃度で培地に添加されて使用され得、通常 5〜20%程度の濃度で培地に添加され て使用される。より好ましくは、血清濃度は、 7〜： 15%程度であり、最も好ましくは、 10 %程度である。  [0094] The types of "serum" that can be used in the present invention include urine fetal serum, urine neonatal serum, pupal serum, horse serum, human serum and the like. Is preferred. Preferably, non-immobilized serum is used. Serum can be used by being added to the medium at a concentration of about 2 to 25%, and is usually added to the medium at a concentration of about 5 to 20%. More preferably, the serum concentration is about 7 to about 15%, and most preferably about 10%.
[0095] 本発明において使用され得る「細胞成長因子」の例としては、線維芽細胞増殖因子  [0095] Examples of "cell growth factor" that can be used in the present invention include fibroblast growth factor
(FGF、酸性 FGF、塩基性 FGF)、血小板由来増殖因子（PDGF_AB、 BB)、イン スリン様増殖因子一 1 (IGF— 1)、表皮増殖因子 (EGF)、肝細胞増殖因子 (HGF)、 グリア細胞株神経栄養因子（GDNF)、神経栄養因子（NF)、ホルモン、サイト力イン 、骨形成因子 (BMP)、トランスフォーミング増殖因子 (TGF)、上皮細胞増殖因子 (E GF)、血管内皮細胞増殖因子 (VEGF)などが挙げられるが、とりわけ、酸性線維芽 細胞増殖因子（酸性 FGF)が好ましい。さらに、へパリンなどが、酸性 FGFの作用を 増強するために添加され得る。細胞成長因子は、培地中で、 0. 05〜20ng/ml程 度の濃度で使用され得、好ましくは、 0. 1〜： 10ng/ml程度の濃度、より好ましくは、 l〜5ng/ml程度の濃度、最も好ましくは、 2ng/ml程度の濃度で使用され得る。へ パリンが酸性 FGFとともに使用される場合、へパリンは、 0.：!〜 20ng/ml程度の濃 度で培地に添加され得、好ましくは、 1〜： 10ng/m 呈度の濃度、より好ましくは、 2〜 7ngZml程度、最も好ましくは 5ng/ml程度の濃度で使用され得る。  (FGF, acidic FGF, basic FGF), platelet-derived growth factor (PDGF_AB, BB), insulin-like growth factor 1 (IGF-1), epidermal growth factor (EGF), hepatocyte growth factor (HGF), glia Cell line neurotrophic factor (GDNF), neurotrophic factor (NF), hormone, cyto force-in, bone morphogenetic factor (BMP), transforming growth factor (TGF), epidermal growth factor (EGF), vascular endothelial cell proliferation Factor (VEGF) and the like can be mentioned, and acidic fibroblast growth factor (acidic FGF) is particularly preferable. In addition, heparin and the like can be added to enhance the action of acidic FGF. The cell growth factor can be used in the medium at a concentration of about 0.05 to 20 ng / ml, preferably 0.1 to about 10 ng / ml, more preferably about 1 to 5 ng / ml. Can be used at a concentration of about 2 ng / ml. When heparin is used with acidic FGF, heparin can be added to the medium at a concentration of about 0.:! To 20 ng / ml, preferably 1 to: a concentration of 10 ng / m, more preferably Can be used at a concentration of about 2-7 ng Zml, most preferably about 5 ng / ml.
[0096] 「抗生物質」としては、例えば、ペニシリン、ストレプトマイシン、ゲンタマイシンなどが 使用され得るが、本発明において使用する抗生物質としては、とりわけ、ペニシリンお よびストレプトマイシンが好ましレ、。本発明において、抗生物質は、 20〜450IUまた は 20〜450mg/ml程度の濃度で使用され得、好ましくは、 50〜400IUまたは 50 〜400mgZml程度の濃度、より好ましくは、 50〜200IUまたは 50〜200mgZml 程度の濃度、さらにより好ましくは、 70〜： 150IUまたは 70〜150mgZml程度の濃 度、最も好ましくは、 100IUまたは 100mg/ml程度の濃度で、使用され得る。 [0096] As the "antibiotic", for example, penicillin, streptomycin, gentamicin and the like can be used, but as the antibiotic used in the present invention, penicillin and streptomycin are particularly preferable. In the present invention, the antibiotic may be used at a concentration of about 20 to 450 IU or 20 to 450 mg / ml, preferably a concentration of about 50 to 400 IU or 50 to 400 mgZml, more preferably 50 to 200 IU or 50 to Concentration on the order of 200 mgZml, even more preferred 70-: 150 IU or a concentration on the order of 70-150 mgZml Degrees, most preferably at concentrations as high as 100 IU or 100 mg / ml.
[0097] 本発明で使用される培地に添加してもよい他の添加物としては、例えば、インスリン 、トランスフェリン、アルブミン、ペプトン、 EGF (EpithelialGrowthFactor)のような タンパク質、アミノ酸（例えば、アルギニン、システィン、グノレタミン、グリシン、ヒスチジ ン、イソロイシン、ロイシン、リジン、メチォニン、フエニノレアラニン、セリン、スレオニン、 トリプトファン、チロシン、ノ リンなど）、ビタミン (例えば、パントテン酸、コリン、葉酸、ィ ノシトール、ニコチン酸アミド、リボフラビン、チアミン、ピリドキシンなど）などが挙げら れる。 [0097] Other additives that may be added to the medium used in the present invention include, for example, insulin, transferrin, albumin, peptone, proteins such as EGF (EpithelialGrowthFactor), amino acids (for example, arginine, cysteine, Gnoretamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenenolealanine, serine, threonine, tryptophan, tyrosine, noline, etc.), vitamins (eg pantothenic acid, choline, folic acid, inositol, nicotinamide, Riboflavin, thiamine, pyridoxine, etc.).
[0098] したがって、本発明で使用される培養培地の典型的な例は、 5-20% ゥシ胎仔血 清、 50〜200IU ペニシリン、 50〜200mgZml ス卜レプ卜マイシン、：!〜 10 μ gZ ml へパリン、および 0. 5〜： 10ng/ml 酸性線維芽細胞増殖因子を補充された M — 199培地である。  [0098] Thus, a typical example of the culture medium used in the present invention is 5-20% urine fetal serum, 50-200 IU penicillin, 50-200 mg Zml sprepmycin,:!-10 μgZ M-199 medium supplemented with ml heparin, and 0.5 to: 10 ng / ml acidic fibroblast growth factor.
[0099] 本発明の ASCの調製方法において、単離した脂肪由来幹細胞の培養に要する期 間は、少なくとも 1週間であり、好ましくは、少なくとも 2週間、より好ましくは、少なくとも 3週間、より好ましくは、少なくとも 4週間、より好ましくは、少なくとも 5週間、より好まし くは、少なくとも 6週間、より好ましくは、少なくとも 7週間、より好ましくは、少なくとも 8 週間、より好ましくは、少なくとも 9週間、より好ましくは、少なくとも 10週間、最も好まし くは、少なくとも 20週間である。長期間培養することによって、所望の細胞を大量に得 ること力 Sできる。  [0099] In the ASC preparation method of the present invention, the period required for culturing the isolated adipose-derived stem cells is at least 1 week, preferably at least 2 weeks, more preferably at least 3 weeks, more preferably At least 4 weeks, more preferably at least 5 weeks, more preferably at least 6 weeks, more preferably at least 7 weeks, more preferably at least 8 weeks, more preferably at least 9 weeks, more preferably At least 10 weeks, most preferably at least 20 weeks. By culturing for a long time, it is possible to obtain a large amount of desired cells.
[0100] 好ましくは、培地交換を少なくとも週 2回、細胞継代を少なくとも週 1回行う。培地は 、通常同じ組成のもので交換するが、これに限定されず、組成を変更した新たな培地 (例えば、血清濃度、細胞性成長因子の濃度等を変更した新たな培地）を用いて培 地交換を行っても良い。  [0100] Preferably, medium exchange is performed at least twice a week and cell passage is performed at least once a week. The medium is usually replaced with one having the same composition, but the medium is not limited to this, and the medium is cultivated using a new medium having a changed composition (for example, a new medium having a changed serum concentration, cellular growth factor concentration, etc.) You may exchange land.
[0101] 通常、上記培養は、哺乳動物の細胞の培養に適する任意の条件で実施することが できるが、好ましくは、 37°C程度 (例えば、 35〜38°C)の温度で、例えば、湿潤大気 中、 5%CO雰囲気下で行われる。  [0101] Usually, the culture can be performed under any conditions suitable for mammalian cell culture, but preferably at a temperature of about 37 ° C (eg, 35-38 ° C), for example, Performed in a humid atmosphere under a 5% CO atmosphere.
2  2
[0102] なお、本発明で使用する ASCの培養方法は、脂肪吸引術によって得られる脂肪吸 引物から単離された脂肪由来幹細胞に対して使用し得るのみならず、他の方法 (例 えば、一般的な外科的切除術)で得た脂肪から単離された脂肪由来幹細胞に対して も使用し得ることは、当業者に理解され得る。また、ヒト由来の脂肪由来幹細胞に限 定されず、他の哺乳動物（例えば、ブタ、サル、チンパンジー、ィヌ、ゥシ、ゥサギ、ラ ット、マウス等）、鳥類、またはは虫類等に由来する脂肪由来幹細胞に対しても、本発 明の方法が適用され得ることは、当業者に理解され得る。 [0102] The ASC culture method used in the present invention can be used not only for adipose-derived stem cells isolated from a liposuction material obtained by liposuction, but also for other methods (eg, It can be understood by those skilled in the art that it can also be used for adipose-derived stem cells isolated from fat obtained by general surgical excision. Moreover, it is not limited to human-derived adipose-derived stem cells, but derived from other mammals (for example, pigs, monkeys, chimpanzees, inu, ushi, magpies, rats, mice, etc.), birds, reptiles, etc. It can be understood by those skilled in the art that the method of the present invention can be applied to the adipose-derived stem cells.
[0103] したがって、本発明は別の態様において、新たに単離された脂肪由来幹細胞を培 養する工程を包含する、 CD34 +および/または CD105+脂肪由来幹細胞の調製方 法を提供する。 [0103] Therefore, in another embodiment, the present invention provides a method for preparing CD34 + and / or CD105 + adipose-derived stem cells, comprising the step of culturing newly isolated adipose-derived stem cells.
[0104] 本明細書中、細胞について、「新たに単離された（freshly isolated)」とは、当該 細胞が、ドナー（例えば、ヒトドナー）から得られた脂肪または脂肪吸引物力 適切な 方法によって単離されたままの細胞であって、培養増殖されていない細胞のことをい うものとする。なお、新たに単離された脂肪由来幹細胞は、脂肪吸引術によって得ら れた脂肪吸引物から単離されたものに限定されない。また、脂肪由来幹細胞は、ヒト 由来のものに限定されなレ、。  [0104] In the present specification, for a cell, "freshly isolated" means that the cell is simply obtained by a suitable method of fat or lipoaspirate obtained from a donor (eg, a human donor). A cell that has been detached and has not been grown in culture. The newly isolated adipose-derived stem cells are not limited to those isolated from lipoaspirate obtained by liposuction. Adipose-derived stem cells are not limited to those derived from humans.
[0105] (組成物）  [0105] (Composition)
1つの局面において、本発明はまた、多能性幹細胞を含む組成物であって、該組 成物は、 CD34、 CD105、またはこれらの両方の細胞表面マーカーを発現し得る多 能性幹細胞を少なくとも 10%含む、組成物を提供する。ここで、本発明は、このような 組成物において、長期保存可能なものを提供することができることを見出した。好まし く ίま、この糸且成物 ίま、 5〜20ο/ο ゥシ月台仔血?青、 50〜400IUまた ίま 50〜400mg/m 1 抗生物質、 1〜： 10 /i g/ml へパリン、および 0. 5〜： 10ng/ml 酸性線維芽細胞 増殖因子を含有する培地を用いて培養された（たとえば、 2〜20週間）ものであり得 る。 In one aspect, the present invention is also a composition comprising pluripotent stem cells, the composition comprising at least pluripotent stem cells capable of expressing CD34, CD105, or both cell surface markers. A composition comprising 10% is provided. Here, it has been found that the present invention can provide such a composition that can be stored for a long period of time. Preferable ί, this thread and adult product ί, 5-20 ο / ο月 tsukidai pupa blood blue, 50-400 IU or ί 50-400 mg / m 1 Antibiotic, 1-: 10 / ig / ml heparin, and 0.5-: 10 ng / ml acidic fibroblasts can be cultured (eg, 2-20 weeks) using a medium containing growth factors.
[0106] 好ましい実施形態では、本発明の組成物は、 CD34、 CD105、またはこれらの両 方の細胞表面マーカーを発現し得る多能性幹細胞を少なくとも 50%含み得る。この ような細胞を含む組成物は、従来得ることが難しかった。  [0106] In a preferred embodiment, the compositions of the invention may comprise at least 50% pluripotent stem cells capable of expressing CD34, CD105, or both cell surface markers. It has been difficult to obtain a composition containing such cells.
[0107] ある実施形態では、本発明の組成物は、 CD105の細胞表面マーカーを発現し得 る多能性幹細胞を少なくとも 50%含むものであり得る。間葉系幹細胞で、このような 特性を有する幹細胞はこれまで提供されてこなかった。このような細胞は、未分化能 を有する細胞の比率が高いとレ、う点で多大な効果を奏する。 [0107] In an embodiment, the composition of the present invention may comprise at least 50% of pluripotent stem cells capable of expressing a cell surface marker of CD105. Such as mesenchymal stem cells No stem cells with properties have been provided so far. Such cells have a great effect in that they have a high proportion of undifferentiated cells.
[0108] ある実施形態では、本発明の組成物は、 CD34の細胞表面マーカーを発現し得る 多能性幹細胞を少なくとも 10%含むものであり得る。  [0108] In certain embodiments, a composition of the invention may comprise at least 10% pluripotent stem cells capable of expressing a cell surface marker of CD34.
間葉系幹細胞で、このような特性を有する幹細胞はこれまで提供されてこなかった。 このような細胞は、未分化能を有する細胞の比率が高いという点で多大な効果を奏 する。  A mesenchymal stem cell having such characteristics has not been provided so far. Such cells have a great effect in that the ratio of undifferentiated cells is high.
[0109] 別の局面において、本発明はまた、 CD34+および/または CD105+脂肪由来幹 細胞を含む組成物を調製する方法であって、 A)新たに単離された脂肪由来幹細胞 を培養する工程を包含する、方法を提供する。ここで、この方法では、脂肪由来幹細 胞を少なくとも 2週間培養することが好ましいがこれに限定されなレ、。あるいは、この 方法では、脂肪由来幹細胞を 1〜2週間培養してもよい。：!〜 2週間の培養により、 C D34陽性細胞を多量に（たとえば、 20%、あるいは 50%)含有する組成物が提供さ れた。 [0109] In another aspect, the present invention also provides a method for preparing a composition comprising CD34 + and / or CD105 + adipose-derived stem cells, comprising A) culturing newly isolated adipose-derived stem cells A method comprising the steps is provided. In this method, the fat-derived stem cells are preferably cultured for at least 2 weeks, but the method is not limited thereto. Alternatively, in this method, adipose-derived stem cells may be cultured for 1 to 2 weeks. :! ~ 2 weeks of culturing provided a composition containing large amounts of CD34 positive cells (eg, 20% or 50%).
[0110] 別の実施形態では、本発明の方法では、脂肪由来幹細胞を少なくとも 10週間、あ るいは少なくとも 20週間培養する。長期培養でも CD34+および/または CD105+ 脂肪由来幹細胞を含む組成物が提供されたことは予想外の顕著な効果といえる。 [0110] In another embodiment, in the method of the present invention, the adipose-derived stem cells are cultured for at least 10 weeks or at least 20 weeks. The provision of a composition containing CD34 + and / or CD105 + adipose-derived stem cells even in long-term culture is an unexpected and remarkable effect.
[0111] 好ましい実施形態では、本発明の組成物生産方法における培養は 5〜20% ゥシ 月台仔血？青、 50〜400IUまた ίま 50〜400mg/ml 抗生物質、：!〜 10 μ g/ml へノヽ° リン、および 0. 5〜： 10ng/ml 酸性線維芽細胞増殖因子を含有する培地を用いて 行われ得る。このような培養環境を用いることによって、長期培養可能という効果を与 えることが予測されることから、これと等価な条件であれば、どのような環境を用いても よい。  [0111] In a preferred embodiment, culturing in the method for producing a composition of the present invention is 5 to 20% Ushiki pupa blood. Blue, 50-400 IU or ί or 50-400 mg / ml antibiotics:! ~ 10 μg / ml hexane phosphorous, and 0.5 ~: 10 ng / ml medium containing acidic fibroblast growth factor Can be done. The use of such a culture environment is expected to give the effect of long-term culture, so any environment may be used as long as the conditions are equivalent to this.
[0112] 1つの実施形態では、脂肪由来幹細胞は、ヒトドナー由来の脂肪吸引物を得る工程 、および脂肪吸引物から、脂肪由来幹細胞を単離する工程  [0112] In one embodiment, the fat-derived stem cells obtain a lipoaspirate from a human donor, and the step of isolating the fat-derived stem cells from the lipoaspirate
によって、単離される。より好ましい実施形態では、この方法で用いられる脂肪吸引 物は、液体部分を含む。液体部分から、 CD34+および/または CD105+脂肪由来 幹細胞を含む組成物を調製することができたのは、驚くべきことである。 [0113] したがって、本発明では、脂肪吸引物を得る工程は、液体部分を分離する工程をさ らに包含する。この液体部分を分離する工程は、本明細書において他の場所におい て詳述されている任意の方法を用いることができる。 Isolated by In a more preferred embodiment, the lipoaspirate used in this method includes a liquid portion. It is surprising that a composition comprising CD34 + and / or CD105 + adipose derived stem cells could be prepared from the liquid part. [0113] Therefore, in the present invention, the step of obtaining the lipoaspirate further includes a step of separating the liquid portion. The step of separating the liquid portion can employ any of the methods detailed elsewhere herein.
(培養増殖された脂肪由来幹細胞）  (Adipose-derived stem cells grown in culture)
本発明の ASCの調製方法により調製された PLA細胞および LAF細胞はいずれも 、長期間培養した後でも CD34や CD105を発現し得る。このような性質は、臨床的な 適用におレ、て非常に有用である。  Both PLA cells and LAF cells prepared by the ASC preparation method of the present invention can express CD34 and CD105 even after long-term culture. This property is very useful for clinical applications.
[0114] 多くの研究によって、ヒト ASCの特徴付けが行われており、新たに単離された ASC 、または 2週間未満の期間培養された ASCが CD34+であるが（例えば、 Planat-B enard et al. Circulation 2004 ; 109 : 656— 663)、従来の培養方法によって 2週間を超えて培養したヒト ASCにおいて、 CD34発現は見出されな力、つたことが報 告されている（例えば、 Zuk et al. Mol Biol Cell 2002 ; 13 : 4279— 4295)。 これに対して、本発明は、 CD34+の ASC力 10〜20週間の培養後においてもなお 10〜20%で存在することを示した（図 5)。 [0114] Numerous studies have characterized human ASCs, although freshly isolated ASCs or ASCs cultured for less than 2 weeks are CD34 + (eg, Planat-B enard et al. Circulation 2004; 109: 656-663), CD34 expression has been reported to be an undiscovered force in human ASCs cultured for more than 2 weeks by conventional culture methods (eg, Zuk). et al. Mol Biol Cell 2002; 13: 4279-4295). In contrast, the present invention showed that CD34 + ASC strength was still present at 10-20% after 10-20 weeks of culture (FIG. 5).
[0115] 本発明は、 CD34+の ASCが長期培養期間にわたって拡大され得ることを、初めて 示すものである。本発明の培養プロトコルを使用して、例えば、脂肪吸引物から得ら れた CD34+の ASCを、 4週間培養することによって、 104〜： 107倍まで拡大すること が可能である。この事実は、細胞ベースの治療への臨床的適用に対するヒト ASCの 価値をさらに高いものとする。 [0115] The present invention is the first to show that CD34 + ASC can be expanded over a long culture period. Using the culture protocol of the present invention, for example, CD34 + ASC obtained from lipoaspirate can be expanded from 10 4 to 10 7 times by culturing for 4 weeks. This fact further enhances the value of human ASC for clinical application in cell-based therapy.
[0116] したがって、本発明はまた、単離された多能性幹細胞または培養された多能性幹 細胞であって、 CD34、 CD105、またはこれらの両方を細胞表面マーカーとして発 現し得る細胞を提供する。より特定的には、本発明は、培養された脂肪由来幹細胞 であって、 CD34、 CD105、またはこれらの両方を細胞表面マーカーとして発現し得 る細胞を提供する。特に、本発明は、長期間（例えば、少なくとも 2週間、より好ましく は少なくとも 3週間、さらに好ましくは少なくとも 4週間、さらに好ましくは少なくとも 5週 間、さらに好ましくは少なくとも 6週間、さらに好ましくは少なくとも 7週間、さらに好まし くは少なくとも 8週間、さらに好ましくは少なくとも 9週間、さらに好ましくは少なくとも 10 週間、さらにより好ましくは少なくとも 20週間）培養増殖されたヒト由来の脂肪由来幹 細胞であって、 CD34、 CD105、またはこれらの両方を細胞表面マーカーとして発 現し得る細胞を提供する。 [0116] Therefore, the present invention also provides isolated pluripotent stem cells or cultured pluripotent stem cells, which can express CD34, CD105, or both as cell surface markers. To do. More specifically, the present invention provides cultured adipose-derived stem cells that can express CD34, CD105, or both as cell surface markers. In particular, the present invention provides a long term (eg, at least 2 weeks, more preferably at least 3 weeks, more preferably at least 4 weeks, more preferably at least 5 weeks, more preferably at least 6 weeks, more preferably at least 7 weeks). More preferably at least 8 weeks, more preferably at least 9 weeks, more preferably at least 10 weeks, even more preferably at least 20 weeks) human-derived fat-derived stems grown in culture. Provided are cells that can express CD34, CD105, or both as cell surface markers.
[0117] (疾患の治療または再生医療における本発明の細胞の使用）  [0117] (Use of cells of the present invention in disease treatment or regenerative medicine)
本発明はまた、別の態様において、多能性幹細胞を被験体に投与する工程を包含 する、ヒトもしくは動物の疾患を治療するため、またはヒトもしくは動物の細胞、組織、 もしくは臓器を再生するための方法を提供する。そのような疾患としては、難治性潰 瘍'褥瘡'熱傷をはじめとする皮膚疾患、骨欠損'骨変性を伴う骨疾患、軟骨変形'欠 損 ·変性を伴う軟骨疾患、脂肪萎縮 ·変性を伴う脂肪疾患、心筋梗塞 ·拡張型心筋症 をはじめとする心臓疾患、デュシェンヌ型筋ジストロフィーをはじめとする筋疾患、脊 髄損傷 *脳梗塞 ·パーキンソン病をはじめとする中枢神経系疾患、美容を目的とする 組織増大'アンチエイジング治療などなどが挙げられる。また、本発明は、脂肪再生、 骨再生、心筋再生、血管新生、軟骨再生、骨髄再生、神経再生、骨格筋再生などの 再生医療に使用され得る。  The present invention, in another aspect, also comprises the step of administering pluripotent stem cells to a subject, to treat a human or animal disease, or to regenerate a human, animal cell, tissue, or organ. Provide a way. Such diseases include skin diseases such as refractory ulcer 'decubitus' burns, bone defects 'bone diseases with bone degeneration, cartilage deformation' defects · cartilage diseases with degeneration, fat atrophy · degeneration Fatty diseases, myocardial infarction · Cardiovascular diseases including dilated cardiomyopathy, muscle diseases including Duchenne muscular dystrophy, spinal cord injury * Cerebral infarction · Central nervous system diseases including Parkinson's disease, beauty Examples include tissue augmentation and anti-aging treatment. In addition, the present invention can be used for regenerative medicine such as fat regeneration, bone regeneration, myocardial regeneration, angiogenesis, cartilage regeneration, bone marrow regeneration, nerve regeneration, and skeletal muscle regeneration.
[0118] 本発明の多能性幹細胞は、様々な方法で被験体に投与することができる。例えば 、胃腸管の壁への局所注入を経て、全身または門脈循環系へ投与することにより、あ るいは任意の他の実践的手段により、移植することができる。ヒトの胃腸障害のような 障害の治療における多能性幹細胞の使用は、動物モデルを使用することにより実証 すること力 Sできる。  [0118] The pluripotent stem cells of the present invention can be administered to a subject by various methods. For example, it can be transplanted by local injection into the wall of the gastrointestinal tract, administration systemically or to the portal circulatory system, or by any other practical means. The use of pluripotent stem cells in the treatment of disorders such as human gastrointestinal disorders can be demonstrated by using animal models.
[0119] 本発明は、宿主に対して異種のドナー組織力 調製された多能性幹細胞の使用を も包含する。し力 ながら一般に、異種移植がうまくいくためには、移植される組織に 対する免疫応答を低減するか、または排除する幾つかの方法を用いることが好まし レ、。したがって、被移植者は、通常、例えば、シクロスポリンのような免疫抑制薬の使 用により、あるいは局所塗布される免疫抑制薬を用いた局所免疫抑制ストラテジーに より、免疫抑制され得る。免疫抑制の方法はこれらに限定されず、他の方法 (例えば 、幹細胞における相同組換えを用いた遺伝子置換またはノックアウト法)も使用され 得る。なお、長期に培養された本発明の ASCにおいてはクラス 2HLA (ヒト白血球抗 原）の発現が低下しており（参考文献： Rodriguez et al, J. Exp. Med. , 20 05)、免疫抑制剤の使用がなくても、 自己以外の ASCの移植拒絶が起こらないことも 予想される。 [0119] The present invention also includes the use of pluripotent stem cells prepared with different donor tissue powers for the host. However, in general, in order for xenotransplantation to be successful, it is preferable to use several methods that reduce or eliminate the immune response to the transplanted tissue. Thus, recipients can usually be immunosuppressed, for example, by the use of immunosuppressive drugs such as cyclosporine, or by local immunosuppressive strategies using locally applied immunosuppressive drugs. The method of immunosuppression is not limited to these, and other methods (for example, gene replacement or knockout method using homologous recombination in stem cells) can also be used. In the ASC of the present invention cultured for a long time, the expression of class 2HLA (human leukocyte antigen) is decreased (reference: Rodriguez et al, J. Exp. Med., 20 05) and immunosuppressant Even if there is no use of ASC, the rejection of transplantation of non-self ASCs may not occur is expected.
[0120] 本発明の多能性幹細胞は、部位特異的な様式 (すなわち、局所的に)あるいは全 身的に、送達され得る。細胞は、周囲領域の完全性を維持する任意の方法を用いて 、好ましくは局所注入により、特定領域に投与され得る。細胞はまた、全身または門 脈循環系に投与され得る。  [0120] The pluripotent stem cells of the invention can be delivered in a site-specific manner (ie, locally) or globally. Cells can be administered to a particular area using any method that maintains the integrity of the surrounding area, preferably by local injection. The cells can also be administered systemically or in the portal circulatory system.
[0121] 本発明はまた、本発明の多能性幹細胞を含有する、ヒトもしくは動物の疾患の治療 、またはヒトもしくは動物の細胞、組織、もしくは臓器の再生に使用するための薬剤を 提供する。この薬剤は、多能性幹細胞に加えて、多能性幹細胞の成長および分化を 促進する成長因子（例えば、塩基性もしくは酸性線維芽細胞増殖因子 (bFGF； aFG F)、血小板由来増殖因子（PDGF_AB、 BB)、インスリン、インスリン様増殖因子— 1 (IGF_ 1)、肝細胞増殖因子 (HGF)、グリア細胞株由来神経栄養因子 (GDNF)、 神経栄養因子（NF)、ホルモン、サイト力イン、骨形成因子（BMP)、トランスフォーミ ング増殖因子 (TGF)、上皮細胞増殖因子 (EGF)、血管内皮細胞増殖因子 (VEGF )など）、免疫抑制薬 (例えば、シクロスポリン)、抗炎症剤等を含むことができる。さら に、所望の場合には、多能性幹細胞は、当業者に周知の技術により、カプセル化す ること力 Sできる。  [0121] The present invention also provides a drug containing the pluripotent stem cells of the present invention for use in the treatment of human or animal diseases or the regeneration of human or animal cells, tissues, or organs. In addition to pluripotent stem cells, this drug is a growth factor that promotes the growth and differentiation of pluripotent stem cells (eg, basic or acidic fibroblast growth factor (bFGF; aFG F), platelet-derived growth factor (PDGF_AB , BB), insulin, insulin-like growth factor—1 (IGF_ 1), hepatocyte growth factor (HGF), glial cell line-derived neurotrophic factor (GDNF), neurotrophic factor (NF), hormone, cytosite in, bone Including formation factor (BMP), transforming growth factor (TGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), immunosuppressive drugs (eg cyclosporine), anti-inflammatory agents, etc. be able to. Further, if desired, pluripotent stem cells can be encapsulated by techniques well known to those skilled in the art.
[0122] 幹細胞はそのままで移植治療に用いることもできるが、所望の細胞へ誘導し、一定 の臓器の形へとデザインしてレ、くために、種々の組織工学的な技術を使用し得る。例 えば、本発明の多能性幹細胞は、足場材料 (細胞外マトリックスの役割を果たす）とと もに被験体に投与され得る。したがって、本発明の再生医療用材料または薬剤は、 多能性幹細胞とともに足場材料をさらに含み得る。足場材料を使用することによって 、インビトロまたはインビボで、幹細胞を一定の形態へと増殖'分化させることを容易 にすることができる。  [0122] Stem cells can be used as they are for transplantation treatment, but various tissue engineering techniques can be used to guide them to the desired cells and design them into a certain organ shape. . For example, the pluripotent stem cells of the present invention can be administered to a subject together with a scaffold material (acting as an extracellular matrix). Therefore, the regenerative medical material or drug of the present invention may further contain a scaffold material together with pluripotent stem cells. The use of scaffold materials can facilitate the growth and differentiation of stem cells into certain forms in vitro or in vivo.
[0123] 本発明において使用し得る足場材料としては、例えば、コラーゲン、アルブミン、フ イブリンなどのタンパク質、ポリ乳酸、ポリダリコール酸、乳酸とグリコール酸との共重 合体、ポリ一 ε—力プロラタトン、 ε—力プロラタトンと乳酸又はダリコール酸との共重 合体、ポリクェン酸、ポリリンゴ酸、ポリ一ひ—シァノアクリレート、ポリ— is—ヒドロキシ 酪酸、ポリトリメチレンォキサレート、ポリテトラメチレンォキサレート、ポリオルソエステ ノレ、ポリオノレソカーボネート、ポリエチレンカーボネート、ポリプロピレンカーボネート、 ポリ γ —べンジルー L グルタメート、ポリ γ—メチルー L グルタメート、ポリ Lーァラニンなどの合成項分子、デンプン、アルギン酸、ヒアノレロン酸、キチン、ぺク チン酸及びそれらの誘導体などの多糖が挙げられる。また、これらの材料の混合物 及び共重合体なども同様に本発明において使用され得る。足場材料の形態は、特 に限定されず、例えば、スポンジ状、メッシュ状、不織布状、ディスク状、フィルム状、 棒状、粒子状、ペースト状などの形態をとることができる。足場材料は、生分解性の材 料でできていてもよぐまた、幹細胞、細胞成長因子等を一定期間（例えば、：!〜 3週 間）にわたつて生体内で徐放可能な材料であってもよレ、。 [0123] Scaffold materials that can be used in the present invention include, for example, proteins such as collagen, albumin, and fibrin, polylactic acid, polydaricholic acid, a copolymer of lactic acid and glycolic acid, poly-one ε -force prolatathon, ε —Copolymers of prolataton and lactic acid or darlicolic acid, polycenoic acid, polymalic acid, polymono-cyanate, poly-is-hydroxybutyric acid, polytrimethylene oxalate, polytetramethylene oxalate, poly Ortho Este Synthetic molecules such as Nore, Polyonolecarbonate, Polyethylene carbonate, Polypropylene carbonate, Poly γ-Benzylu L glutamate, Poly γ-Methyl-L glutamate, Poly L-alanine, Starch, Alginic acid, Hyanorenoic acid, Chitin, Pectinic acid And polysaccharides such as their derivatives. Also, mixtures and copolymers of these materials can be used in the present invention as well. The form of the scaffold material is not particularly limited, and can be, for example, a sponge form, a mesh form, a non-woven form, a disk form, a film form, a rod form, a particle form, a paste form, or the like. The scaffold material may be made of a biodegradable material, and it is a material that can release stem cells, cell growth factors, etc. for a certain period of time (eg:! ~ 3 weeks) in vivo. It ’s okay.
[0124] (分化細胞を調製するための方法）  [0124] (Method for preparing differentiated cells)
本発明の幹細胞を用いて、種々の分化細胞および/または前駆細胞を得ることが できる。具体的な手順は、以下に説明される。  Using the stem cells of the present invention, various differentiated cells and / or progenitor cells can be obtained. A specific procedure is described below.
[0125] 1つの局面において、本発明は、幹細胞から分化細胞を調製するための方法を提 供する。幹細胞から得られる分化細胞としては、最終的に分化した細胞のみならず、 前駆細胞も含まれる。  [0125] In one aspect, the present invention provides a method for preparing differentiated cells from stem cells. Differentiated cells obtained from stem cells include not only finally differentiated cells but also progenitor cells.
[0126] 脂肪吸引物由来の幹細胞から分化細胞を調製する方法としては、（1)培養培地中 に分化誘導剤（例えば、デキサメサゾン)を添加する方法、および（2)所望の部位に 対応する分化細胞とともに培養する方法が挙げられる。  [0126] Methods for preparing differentiated cells from lipoaspirate-derived stem cells include (1) a method of adding a differentiation inducer (eg, dexamethasone) to the culture medium, and (2) differentiation corresponding to a desired site. A method of culturing with cells is mentioned.
[0127] (培養培地中に分化誘導剤を添加することにより、幹細胞の分化を誘導する方法） 本発明の細胞混合物は、所望の部位に対応する分化細胞への分化を促進する因 子をさらに含み得る。この因子は、所望の部位に対応する分化細胞への分化を促進 することが知られているかまたは確認されている任意の因子であり得る。好ましい分 化誘導剤の例としては、デキサメタゾンなどの副腎皮質ステロイド、インスリン、グノレコ ース、インドメタシン、イソブチル一メチルキサンチン（ΙΒΜΧ)、ァスコルべート一 2_ ホスフェート（ascorbate_ 2_phosphate)、ァスコルビン酸およびその誘導体、グリ セロホスフェート、エストロゲンおよびその誘導体、プロゲステロンおよびその誘導体、 アンドロゲンおよびその誘導体、 ひ FGF、 β FGF、 EGF、 IGF、 TGF_ β 、 ECGF、 BMP、 PDGFをはじめとする増殖因子、下垂体エキス、松果体エキス、レチノイン酸 、ビタミン D、甲状腺ホルモン、仔ゥシ血清、ゥマ血清、ヒト血清、へパリン、炭酸水素 ナトリウム、 HEPES、アルブミン、トランスフェリン、セレン酸（例えば、亜セレン酸ナト リウムなど）、リノレン酸、 3—イソブチルー 1ーメチルキサンチン、 5—ァザンシチジン などの脱メチル化斉 1J、トリコスタチンなどのヒストン脱ァセチル化剤、ァクチビン、 LIF、 IL_ 2、 IL_ 6などのサイト力イン、へキサメチレンビスァセトアミド（HMBA)、ジメチ ノレァセトアミド（DMA)、ジブチル cAMP (dbcAMP)、ジメチルスルホキシド（DMS O)、ョードデォキシゥリジン（IdU)、ヒドロキシゥレア（HU)、シトシンァラビノシド（Ara C)、マイトマイシン C (MMC)、酪酸ナトリウム（NaBu)、ポリプレン、セレニウムなどが 挙げられる力 これらに限定されない。 (Method of Inducing Stem Cell Differentiation by Adding Differentiation Inducing Agent in Culture Medium) The cell mixture of the present invention further comprises a factor that promotes differentiation into a differentiated cell corresponding to a desired site. May be included. This factor can be any factor known or confirmed to promote differentiation into differentiated cells corresponding to the desired site. Examples of preferred differentiation-inducing agents include corticosteroids such as dexamethasone, insulin, gnolecose, indomethacin, isobutyl monomethylxanthine (ΙΒΜΧ), ascorbate 2_phosphate, ascorbic acid and its derivatives. Glycerophosphate, estrogen and its derivatives, progesterone and its derivatives, androgen and its derivatives, FGF, β FGF, EGF, IGF, TGF_β, ECGF, BMP, PDGF and other growth factors, pituitary extract, pine Fruit extract, retinoic acid , Vitamin D, thyroid hormone, baby serum, horse serum, human serum, heparin, sodium bicarbonate, HEPES, albumin, transferrin, selenate (eg, sodium selenite), linolenic acid, 3- Demethylation chirality 1J such as isobutyl-1-methylxanthine, 5-azazancitidine, histone deacetylating agents such as trichostatin, cytosine inactivins such as activin, LIF, IL_2, IL_6, hexamethylenebisacetamide ( HMBA), dimethylolacetamide (DMA), dibutyl cAMP (dbcAMP), dimethyl sulfoxide (DMS O), ododeoxyuridine (IdU), hydroxyurea (HU), cytosine arabinoside (Ara C) , Mitomycin C (MMC), sodium butyrate (NaBu), polyprene, selenium and the like.
本発明の細胞混合物については、混合される細胞の維持および所望の部位に対 応する分化細胞への分化を維持する限り、任意の培養液を用いることができる。その ような培養液の例としては、 DMEM、 Ml 99、 MEM、 HBSS (Hanks' Balanced Salt Solution) , Ham' s F12、 BME、 RPMI1640、 MCDB104、 MCDB153 ( KGM)などが挙げられる力 これらに限定されなレ、。このような培養液には、デキサメ タゾン（dexamethasone)などの副腎皮質ステロイド、インスリン、グルコース、インド メタシン、イソブチルーメチルキサンチン（IBMX)、ァスコルべ一トー 2—ホスフェート (ascorbate - 2 - phosphate) ,ァスコルビン酸およびその誘導体、グリセ口ホスフエ ート、エストロゲンおよびその誘導体、プロゲステロンおよびその誘導体、アンドロゲン およびその誘導体、 a FGF、 β FGF、 EGF、 IGF、 TGF β、 ECGF、 BMP, PDGF をはじめとする増殖因子、下垂体エキス、松果体エキス、レチノイン酸、ビタミン D、甲 状腺ホルモン、仔ゥシ血清、ゥマ血清、ヒト血清、へパリン、炭酸水素ナトリウム、 HEP ES、ァノレブミン、トランスフェリン、セレン酸（例えば、亜セレン酸ナトリウムなど）、リノ レン酸、 3 _イソブチル _ 1—メチルキサンチン、 5—ァザンシチジンなどの脱メチル 化剤、トリコスタチンなどのヒストン脱ァセチル化斉 'j、ァクチビン、 LIF、 IL_ 2、 IL-6 などのサイト力イン、へキサメチレンビスァセトアミド（HMBA)、ジメチルァセトアミド（ DMA)、ジブチル cAMP (dbcAMP)、ジメチルスルホキシド（DMSO)、ョードデォ キシゥリジン（IdU)、ヒドロキシゥレア（HU)、シトシンァラビノシド（AraC)、マイトマイ シン C (MMC)、酪酸ナトリウム（NaBu)、ポリブレン、セレニウムなどの 1つまたはそ の組み合わせを含ませてぉレ、てもよレ、。 For the cell mixture of the present invention, any culture solution can be used as long as the cells to be mixed and the differentiation into differentiated cells corresponding to the desired site are maintained. Examples of such cultures include DMEM, Ml 99, MEM, HBSS (Hanks' Balanced Salt Solution), Ham's F12, BME, RPMI1640, MCDB104, MCDB153 (KGM), etc. Nare ,. Such cultures include corticosteroids such as dexamethasone, insulin, glucose, indomethacin, isobutyl-methylxanthine (IBMX), ascorbate 2-phosphate, ascorbine Growth factors including acids and their derivatives, glyceguchi phosphate, estrogens and their derivatives, progesterone and its derivatives, androgens and their derivatives, a FGF, β FGF, EGF, IGF, TGF β, ECGF, BMP, PDGF Pituitary extract, pineal gland extract, retinoic acid, vitamin D, thyroid hormone, feline serum, horse serum, human serum, heparin, sodium bicarbonate, HEP ES, anolebumin, transferrin, selenate ( For example, sodium selenite), linolenic acid, 3_isobutyl_1-methyl Demethylating agents such as xanthine and 5-hazancytidine, histone deacetylating agents such as trichostatin, site forces such as activin, LIF, IL_2 and IL-6, hexamethylenebisacetamide (HMBA) , Dimethylacetamide (DMA), dibutyl cAMP (dbcAMP), dimethylsulfoxide (DMSO), pseudodeoxyuridine (IdU), hydroxyurea (HU), cytosinearabinoside (AraC), mitomycin C (MMC), One or more of sodium butyrate (NaBu), polybrene, selenium, etc. Including the combination of ぉ les, even ok.
[0129] 脂肪吸引物由来の幹細胞の脂肪細胞への分化誘導の条件としては、例えば、イソ プチルーメチルキサンチン、デキサメタゾン、インスリン、およびインドメタシンを添カロ した DMEM培地中でその幹細胞を培養することが挙げられる力 S、これに限定されな レ、。  [0129] As a condition for inducing differentiation of a lipoaspirate-derived stem cell into an adipocyte, for example, culturing the stem cell in a DMEM medium supplemented with isoptirumethylxanthine, dexamethasone, insulin, and indomethacin. The power S that can be mentioned, and not limited to this.
[0130] 脂肪吸引物由来の幹細胞の軟骨細胞への分化誘導の条件としては、例えば、イン スリン、ァスコルべート一 2 _ホスフェート、および TGF— β 1を添加した DMEM培地 中でその幹細胞を培養することが挙げられるが、これに限定されない。  [0130] The conditions for induction of differentiation of lipoaspirate-derived stem cells into chondrocytes include, for example, the stem cells in DMEM medium supplemented with insulin, ascorbate 2-phosphate, and TGF-β1. Although culture | cultivation is mentioned, it is not limited to this.
[0131] 脂肪吸引物由来の幹細胞の骨細胞への分化誘導の条件としては、例えば、デキサ メタゾン、ァスコルべート一 2_ホスフェート、 β—グリセ口ホスフェートを添加した DM EM培地中でその幹細胞を培養することが挙げられるが、これに限定されない。  [0131] As a condition for inducing differentiation of a lipoaspirate-derived stem cell into a bone cell, for example, the stem cell in a DM EM medium supplemented with dexamethasone, ascorbate 1_2 phosphate, β-glyce mouth phosphate However, it is not limited to this.
[0132] 脂肪吸引物由来の幹細胞の筋細胞への分化誘導の条件としては、例えば、 10% の仔ゥシ血清（FBS)と 5%のゥマ血清（HS)とを含む DMEM培地に、デキサメタゾ ン、ハイド口コルチゾンを添加した培地中でその幹細胞を培養することが挙げられる 1S これに限定されない。  [0132] The conditions for inducing differentiation of lipoaspirate-derived stem cells into myocytes include, for example, a DMEM medium containing 10% feline serum (FBS) and 5% horse serum (HS). Examples include culturing the stem cells in a medium supplemented with dexamethasone and hyodecortisone 1S, but is not limited thereto.
[0133] (幹細胞と、所望の部位に対応する分化細胞とを、同時に培養することによる分化 誘導）  [0133] (Induction of differentiation by simultaneously culturing stem cells and differentiated cells corresponding to the desired site)
この幹細胞と所望の部位に対応する分化細胞との同時培養により、所望の性質を、 好ましくは均質に含有する分化細胞を一定量以上で提供することができる。この方法 は、 A) a)脂肪由来前駆細胞と、 b)所望の部位に対応する分化細胞とを混合して混 合物を得る工程;および B)この脂肪由来前駆細胞の分化が生じるに十分な条件で、 この混合物を培養する工程、を包含する。  By co-culturing the stem cells and differentiated cells corresponding to the desired site, differentiated cells containing the desired properties, preferably homogeneously, can be provided in a certain amount or more. This method comprises: A) a) mixing adipose-derived progenitor cells with b) differentiated cells corresponding to the desired site to obtain a mixture; and B) sufficient to cause differentiation of the adipose-derived progenitor cells And culturing the mixture under various conditions.
[0134] 所望の部位に対応する分化細胞もまた、当該分野において周知の技法を用いて 調製すること力 Sできる。あるいは、そのような分化細胞は、市販の細胞株 (例えば、 A TCCなどから分譲される細胞株など）を用いることができる。分化細胞は、移植を目 的とする被検体から初代培養された細胞 (例えば、肝細胞、腎細胞、脂肪細胞、骨細 胞、軟骨細胞など）を用いてもよい。そのような初代培養および細胞株の培養方法は 、当該分野において周知であり、例えば、 AMBOマニュアル 細胞研究法 [AMBO Manual of Cell Study Method]、畠中 寛'淺野 朗 共編、 TaKaRa ;バイ ォ実験イラストレイテッド（6)すくすく育て細胞培養 [Illustrated Culture Experi ments - Cells grow quickly]、渡邊利雄編、秀潤社（1996)などに記載されて おり、本明細書においてその内容が援用される。 [0134] Differentiated cells corresponding to the desired site can also be prepared using techniques well known in the art. Alternatively, as such differentiated cells, commercially available cell lines (for example, cell lines distributed from ATCC and the like) can be used. Differentiated cells may be cells cultured primarily from a subject intended for transplantation (eg, hepatocytes, kidney cells, adipocytes, bone cells, chondrocytes, etc.). Such primary cultures and cell line culture methods are well known in the art, for example, the AMBO Manual Cell Research Method [AMBO Manual of Cell Study Method], Hiroshi Hatanaka, Akira Anno, TaKaRa; Bio-Experimental Illustrated (6) Illustrated Culture Experiments-Cells grow quickly, Toshio Watanabe, Shujunsha (1996) The contents of which are incorporated herein by reference.
[0135] 本発明において、分化細胞としては、所望の部位に対応するものであれば、どのよ うな細胞を用いてもよいが、好ましくは、間葉系細胞が用いられ、そのような間葉系細 胞としては、例えば脂肪細胞、骨髄細胞、骨芽細胞、軟骨細胞、線維芽細胞、筋線 維芽細胞、神経細胞、骨格筋細胞、心筋細胞、血管内皮細胞、血管平滑筋細胞、 肝細胞、膝細胞などが挙げられるが、これらに限定されない。そのような分化細胞は 、同定された細胞であってもよレ、が、その細胞の性質が未知であったとしても、例え ば FACSのような分別技術を用いて所望の部位に対応する細胞を調製するために 使用され得る。 [0135] In the present invention, any cell may be used as a differentiated cell as long as it corresponds to a desired site. Preferably, a mesenchymal cell is used, and such a mesenchymal cell is used. Examples of the systemic cells include adipocytes, bone marrow cells, osteoblasts, chondrocytes, fibroblasts, myofibroblasts, neurons, skeletal muscle cells, cardiomyocytes, vascular endothelial cells, vascular smooth muscle cells, liver Examples include, but are not limited to, cells and knee cells. Such a differentiated cell may be an identified cell, but even if the nature of the cell is unknown, for example, a cell corresponding to a desired site using a sorting technique such as FACS. Can be used to prepare
[0136] 所望の部位に対応する細胞の決定に有用なマーカーの例としては、（1)脂肪：細 胞質内におけるトリグリセリドの存在、 OilRed—O染色、グリセ口ホスフェートデヒドロ ゲナーゼ（Glycerophosphate dehydrogenase = GPDH)活性、細胞質内の GL UT4 、 Ap2 (脂肪酸結合タンパク質）、 LPL (リポタンパク質リパーゼ）、 PPAR y 1 , 2 (ペルォキシソーム増殖活性化レセプター γ 1 , 2)、およびレプチン（Leptin)の発 現；（2)骨細胞、骨組織:アルカリホスファターゼの存在、骨石灰化 (カルシウムの沈 着）の程度を確認する；ォステオカルシン（Osteocalcin)、ォステオポンチン（Osteo pontin)、ォステオネクチン（Osteonectin)の発現；（3)軟骨細胞、軟骨組織：ムコ 多糖の存在、 II型コラーゲン、コンドロイチン一 4—サルフェート（chondroitin— 4— sulfate)の発現/存在；（4)骨格筋細胞：細胞質内のミオシンの豊富な存在などが 挙げられる力 これらに限定されなレ、。 FACSの使用方法は、フローサイトメトリー自 由自在 [Master of Flow cytometry] 細胞工学別冊（秀潤社）、中内編、 199 9などに記載されており、本明細書においてその内容を参考として援用する。  [0136] Examples of useful markers for determining the cell corresponding to the desired site include: (1) Fat: presence of triglycerides in the cytoplasm, OilRed-O staining, glycephosphate phosphate dehydrogenase (Glycerophosphate dehydrogenase = GPDH ) Activity, cytoplasmic expression of GLUT4, Ap2 (fatty acid binding protein), LPL (lipoprotein lipase), PPAR y 1, 2 (peroxisome proliferator activated receptor γ 1, 2), and leptin (Leptin); 2) Bone cells, bone tissue: Check for the presence of alkaline phosphatase and the degree of bone mineralization (calcium deposition); Osteocalcin, Osteopontin, Osteonectin expression; (3) Cartilage Cell, cartilage tissue: presence of mucopolysaccharide, type II collagen, chondroitin 4-sulfate expression / presence; (4) bone Muscle Cells: Power like presence of abundant myosin within the cytoplasm and the like, such limitation les. The use of FACS is described in the flow of flow cytometry [Master of Flow cytometry] Cell Engineering separate volume (Shujunsha), Nakauchi edition, 1999, etc., which is incorporated herein by reference. To do.
[0137] 本発明の細胞混合物には、所望の部位に対応する分化細胞への分化を促進する 因子がさらに含有され得る。そのような因子は、所望の分化細胞への分化を促進する ことが知られている力、または確認された任意の因子であり得る。好ましい分化促進因 子としては、例えば、デキサメタゾンなどの副腎皮質ステロイド、インスリン、ダルコ一 ス、インドメタシン、イソブチルーメチルキサンチン（IBMX)、ァスコルべ一トー 2—ホ スフェート（ascorbate— 2— phosphate)、ァスコルビン酸およびその誘導体、グリセ 口ホスフェート、エストロゲンおよびその誘導体、プロゲステロンおよびその誘導体、ァ ンドロゲンおよびその誘導体、 ひ FGF. β FGF - EGF - IGF - TGF - β -ECGF- BM P' PDGFをはじめとする増殖因子、下垂体エキス、松果体エキス、レチノイン酸、ビタ ミン D、甲状腺ホルモン、仔ゥシ血清、ゥマ血清、ヒト血清、へパリン、炭酸水素ナトリ ゥム、 HEPES、ァノレブミン、トランスフェリン、セレン酸（亜セレン酸ナトリウムなど）、リ ノレン酸、 3 _イソブチル _ 1—メチルキサンチン、 5—ァザンシチジンなどの脱メチル 化剤、トリコスタチンなどのヒストン脱ァセチル化斉 'j、ァクチビン、 LIF、 IL_ 2、 IL-6 などのサイト力イン、へキサメチレンビスァセトアミド（HMBA)、ジメチルァセトアミド（ DMA)、ジブチル cAMP (dbcAMP)、ジメチルスルホキシド（DMSO)、ョードデォ キシゥリジン（IdU)、ヒドロキシゥレア（HU)、シトシンァラビノシド（AraC)、マイトマイ シン C (MMC)、酪酸ナトリウム（NaBu)、ポリブレン、セレニウムが挙げられるがそれ らに限定されない。 [0137] The cell mixture of the present invention may further contain a factor that promotes differentiation into differentiated cells corresponding to a desired site. Such factors can be forces known to promote differentiation into the desired differentiated cells, or any factor identified. Preferred differentiation promoting factor Examples of such children include corticosteroids such as dexamethasone, insulin, dalcos, indomethacin, isobutyl-methylxanthine (IBMX), ascorbate 2-phosphate, ascorbic acid and its derivatives. Glycate oral phosphate, estrogen and its derivatives, progesterone and its derivatives, androgen and its derivatives, FGF. Β FGF-EGF-IGF-TGF-β-ECGF-BM P 'PDGF and other growth factors, pituitary gland Extract, pineal extract, retinoic acid, vitamin D, thyroid hormone, baby serum, horse serum, human serum, heparin, sodium bicarbonate, HEPES, anolebumin, transferrin, selenate (selenite) Sodium), linolenic acid, 3_isobutyl_1-methylxanthine, 5-azane Demethylating agents such as thymidine, histone deacetylating compounds such as trichostatin, cytosines such as activin, LIF, IL_2, and IL-6, hexamethylenebisacetamide (HMBA), dimethylacetate Amide (DMA), Dibutyl cAMP (dbcAMP), Dimethyl sulfoxide (DMSO), Rhododeoxyuridine (IdU), Hydroxyurea (HU), Cytosine arabinoside (AraC), Mitomycin C (MMC), Sodium butyrate (NaBu ), Polybrene, and selenium, but are not limited thereto.
本発明の細胞混合物については、混合される細胞の維持および所望の部位に対 応する分化細胞への分化を維持する限り、任意の培養液を用いることができる。その ような培養液の例としては、 DMEM、 Ml 99、 MEM、 HBSS (Hanks' Balanced Salt Solution) , Ham' s F12、 BME、 RPMI1640、 MCDB104、 MCDB153 ( KGM)などが挙げられる力 これらに限定されなレ、。このような培養液には、デキサメ タゾン（dexamethasone)などの副腎皮質ステロイド、インスリン、グルコース、インド メタシン、イソブチル一メチルキサンチン（IBMX)、ァスコルべート一 2_ホスフェート (ascorbate - 2 - phosphate) ,ァスコルビン酸およびその誘導体、グリセ口ホスフエ ート、エストロゲンおよびその誘導体、プロゲステロンおよびその誘導体、アンドロゲン およびその誘導体、 ひ FGF、 β FGF、 EGF、 IGF、 TGF— β 、 ECGF、 BMP, PD GFをはじめとする増殖因子、下垂体エキス、松果体エキス、レチノイン酸、ビタミン D 、甲状腺ホルモン、仔ゥシ血清、ゥマ血清、ヒト血清、へパリン、炭酸水素ナトリウム、 HEPES、アルブミン、トランスフェリン、セレン酸（亜セレン酸ナトリウムなど）、リノレン 酸、 3—イソブチルー 1ーメチルキサンチン、 5—ァザンシチジンなどの脱メチル化剤 、トリコスタチンなどのヒストン脱ァセチル化斉 IJ、ァクチビン、 LIF、 IL— 2、 IL— 6など のサイト力イン、へキサメチレンビスァセトアミド（HMBA)、ジメチルァセトアミド（DM A)、ジブチル cAMP (dbcAMP)、ジメチルスルホキシド（DMS〇）、ョードデォキシ ゥリジン（IdU)、ヒドロキシゥレア（HU)、シトシンァラビノシド（AraC)、マイトマイシン C (MMC)、酪酸ナトリウム（NaBu)、ポリブレン、セレニウムなどを 1つまたはその組 み合わせを含ませてぉレ、てもよレ、。 As for the cell mixture of the present invention, any culture solution can be used as long as the cells to be mixed and the differentiation into differentiated cells corresponding to the desired site are maintained. Examples of such cultures include DMEM, Ml 99, MEM, HBSS (Hanks' Balanced Salt Solution), Ham's F12, BME, RPMI1640, MCDB104, MCDB153 (KGM), etc. Nare ,. Such cultures include corticosteroids such as dexamethasone, insulin, glucose, indomethacin, isobutyl monomethylxanthine (IBMX), ascorbate 2_phosphate, Ascorbic acid and its derivatives, glyceguchi phosphate, estrogen and its derivatives, progesterone and its derivatives, androgen and its derivatives, FGF, β FGF, EGF, IGF, TGF—β, ECGF, BMP, PD GF and others Growth factor, pituitary extract, pineal extract, retinoic acid, vitamin D, thyroid hormone, feline serum, horse serum, human serum, heparin, sodium bicarbonate, HEPES, albumin, transferrin, selenate ( Sodium selenite), linolene Demethylating agents such as acid, 3-isobutyl-1-methylxanthine and 5-azazancydine, histone deacetylated chiral compounds such as trichostatin IJ, activin, LIF, IL-2, IL-6 Methylenebisacetamide (HMBA), dimethylacetamide (DM A), dibutyl cAMP (dbcAMP), dimethyl sulfoxide (DMS0), ododeoxyuridine (IdU), hydroxyurea (HU), cytosine arabinoside ( AraC), mitomycin C (MMC), sodium butyrate (NaBu), polybrene, selenium, etc., or a combination thereof.
[0139] 従って、別の局面において、本発明は、脂肪吸引物由来の幹細胞と、所望の部位 に対応する分化細胞とを含む、細胞混合物を提供する。このような混合物は、細胞移 植に有用であり、従来の細胞単独を用いた移植に比べて、各々の成分が少なくてす むという利点もある。さらに、従来技術と比べて有利な点としては、例えば、 （1)組織 外で再生組織を作る必要がない；（2)より確実に、より大きな組織の再生が可能であ る；（3)簡便かつ短時間の処理により実現が可能である；（4)皮膚など臓器を切開す る手術を必要とせず、針を用いるだけで細胞および/または組織を投与 (移植)する ことが可能であることが挙げられる力 これらに限定されない。  [0139] Therefore, in another aspect, the present invention provides a cell mixture comprising stem cells derived from lipoaspirate and differentiated cells corresponding to a desired site. Such a mixture is useful for cell transplantation, and has an advantage that each component is less than the conventional transplantation using cells alone. In addition, for example, (1) there is no need to create a regenerative tissue outside the organization; (2) a larger tissue can be regenerated more reliably; (3) Can be realized by simple and short-time processing; (4) Cells and / or tissues can be administered (transplanted) with the use of a needle without the need to perform an operation to open an organ such as the skin. It is not limited to these.
[0140] 本発明の細胞混合物は、好ましくは脂肪の幹細胞の分化が生じるに十分な条件に 供された後のものであるが、本発明はそれに限定されない。分化条件に供された後 の細胞混合物は、そのまま移植に使用されても、組織または器官に分化させてもよい  [0140] The cell mixture of the present invention is preferably after being subjected to conditions sufficient for the differentiation of adipose stem cells, but the present invention is not limited thereto. The cell mixture after being subjected to differentiation conditions may be used for transplantation as it is, or may be differentiated into a tissue or organ.
[0141] (細胞移植組成物） [0141] (Cell transplant composition)
別の局面において、本発明は、細胞移植のための組成物（例えば、組織片、およ び移植片、ならびにこれらを含む組成物）を提供する。この移植は、所望の部位に対 応する分化細胞の欠損または劣化などに伴う疾患、障害または異常状態を処置また は予防するため、あるいは美容状態を処置または改善するためであれば、任意の目 的で使用することができる。このような移植の場合、好ましくは、移植は、所望の部位 に移植されるがそれに限定されず、最終的に所望の部位の処置または予防が可能 なのであれば、本発明の細胞含有組成物は、任意の部位に投与または移植すること ができる。 [0142] 脂肪吸引物由来の幹細胞と、分化細胞との混合比率は、所望の分化が生じる限り 、どのような比率でもよいが、通常、約 1 : 10〜約 10 : 1であり、好ましくは、約 1 : 5〜約 5 : 1であり、より好ましくは、約 1 : 2〜約 2 : 1であり、最も好ましくはほぼ等量で含有さ れる。この細胞組成物において、移植のために使用される分化細胞および脂肪吸引 物由来の幹細胞は、本明細書において、「分化細胞を調製するための方法」におい て説明したような任意の形態または態様をとり得る。 In another aspect, the present invention provides compositions for cell transplantation (eg, tissue pieces and grafts, and compositions comprising them). This transplantation may be performed by any eye as long as it treats or prevents a disease, disorder or abnormal condition associated with the loss or deterioration of differentiated cells corresponding to a desired site, or treats or improves a cosmetic condition. Can be used. In the case of such transplantation, preferably, the transplantation is transplanted to a desired site, but is not limited thereto. If the desired site can be finally treated or prevented, the cell-containing composition of the present invention can be used. It can be administered or transplanted at any site. [0142] The mixing ratio of the lipoaspirate-derived stem cells and differentiated cells may be any ratio as long as desired differentiation occurs, but is usually about 1:10 to about 10: 1, preferably About 1: 5 to about 5: 1, more preferably about 1: 2 to about 2: 1 and most preferably about equal amounts. In this cell composition, the differentiated cells used for transplantation and the stem cells derived from lipoaspirates are in any form or embodiment as described in “Method for preparing differentiated cells” in the present specification. Can take.
[0143] この分化細胞および脂肪由来前駆細胞は、移植されるべき宿主に対して、それぞ れ異種、同種異系または同系である。好ましくは、同種異系または同系であり、より好 ましくは同系であるが、本発明はそれに限定されない。理論に束縛されることは望ま ないが、同系であれば、免疫拒絶反応が抑制できるからである。しかし、拒絶反応が 予測される場合は、本発明は、拒絶反応を回避する工程をさらに包含し得る。拒絶 反応を回避する手順は、当該分野において公知である（例えば、新外科学体系、第 12卷、臓器移植 (心臓移植 ·肺移植、技術的 ·倫理的整備から実施に向けて [New Whole Surgery, Vol. 12, Organ Transplantation (Heart Transplantatio n · Lung Transplantation From Technical ana Ethical Improvements to Oractice) ] (改訂第 3版）、中山書店などを参照のこと）。そのような方法の例とし ては、免疫抑制剤、ステロイド剤の使用などの方法が挙げられる力 これらに限定さ れない。拒絶反応を予防する免疫抑制剤は、現在、「シクロスポリン」（サンデイミユン /ネオ一ラル）；「タクロリムス」（プログラフ）；「ァザチォプリン」（イムラン）；「ステロイド ホルモン」（プレドニン、メチルプレドニン）；「T細胞抗体」（OKT3、 ATGなど）がある 。予防的免疫抑制療法として世界中で行われている方法は、シクロスポリン、ァザチ ォプリン、ステロイドホルモンの 3剤併用である。免疫抑制剤は、本発明の薬剤と同時 期に投与されることが望ましいが、本発明はこれに限定されない。免疫抑制効果が達 成される限り、免疫抑制剤は本発明の再生法 Ζ治療法の前または後にも投与され得 る。  [0143] These differentiated cells and adipose-derived progenitor cells are different from each other, allogeneic or syngeneic with respect to the host to be transplanted. Preferably, they are allogeneic or syngeneic, more preferably syngeneic, but the present invention is not limited thereto. Although not wishing to be bound by theory, it is because immune rejection can be suppressed if the same strain is used. However, if rejection is expected, the present invention may further include a step of avoiding rejection. Procedures to avoid rejection are well known in the art (eg, New Surgery System, Section 12, Organ Transplantation (from Heart Transplantation / Lung Transplantation, Technical / Ethical Development to Implementation [New Whole Surgery , Vol. 12, Organ Transplantation (Heart Transplantation · Lung Transplantation From Technical ana Ethical Improvements to Oractice)] (Ref. 3rd edition, Nakayama Shoten, etc.). Powers including, but not limited to, methods such as the use of inhibitors, steroids, etc. Immunosuppressants to prevent rejection are currently “cyclosporine” (Sandy Miyun / Neo-Iral); “Tacrolimus” (Prograf) "Azathioprine" (Imran); "steroid hormones" (predonin, methylpredonin); "T cell antibodies" (OKT3, ATG, etc.) Prophylactic immunosuppressive therapy The method used in the world is a combination of cyclosporine, azathioprine, and steroid hormones, and it is desirable that the immunosuppressant be administered at the same time as the drug of the present invention. The immunosuppressive agent can be administered before or after the regenerative method / therapeutic method of the present invention as long as an immunosuppressive effect is achieved.
[0144] 上記分化細胞および脂肪由来前駆細胞は、それぞれ異種、同種異系または同系 であり、好ましくは、同種異系または同系であり、より好ましくは同系である。理論に束 縛されることは望まないが、同種異系または同系（好ましくは同系）の方が、分化細胞 と脂肪吸引物由来の幹細胞とが均一の細胞集団と形成しやすいからである。 [0144] The differentiated cells and adipose-derived progenitor cells are each different, allogeneic or syngeneic, preferably allogeneic or syngeneic, and more preferably syngeneic. Although not wishing to be bound by theory, allogeneic or syngeneic (preferably syngeneic) are more differentiated cells. This is because stem cells derived from lipoaspirates tend to form a uniform cell population.
[0145] 上記の細胞混合物または組成物は、医薬として提供され得る。このような医薬は、 所望の部位に対応する分化細胞の欠損または劣化に関連する疾患、障害または異 常状態を処置または予防するため、あるいは美容状態を処置または改善するために 使用され得る。本発明の医薬には、細胞混合物またはそれを含む組成物のほか、薬 学的に受容可能なキャリアが含まれていてもよい。そのようなキャリアとしては、本明 細書において記載される任意のキャリアが選択され得、その用途に応じて当業者に より使用され得る。当業者は、本発明に含ませるべき成分を改変することができる。  [0145] The cell mixture or composition described above may be provided as a medicament. Such medicaments can be used to treat or prevent a disease, disorder or abnormal condition associated with the loss or deterioration of differentiated cells corresponding to the desired site, or to treat or ameliorate a cosmetic condition. The medicament of the present invention may contain a pharmaceutically acceptable carrier in addition to the cell mixture or the composition containing it. As such a carrier, any carrier described in the present specification can be selected, and can be used by those skilled in the art depending on its use. One skilled in the art can modify the ingredients to be included in the present invention.
[0146] (細胞混合を用いた治療および予防法）  [0146] (Treatment and prevention using mixed cells)
別の局面において、本発明は、分化細胞の欠損に起因する疾患、障害または異常 状態を処置または予防するための方法、および美容状態を処置または改善するため の方法を提供する。これらの方法は、 A) a)脂肪由来前駆細胞；および b)所望の部 位に対応する分化細胞、を含む組成物を提供する工程、ならびに B)被検体にその 組成物を投与する工程、を包含する。この方法において、移植に使用される分化細 胞および脂肪吸引物由来の幹細胞は、本明細書において、「分化細胞を調製するた めの方法」において説明したような任意の形態および態様をとり得る。  In another aspect, the present invention provides a method for treating or preventing a disease, disorder or abnormal condition resulting from a defect in differentiated cells, and a method for treating or ameliorating a cosmetic condition. These methods comprise the steps of: A) providing a composition comprising a) adipose-derived progenitor cells; and b) differentiated cells corresponding to a desired site, and B) administering the composition to a subject, Is included. In this method, the stem cells derived from differentiated cells and lipoaspirates used for transplantation can take any form and embodiment as described in “Methods for preparing differentiated cells” herein. .
[0147] 本発明の技術分野において公知な投与方法を含む任意の投与方法が、使用され 得る。例えば、上記組成物は、シリンジ、カテーテル、チューブなどを用いて注入され る力 これらに限定されない。好ましくは、投与経路の例としては、局所注入 (皮下注 入、筋肉や脂肪など臓器内注入)、静脈内注入、動脈内注入、組織上投与などが挙 げられる力 S、これらに限定されない。本発明のこの移植による処置方法または予防方 法は、例えば、以下の従来技術と比べて有利な点を有するが、これらに限定されない ： (1)組織外で再生組織を作る（ェキソビボ産生)必要がなレ、；（2)より確実に、より大 きな組織の再生が可能である；（3)簡便かつ短時間の処理により実現が可能である； (4)皮膚など臓器を切開する手術を必要とせず、針を刺すことによって細胞および組 織を投与 (移植)することが可能である、など。  [0147] Any method of administration can be used, including those known in the art of the invention. For example, the above composition is not limited to the force injected using a syringe, catheter, tube, or the like. Preferably, examples of administration routes include, but are not limited to, local injection (subcutaneous injection, intramuscular or intramuscular injection), intravenous injection, intraarterial injection, tissue administration, and the like. This method of treatment or prevention by transplantation of the present invention has advantages over, for example, the following conventional techniques, but is not limited to these: (1) Necessary to create regenerative tissue outside the tissue (ex vivo production) (2) Larger tissue regeneration is possible more reliably; (3) Can be achieved by simple and short processing; (4) Surgery for incising an organ such as the skin It is possible to administer (transplant) cells and tissues by puncturing a needle without the need for a device.
[0148] (使用）  [0148] (Use)
別の局面において、本発明は、 a)脂肪由来前駆細胞と、 b)所望の部位に対応する 分化細胞との混合物の、分化細胞の欠損に起因する疾患、障害または異常状態を 処置または予防するため、あるいは美容状態を処置または改善するための医薬の調 製のための使用を提供する。移植のための細胞混合物において用いられる分化細 胞および脂肪由来前駆細胞は、本明細書において、「分化細胞を調製するための方 法」において説明したような任意の形態をとり得る。 In another aspect, the present invention corresponds to a) adipose-derived progenitor cells, b) desired sites Provided is the use of a mixture with differentiated cells for the preparation of a medicament for treating or preventing a disease, disorder or abnormal condition resulting from a defect in differentiated cells, or for treating or ameliorating a cosmetic condition. Differentiated cells and adipose-derived progenitor cells used in the cell mixture for transplantation can take any form as described herein in “Methods for Preparing Differentiated Cells”.
(一般技術）  (General technology)
本明細書において用いられる分子生物学的手法、生化学的手法、微生物学的手 法は、当該分野において周知であり慣用されるものであり、例えば、 Maniatis, T. e t al. (1989) . Molecular Cloning： A Laboratory Manual, Cold Spring Molecular biological techniques, biochemical techniques, and microbiological techniques used herein are well known and commonly used in the art, for example, Maniatis, T. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring
Harborおよびその 3rd Ed. (2001)； Ausubel, F. M. (1987) . Current Pro tocols in Molecular Biology, Greene Pub. AssociatESand Wiley— Inte rscience ; Ausubel, F. M. (1989) . Short Protocols in Molecular Biolog y : A Compendium of Methods from Current Protocols in Molecul ar Biology, Greene Pub. Associates and Wiley— Interscience； Sambroo k, J. et al. (1989) . Molecular Cloning： A Laboratory Manual, Cold Spring Harbor ; Innis, M. A. (1990) . PCR Protocols： A Guide to Me thods and Applications, Academic Press ; Ausubel, F. M. " 992) . Shor t Protocols in Molecular Biology : A Compendium of Methods fro m Current Protocols in Molecular Biology, Greene Pub. Associates； Ausubel, F. M. (1995) . Short Protocols in Molecular Biology : A Co mpendium of Methods from Current Protocols in Molecular Biolog y, Greene Pub. Associates； Innis, M. A. et al. (1995) . PCR Strategies , Academic Press ; Ausubel, F. M. (1999) . Short Protocols in Molecul ar Biology : A Compendium of Methods from Current Protocols inHarbor and its 3rd Ed. (2001); Ausubel, FM (1987). Current Pro tocols in Molecular Biology, Greene Pub. Associat ES and Wiley— Internet science; Ausubel, FM (1989). Short Protocols in Molecular Biolog y: A Compendium of Methods from Current Protocols in Molecul ar Biology, Greene Pub. Associates and Wiley— Interscience; Sambrook, J. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor; Innis, MA (1990). PCR Protocols A Guide to Methods and Applications, Academic Press; Ausubel, FM "992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Biology, Greene Pub. Associates; Ausubel, FM (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biolog y, Greene Pub. Associates; Innis, MA et al. (1995). PCR Strategies, Academic Press; Ausubel, FM (1999). Short Protocols in Molecul ar Biology: A Compendium of Methods from Current Protocols in
Molecular Biology, Wiley, and annual updates ; Sninsky, J. J. et al. (1 999) . PCR Applications： Protocols for Functional Genomics, Acade mic Press、別冊実験医学「遺伝子導入 &発現解析実験法」羊土社、 1997などに 記載されており、これらは本明細書において関連する部分 (全部であり得る）が参考と して援用される。 Molecular Biology, Wiley, and annual updates; Sninsky, JJ et al. (1 999). PCR Applications: Protocols for Functional Genomics, Academic Press, Separate Experimental Medicine “Gene Transfer & Expression Analysis Experiments” Yodosha, 1997, etc. These are described in the relevant part of this specification (which may be all). Incorporated.
[0150] 本明細書において引用された、科学文献、特許、特許出願などの参考文献は、そ の全体力 各々具体的に記載されたのと同じ程度に本明細書において参考として援 用される。  [0150] References such as scientific literature, patents, and patent applications cited herein are incorporated herein by reference to the same extent as if they were specifically described. .
[0151] 以下に、実施例に基づいてより具体的に本発明を説明する。以下に説明される実 施例は、例示目的のみのために提供される。従って、本発明の範囲は、上記発明の 詳細な説明によっても下記実施例によっても限定されるものではなぐ特許請求の範 囲によってのみ限定される。  [0151] Hereinafter, the present invention will be described more specifically based on examples. The examples described below are provided for illustrative purposes only. Accordingly, the scope of the present invention is limited only by the scope of the claims which are not limited by the above detailed description of the invention nor by the following examples.
実施例  Example
[0152] 以下の実施例において使用した試薬は、特に言及しない限り和光純薬または Sig maから得た。動物の飼育は、 National Society for Medical Researchにより 作成 れた「Principles of Laboratory Animal Care」およひ 'Institute of L aboratory Animal Resource力 M乍成、 National Institute of Health力 s公表 した「Guide for the Care and Use of Laboratory AnimalsJ (NIH Pu blication, No. 86— 23, 1985,改訂）に遵つて、動物愛護精神に則って行つ た。ヒトを対象とする場合は、事前に同意を得た上で実験を行った。 [0152] The reagents used in the following examples were obtained from Wako Pure Chemicals or Sigma unless otherwise stated. Animal breeding, National Society for Medical was created by Research "Principles of Laboratory Animal Care" Oyohi 'Institute of L aboratory Animal Resource force M乍成, announced National Institute of Health force s "Guide for the Care and In accordance with the Use of Laboratory AnimalsJ (NIH Publication, No. 86—23, 1985, revised), we performed in accordance with the spirit of animal welfare. Went.
[0153] (実施例 1：幹細胞の調製および同定）  [Example 1: Preparation and identification of stem cells]
1.材料および方法  1. Materials and methods
(ヒト組織のサンプリング）  (Sampling of human tissue)
本発明者らは、 IRB推奨のプロトコルを用いたインフォームドコンセントの後に、腹 部および大腿部の脂肪吸引術を受けている 21歳から 59歳までの健常女性のドナー 力 の脂肪吸引物（liposuction aspirates)を得た。脂肪吸引物は 2つの部分、す なわち、浮遊脂肪部分（吸引脂肪 (lipoaspirates)とも呼ばれる）および残りの液体 部分に分けられた（図 laを参照）。いずれの部分も、それぞれ、 PLA細胞および LA F細胞のための細胞供給源として使用した。  We have informed consent using the IRB recommended protocol followed by liposuction of donor strength in healthy women aged 21-59 undergoing abdominal and thigh liposuction ( liposuction aspirates). The lipoaspirate was divided into two parts, a floating fat part (also called lipoaspirates) and the remaining liquid part (see figure la). Both parts were used as cell sources for PLA cells and LA F cells, respectively.
[0154] (細胞の単離） [0154] (Isolation of cells)
全ての化学薬品を、特に断らない限り、 Wako Pure Chemicals, Osaka, Japan 力 購入した。 [0155] PLA細胞を、脂肪吸引物の脂肪部分から、 Zukおよびその共同研究者 (Zuk et al. Tissu Eng 7 : 211—28 ; 2001)の方法を改変した手順を使用して分離した 。簡単に説明すると、吸引した脂肪を、シェーカー上で 0. 075%コラゲナーゼを含有 する PBS中、 37°Cで 30分間消化した。成熟脂肪細胞および結合組織を遠心分離（ 800g、 10分間）によってペレットから分離した。ペレットを、赤血球溶解緩衝液（155 mM NH Cl、 10mM KHCO、 0. ImM EDTA)中にて、 5分間室温で再懸濁 All chemicals were purchased from Wako Pure Chemicals, Osaka, Japan unless otherwise noted. [0155] PLA cells were isolated from the fat portion of the lipoaspirate using a modified procedure of the method of Zuk and coworkers (Zuk et al. Tissu Eng 7: 211-28; 2001). Briefly, aspirated fat was digested for 30 minutes at 37 ° C in PBS containing 0.075% collagenase on a shaker. Mature adipocytes and connective tissue were separated from the pellets by centrifugation (800 g, 10 minutes). Resuspend pellet in erythrocyte lysis buffer (155 mM NH 4 Cl, 10 mM KHCO, 0. ImM EDTA) for 5 minutes at room temperature
4 3  4 3
した。そのペレットを再懸濁し、 lOO x mのメッシュ（Millipore, MA, USA)で濾過し た。  did. The pellet was resuspended and filtered through a lOO x m mesh (Millipore, MA, USA).
[0156] LAF細胞を、脂肪吸引物の液体部分から取った。その吸引液を、遠心分離し (40 0g、 10分間）、そして得られたペレットを、赤血球溶解緩衝液中で、 5分間室温で再 懸濁した後、 100 x mのメッシュで濾過した。  [0156] LAF cells were taken from the liquid portion of the lipoaspirate. The aspirate was centrifuged (400 g, 10 minutes) and the resulting pellet was resuspended in erythrocyte lysis buffer for 5 minutes at room temperature and then filtered through a 100 × m mesh.
[0157] 次いで、それらのペレットを密度勾配遠心分離のために、 Ficoll (Amersham Bio sciences, NJ, USA)で処理した。遠心分離（800g、 20分間）の後、勾配界面にあ る細胞を回収し、 PBS中で洗浄し、そして 100 μ ΐηメッシュで濾過した。新たに単離し た LAF細胞のフローサイトメトリー用には、密度勾配遠心分離を行わなかった。有核 糸田胞数を、 NucleoCounter (Chemometec, Denmark) 用いて g十数した。  [0157] The pellets were then treated with Ficoll (Amersham Biosciences, NJ, USA) for density gradient centrifugation. After centrifugation (800 g, 20 minutes), cells at the gradient interface were collected, washed in PBS, and filtered through 100 μΐη mesh. Density gradient centrifugation was not performed for flow cytometry of freshly isolated LAF cells. Nucleated cysts were counted using the NucleoCounter (Chemometec, Denmark).
[0158] (接着細胞の細胞培養）  [0158] (Cell culture of adherent cells)
新たに単離した PLA細胞または LAF細胞を、 5 X 106有核細胞/ 100mmゼラチ ン被覆ディッシュの密度で培地にプレートし、 37°Cで湿潤大気中 5%CO雰囲気下 Freshly isolated PLA or LAF cells are plated on medium at a density of 5 x 10 6 nucleated cells / 100 mm gelatin-coated dish and are at 37 ° C in a humidified atmosphere with 5% CO
2 で培養した。その培養培地は、 10% ゥシ胎仔血清（FBS)、 100IU ペニシリン、 1 OOmg/ml ストレプトマイシン、 5 μ g/ml へパリン、 2ng/ml 酸性 FGFを含有 する M— 199であった。倍加時間の測定のためだけに、 15%FBSを含有する異なる 血清濃度を有する同じ培地もまた使用した。初代細胞を、それらがコンフルエンスに 達するまで 7日間培養し、そして「継代 0」と規定した。培地を 2日おきに取り替え、細 胞を毎週継代した。 7日間の初代培養の後、付着した細胞をトリプシン処理によって 継代し、同じ培地中に 2， 000細胞 Zcm2の密度でプレートした。倍加時間を、播種 の 3日後および 5日後の接着細胞数を用いて計算した。 Incubated in 2. The culture medium was M-199 containing 10% ushi fetal serum (FBS), 100 IU penicillin, 1 OO mg / ml streptomycin, 5 μg / ml heparin, and 2 ng / ml acidic FGF. The same medium with different serum concentrations containing 15% FBS was also used only for doubling time measurements. Primary cells were cultured for 7 days until they reached confluence and defined as “passage 0”. The medium was changed every 2 days and the cells were passaged weekly. After 7 days of primary culture, the attached cells were passaged by trypsinization and plated at a density of 2,000 cells Zcm 2 in the same medium. The doubling time was calculated using the number of adherent cells 3 and 5 days after seeding.
[0159] (培養した PLA細胞または LAF細胞の分化の誘導） 脂肪生成、軟骨形成、および骨形成の系統に沿って分化をする能力を、試験した。 PLA細胞または LAF細胞を継代 3〜5で播種してから 7日後、細胞分化を培養培地 を交換することによって開始した。 10%FBSを含有するコントロール培地（100IUの ペニシリンおよび 100mg/ml ストレプトマイシンを含有する DMEM)に維持した細 胞を、ネガティブコントロールとして使用した。脂肪生成の分化のために、コンフルェ ントな培養物を、 0. 5mM イソブチノレメチノレキサンチン（isobutyl— methylxanthi ne) (Sigma, MO)、 1 μ M デキサメタゾン、 10 μ M インスリン（Sigma)、および 2 00 μ Μ インドメタシンを補充した 10%FBSを含有するコントロール培地とともに、 4 週間インキュベートした。固定した細胞（4% パラフオルムアルデヒドで 10分間）を、 60% イソプロパノール中で洗浄し、引き続いて、 15分間〇il _Red 〇とともにイン キュペートし、脂質の液滴を可視化した。次いで、細胞をイソプロパノールで洗浄し、 へマトキシリンで対比染色した。軟骨形成の分化のために、コンフルェントな培養物 を、 6. 25 /i g/ml インシュリン、 10ng/ml TGF j3 1、および 50nM ァスコノレビ ン酸ー2—ホスフヱート（&3(：01¾& 6 2— 1103 11& 6)を補充した1 %?83を含有す るコントロール培地とともに、 4週間インキュベートした。 固定した細胞 (4%パラフオル ムアルデヒドで 10分間）を、 3%酢酸で洗浄し、引き続いて、 1 % Alcian Blue 8 GX (Sigma)、 3 %酢酸とともに 30分間インキュベートし、細胞外マトリックスを可視化 した。次いで、細胞を 3%酢酸で洗浄し、そして 0. 1 % Nuclear Fast Red, 5% Al (SO ) 溶液で対比染色した。骨形成の分化のために、細胞を、 0. Ι / M デキ[0159] (Induction of differentiation of cultured PLA cells or LAF cells) The ability to differentiate along adipogenic, chondrogenic, and osteogenic lines was tested. Seven days after seeding PLA or LAF cells at passage 3-5, cell differentiation was initiated by changing the culture medium. Cells maintained in control medium containing 10% FBS (DMEM containing 100 IU penicillin and 100 mg / ml streptomycin) were used as negative controls. For adipogenic differentiation, confluent cultures were treated with 0.5 mM isobutyl-methylxantine (Sigma, MO), 1 μM dexamethasone, 10 μM insulin (Sigma), and 200 μΜ incubated with control medium containing 10% FBS supplemented with indomethacin for 4 weeks. Fixed cells (10% with 4% paraformaldehyde) were washed in 60% isopropanol and subsequently incubated with 0il_Red for 15 minutes to visualize lipid droplets. Cells were then washed with isopropanol and counterstained with hematoxylin. For differentiation of chondrogenesis, confluent cultures were treated with 6.25 / ig / ml insulin, 10ng / ml TGF j31, and 50nM wasconolevic acid-2-phosphate (& 3 (： 01¾ & 6 2—1103 11 & 6 Incubate for 4 weeks with control medium containing 1%? 83 supplemented with 3% fixed cells (10% with 4% paraformaldehyde), followed by washing with 3% acetic acid followed by 1% Alcian Blue 8 Incubate with GX (Sigma), 3% acetic acid for 30 min to visualize extracellular matrix, then wash cells with 3% acetic acid and contrast with 0.1% Nuclear Fast Red, 5% Al (SO) solution For the differentiation of bone formation, the cells were stained with 0.
2 4 3 2 4 3
サメタゾン、 50 μ Μ ァスコルビン酸一 2 ホスフェート、および 10mM グリセ口 ホスフェート（Nacalai Tesque, Kyoto, Japan)を補充した 10%FBSを含有するコ ントロール培地中で、 4週間インキュベートした。固定した細胞（4%パラフオルムアル デヒドで 10分間）を洗浄し、喑所で 2. 5%硝酸銀とともに 20分間インキュベートした。 次いで、細胞を洗浄し、 15分間照明下に置き、 2分間 0. 5%ヒドロキノンとともにイン キュペートし、そして 2分間 5%チォ硫酸ナトリウムとともにインキュベートして、石灰化 した沈着物を可視化した。次いで、細胞を洗浄し、 0. 1 % Nuclear Fast Red, 5 % A12 (S04) 3溶液で対比染色した。 Incubation was performed for 4 weeks in control medium containing 10% FBS supplemented with samethasone, 50 μΜ ascorbic acid 1-2 phosphate, and 10 mM glycerol mouth phosphate (Nacalai Tesque, Kyoto, Japan). Fixed cells (4% paraformaldehyde for 10 minutes) were washed and incubated at room with 2.5% silver nitrate for 20 minutes. Cells were then washed, placed under illumination for 15 minutes, incubated with 0.5% hydroquinone for 2 minutes, and incubated with 5% sodium thiosulfate for 2 minutes to visualize calcified deposits. Cells were then washed and counterstained with 0.1% Nuclear Fast Red, 5% A12 (S04) 3 solution.
軟骨形成の分化に関して、コロニー形成ユニット分析もまた、細胞内脂質を用いて コロニーを定量化するために行った。 150個の糸田胞を、 100mmの培養皿にプレート し、 10〜14日間培養し、続いて、 2週間、軟骨形成の培地とともにインキュベートし、 Oil -Red Oで上記のように染色した。染色に関して陽性または陰性のコロニーの 数を、顕微鏡下で計数した。 With respect to chondrogenic differentiation, colony forming unit analysis also uses intracellular lipids. This was done to quantify the colonies. 150 Itoda vesicles were plated on 100 mm culture dishes and cultured for 10-14 days, followed by incubation with chondrogenic medium for 2 weeks and stained with Oil-Red O as described above. The number of positive or negative colonies for staining was counted under a microscope.
[0161] (フローサイトメトリーおよび選別） [0161] (Flow cytometry and sorting)
新たに単離した PLA細胞または LAF細胞を、表面分子または細胞内分子の発現 について、フローサイトメトリーを使用して試験した。培養した PLA細胞または LAF細 胞もまた、 1、 2、 4、 6、 8、 10、または 20週目で試験した。蛍光色素と結合させた下 記のモノクローナル抗体（MAbs)を使用した：抗 CD4_FITC、 CD10_PE、 CD1 3— PE、 CD16— PE、 CD29— PE、 CD31— PE、 CD34— PE、 CD34— FITC、 C D36— PE、 CD44— PE、 CD45— PE、 CD45— FITC、 CD49d— PE、 CD49e— PE、 CD54_PE、 CD56 _PE、 CD57_FITC、 CD62E_PE、 CD62P_PE、 C D69— FITC、 CD73— PE、 CD90— PE、 CD106— FITC、 CD117— PE、 CD13 5— PE、 CD144— PE、 CD— 151— PE、 HLA— A, B, C— PE、 Tie— 2— PE、 ( BD Biosciences, San Diego, CA, USA)、 CD59 -PE (Ancell, Bayport, MN, USA) , CD71— PE、 CD105— PE (Serotec, Oxford, UK) , CD133— P E、 Flk- l -PE (Techne, NJ, USA)。無関係のコントロール MAbsを、全ての蛍 光色素に対してインキュベートした。細胞を、直接結合体化させた MAbsで 30分間コ 一ティングし、洗浄し、そして 1%パラフオルムアルデヒド中で固定した。細胞を、 LSR Freshly isolated PLA or LAF cells were tested for expression of surface or intracellular molecules using flow cytometry. Cultured PLA or LAF cells were also tested at 1, 2, 4, 6, 8, 10, or 20 weeks. The following monoclonal antibodies (MAbs) conjugated with fluorescent dyes were used: anti-CD4_FITC, CD10_PE, CD1 3—PE, CD16—PE, CD29—PE, CD31—PE, CD34—PE, CD34—FITC, CD36 — PE, CD44—PE, CD45—PE, CD45—FITC, CD49d—PE, CD49e—PE, CD54_PE, CD56_PE, CD57_FITC, CD62E_PE, CD62P_PE, C D69—FITC, CD73—PE, CD90—PE, CD106—FITC , CD117—PE, CD13 5—PE, CD144—PE, CD—151—PE, HLA—A, B, C—PE, Tie—2—PE, (BD Biosciences, San Diego, CA, USA), CD59- PE (Ancell, Bayport, MN, USA), CD71—PE, CD105—PE (Serotec, Oxford, UK), CD133—PE, Flk-PE (Techne, NJ, USA). Irrelevant control MAbs were incubated for all fluorescent dyes. Cells were coated with direct conjugated MAbs for 30 minutes, washed and fixed in 1% paraformaldehyde. Cells, LSR
Πフローサイトメーター（Becton Dickinson, San Jose, CA, USA)を使用して 分析した。次いで、データ収集および分析を行った（Cell Questソフトウェア、 Beet on Dickinson)。無関係の抗体を使用して陽性である細胞が 0. 1%を越えないよう に、関連する MAbsおよび無関係の MAbsの組み合わせを用いた染色に基づいて、 ゲートを設定した。細胞の選別およびその後の分析を、 FACSAriaセルソーター（B ecton Dickinson)を使用して行つ 7こ。 Analysis was performed using a flow cytometer (Becton Dickinson, San Jose, CA, USA). Data collection and analysis were then performed (Cell Quest software, Beet on Dickinson). A gate was set based on staining with a combination of related and irrelevant MAbs so that no more than 0.1% of cells were positive using an irrelevant antibody. Perform cell sorting and subsequent analysis using a FACSAria cell sorter (Becton Dickinson).
[0162] (統計学的分析） [0162] (Statistical analysis)
結果を、平均値土 SEMとして表した。 Welchの t—テストを使用して、各パラメータ 一を比較した。 [0163] 2.脂肪吸引物の脂肪部分および液体部分由来の間質細胞の単離および細胞拡大 脂肪吸引物の脂肪部分と比較して、液体部分は、脂肪吸引術の際に吸引部位から 放出された末梢血をより多量に含んでおり、その容量は、患者間で変動していた（図 la)。脂肪部分をコラゲナーゼ消化に供した後、細胞外マトリックス (ECM)断片およ び切片を除去するためにフィルターにかけた。一方、液体部分を遠心分離し、混入し ている赤血球を溶解するために処理した。培養皿にプレートした後、培養培地を交 換することによって非接着細胞を捨てた。接着 PLAおよび接着 LAF細胞の両方が、 線維芽細胞様の形態を示し、類似の倍カ卩時間で増殖することが見出されたが、より 少数の接着細胞が、 PLA細胞よりも LAF細胞から回収された（図 lb)。 The results were expressed as mean soil SEM. Welch t-test was used to compare each parameter one. [0163] 2. Isolation and expansion of stromal cells from the fat and liquid parts of the lipoaspirate As compared to the fat part of the lipoaspirate, the liquid part is released from the suction site during liposuction Higher amounts of peripheral blood were included, and their volume varied between patients (Figure la). The fat portion was subjected to collagenase digestion and then filtered to remove extracellular matrix (ECM) fragments and sections. On the other hand, the liquid portion was centrifuged and treated to lyse contaminating red blood cells. After plating on culture dishes, non-adherent cells were discarded by changing the culture medium. Both adherent PLA and adherent LAF cells were found to show fibroblast-like morphology and proliferate with similar doubling times, but fewer adherent cells were more prone to LAF cells than PLA cells. It was recovered (Figure lb).
[0164] 新たに単離された PLA細胞（n = 28)および LAF細胞（n=28)の正規化された数 を計数し、それらはサンプル (脂肪吸引物）（1リットルの脂肪部分を含む）当たり、そ れぞれ 1.31±0.50X109(平均土 S. E. )ぉよび 1.55±0.79X109(P = 0.40 1)であったが（図 lc)、細胞数は、患者間でかなり変動していた。細胞収量は、単離 された細胞数を、処理された脂肪吸引物に含まれる脂肪部分の容量 (リットル)で割り 算することによって正規化した。赤血球の混入は、新たに単離された PLA細胞およ び LAF細胞の両方において見られた力 LAF細胞は、赤血球溶解緩衝液または密 度勾配遠心分離で処理した後でさえ、明らかに末梢血に由来する細胞をより多く含 んでいた。細胞培養の 1週間後、接着 PLA細胞 (n=28)および接着 LAF細胞 (n= 28)の正規ィ匕した数は、それぞれ、 9.7±1.7X107および 3.0±0.6X107(P<0 .001)であった（図 Id)。したがって、細胞収量においては、新たに単離された PLA 細胞と LAF細胞との間に有意な差違はなかった力 S、 1週間培養された接着 PLA細 胞と接着 LAF細胞との間には、有意な差違が存在した。 [0164] The normalized numbers of freshly isolated PLA cells (n = 28) and LAF cells (n = 28) are counted and they are sample (lipoaspirate) (contains 1 liter of fat part) ) Was 1.31 ± 0.50X10 9 (mean soil SE) and 1.55 ± 0.79X10 9 (P = 0.40 1), respectively (Figure lc), but the number of cells varied considerably between patients. It was. Cell yield was normalized by dividing the number of isolated cells by the volume (liter) of the fat fraction contained in the treated lipoaspirate. Erythrocyte contamination is the force seen in both freshly isolated PLA and LAF cells. LAF cells clearly become peripheral blood, even after treatment with erythrocyte lysis buffer or density gradient centrifugation. Contained more cells derived from. After one week of cell culture, the normal numbers of adherent PLA cells (n = 28) and adherent LAF cells (n = 28) were 9.7 ± 1.7X10 7 and 3.0 ± 0.6X10 7 (P <0. 001) (Fig. Id). Therefore, in cell yield, there was no significant difference between newly isolated PLA cells and LAF cells, and between adherent and cultured LAF cells cultured for 1 week, There were significant differences.
[0165] PLA細胞および LAF細胞を、 10% (各々、 n= 14)または 15%の FBS (各々、 n = 15)を含有する培養培地において培養し、継代 0の細胞を使用して倍加時間を測定 した。 PLA細胞および LAF細胞の倍加時間は、 15%FBSと共に培養した場合に 28 .5±1.7日寺間および 31.0±2.6日寺間、 10%FBSと共に培養した場合に、 43.3± 2.8時間および 40.2 ±3.0時間であった。統計学的な有意性は、 PLA細胞（pく 0 .001)および LAF細胞（p<0.05)の両方において、 2つの血清濃度間で見出され た力 いずれの血清濃度においても PLA細胞と LAF細胞との間には見出されなか つた（図 le)。 [0165] PLA and LAF cells are cultured in culture medium containing 10% (each n = 14) or 15% FBS (each n = 15) and doubled using passage 0 cells. Time was measured. The doubling time for PLA and LAF cells is 28.5 ± 1.7 days and 31.0 ± 2.6 days when cultured with 15% FBS, 43.3 ± 2.8 hours and 40.2 ± when cultured with 10% FBS. It was 3.0 hours. Statistical significance was found between the two serum concentrations in both PLA cells (p 0.001) and LAF cells (p <0.05). It was not found between PLA and LAF cells at any serum concentration (Figure le).
[0166] 3. PLA細胞および LAF細胞のインビトロでの分化  [0166] 3. Differentiation of PLA and LAF cells in vitro
PLA細胞および LAF細胞の多分化能を比較するために、細胞分化を、継代 3〜5 の細胞を用いて、その細胞を 4週間、脂肪生成、軟骨形成、または骨形成のための 培地で培養することによって誘導した。結果は、両方の細胞集団が、脂肪生成（図 2a )、軟骨形成（図 2b)、および骨形成（図 2c)の系統に沿って類似の分化能を有する ことを示した。コロニー形成ユニット分析により、 Oil-Red Oに対して陽性の染色を 示す細胞の割合（％)が、 PLA細胞の 29. 0± 7. 6%、および LAF細胞の 24. 1 ±4 . 3%であることが示された（P = 0. 12) (図 2d)。  In order to compare the pluripotency of PLA and LAF cells, cell differentiation was performed using cells from passage 3 to 5 in medium for adipogenesis, cartilage formation, or bone formation for 4 weeks. Induced by culturing. The results showed that both cell populations had similar differentiation potential along the lineage of adipogenesis (Figure 2a), chondrogenesis (Figure 2b), and bone formation (Figure 2c). According to the colony forming unit analysis, the percentage of cells that stained positive for Oil-Red O was 29.0 ± 7.6% for PLA cells and 24.1 ± 4.3% for LAF cells. (P = 0.12) (Figure 2d).
[0167] 4.新たに単離された力、または培養された PLA細胞および LAF細胞のフローサイトメ トリーによる分析  [0167] 4. Analysis by flow cytometry of newly isolated force or cultured PLA and LAF cells
フローサイトメトリー分析により、新たに単離した LAF細胞力 細胞表面マーカーの 発現において、新たに単離した PLA細胞とは有意に異なることが明らかになった。新 たな PLA細胞は、 CD29、 CD34、および CD90発現に関して陽性の細胞をより高 い割合で含有し、造血性起源の CD45+細胞はわずかにしか含有しない。一方、新 たな LAF細胞においては、 CD31 +細胞および CD45+細胞がより高い割合で存在 し、このことは、新たな LAF細胞力 新たな PLA細胞よりも、末梢血由来の血管内皮 細胞および細胞集団を多数含有することを示唆する。新たな LAF細胞の前方および 測方散乱特性（FSCおよび SSC)によってプロットした FACS図において、顆粒球、 単球、およびリンパ球に対応する 3つの CD45+細胞クラスターが存在する（図 3a)。 Flow cytometric analysis revealed that the expression of freshly isolated LAF cell force cell surface markers was significantly different from freshly isolated PLA cells. The new PLA cells contain a higher percentage of cells positive for CD29, CD34, and CD90 expression, and few CD45 + cells of hematopoietic origin. On the other hand, the new LAF cells have a higher proportion of CD31 + and CD45 + cells, which means that the new LAF cell power is higher than peripheral PLA-derived vascular endothelial cells and cells. Suggests containing a large number of populations. In the FACS plot plotted by the forward and square scatter properties of new LAF cells (FSC and SSC), there are three CD45 + cell clusters corresponding to granulocytes, monocytes, and lymphocytes (Figure 3a).
CD34+細胞および CD45_細胞は、単球に対応するクラスター上に位置する（図 3a CD34 + and CD45_ cells are located on clusters corresponding to monocytes (Figure 3a).
[0168] LAF細胞に含まれている接着間質細胞の起源をさらに調べるために、 CD34およ び CD45についての二重色分析を行った。それにより、 CD34+細胞のほとんどは、 CD45_であることが明らかになった。これは、 CD34+細胞の大部分が、末梢血由来 ではなぐ脂肪組織由来であることを示唆する（図 3b)。さらに、 CD34+CD45_細胞 は、培養皿上で増殖したが、 CD34+CD45+細胞は増殖しな力 た。これは、新たに 単離した LAF細胞が、脂肪組織由来の接着間質細胞を含有することを示唆する（図 3b)。 [0168] To further investigate the origin of adherent stromal cells contained in LAF cells, a double color analysis was performed for CD34 and CD45. This revealed that most of the CD34 + cells were CD45_. This suggests that the majority of CD34 + cells are derived from adipose tissue rather than from peripheral blood (Figure 3b). Furthermore, CD34 + CD45_ cells grew on the culture dish, but CD34 + CD45 + cells did not grow. This is a new This suggests that isolated LAF cells contain adherent stromal cells derived from adipose tissue (FIG. 3b).
[0169] 図 4は、新たに単離したかまたは培養した PLA細胞および LAF細胞の代表的な F ACSデータを示す。 5人の患者からのサンプノレを用いた。示されるように、新たに単 離した PLA細胞および LAF細胞は、細胞表面マーカーの明確に区別されるプロフィ ールを示した。これに対して、 2週間培養した PLA細胞および LAF細胞は、極めて 類似した発現プロフィールを示した。これらの差違は、新たに単離した LAF細胞が、 末梢血由来の細胞を大きな割合で含んでいるという事実から生じると考えられる。  [0169] Figure 4 shows representative FACS data for freshly isolated or cultured PLA and LAF cells. Sampu Nore from 5 patients was used. As shown, newly isolated PLA and LAF cells showed a well-defined profile of cell surface markers. In contrast, PLA and LAF cells cultured for 2 weeks showed very similar expression profiles. These differences are thought to arise from the fact that freshly isolated LAF cells contain a large percentage of peripheral blood-derived cells.
[0170] 図 5は、細胞表面マーカーの発現の連続的な変化を示す。プレートした後、 PLA細 胞および LAF細胞の両方の表面マーカー発現プロフィールが著しく変化し、 2つの 細胞集団の接着細胞が、極めて類似した発現プロフィールを示した。 CD34+細胞は 、 PLA細胞において割合が増大し、 CD13, CD29 ( j3 1インテグリン）、 CD44、 CD 73、および CD90 (Yhy— 1)のような間葉マーカーを均一に発現した。 CD10、 CD 49e、 CD59、および CD151もまた、 1週間よりも長く培養された PLA細胞において 均一に発現された。新たに単離した PLAまたは LAF細胞の 1週間培養物では、それ ぞれ、間葉系幹細胞関連マーカーの 1つである CD105 (エンドダリン (Endoglin) ) の、 1. 2 ± 0. 6力ら 64. 1 ± 9. 7へ（P< 0. 001)、または 1. 4± 0. 7力ら 74. 6 ± 7 . 6へ (ρ< 0· 001)の劇的な変化が生じた（図 5)。 CD105発現の統計学的な有意 性は、接着 PLA細胞と接着 LAf細胞との間には観察されな力 た。 CD45、 Flk—l (VEGFR— 2)、Tie— 2、 CD31 (PECAM— 1)、 CD117 (c— kit)、および CD13 3/AC133 (幹細胞関連マーカーの 1つ）は、 PLA細胞および LAF細胞において 有意に割合が減少した。いずれの細胞集団も、 1週目において CD4、 CD45、 CD6 2E (Eセレクチン）、 CD62P (Pセレクチン）、 CD69、 CD135、 CD144 (VEカドヘリ ン）について、 2週目を過ぎた培養後 ίこおレヽて CD16、 CD31、 CD57、 CD106 (VC AM_ 1)、 CD133、 Flk_ l、Tie_ 2について、陰性であった。 [0170] FIG. 5 shows a continuous change in the expression of cell surface markers. After plating, the surface marker expression profiles of both PLA and LAF cells changed significantly, and the adherent cells of the two cell populations showed very similar expression profiles. CD34 + cells increased in proportion in PLA cells and uniformly expressed mesenchymal markers such as CD13, CD29 (j31 integrin), CD44, CD73, and CD90 (Yhy-1). CD10, CD49e, CD59, and CD151 were also uniformly expressed in PLA cells cultured for longer than 1 week. For each weekly culture of freshly isolated PLA or LAF cells, each of CD105 (Endoglin), one of the mesenchymal stem cell-related markers, 1.2 ± 0. 6 force et al. 64 A dramatic change of 1 ± 9. 7 (P <0. 001) or 1.4 ± 0. 7 force et al. To 74. 6 ± 7. 6 (ρ <0 · 001) occurred (Figure Five). Statistical significance of CD105 expression was not observed between adherent and adherent LAf cells. CD45, Flk—l (VEGFR—2), Tie—2, CD31 (PECAM—1), CD117 (c—kit), and CD13 3 / AC133 (one of the stem cell-related markers) are found in PLA and LAF cells. The rate decreased significantly. All cell populations were tested for CD4, CD45, CD6 2E (E-selectin), CD62P (P-selectin), CD69, CD135, and CD144 (VE cadherin) in the first week after incubation. The results were negative for CD16, CD31, CD57, CD106 (VC AM_1), CD133, Flk_l and Tie_2.
[0171] 培養時間の経過とともに、 CD34発現は減少した力 10〜20%の細胞は、 PLA糸田 胞および LAF細胞における 10週間の培養後においてさえ、 CD34発現を維持して いた（図 5)。 CD34+細胞を選別し、同じ培養プロトコルで培養した力 細胞の約半分 力 培養 2週間後に CD34に対して陰性であることが見出された（図 3b)。これは、 C D34+細胞の中には、培養によって CD34発現を失ったものと、 CD34発現を失わず に増殖したものがあることを示唆する。接着 PLA細胞および接着 LAF細胞における CD34発現は、それぞれ、 20週（継代 19)および 10週（継代 9)まで確認した。さらに 、間葉マーカー（CD13、 CD29、 CD44、 CD73、および CD90)ならびに他のマー カー（CD49d、 CD59、 CD105、および CD151)の発現の維持力 接着 PLA細胞 および接着 LAF細胞において、それぞれ 20週および 10週まで観察された。表面マ 一力一発現における顕著な変化は、接着 PLA細胞と接着 LAF細胞との間で、最初 の 2週間の後の培養期間を通じて観察されなかった。これらをまとめると、接着 PLA 細胞および接着 LAF細胞は、本発明の培養方法によって、幹細胞性（sternness) に関連する表面マーカーを失うことなぐ拡大され得ることが示唆される。これらの事 実は、接着 PLA細胞および接着 LAF細胞が、極めて類似した表面マーカー発現プ ロフィールを最初の 1〜2週間の後の培養期間を通じて有することを明らかにし、両方 の細胞集団が ASCと呼ばれ得ることを示唆する。したがって、 ASCは、脂肪吸引物 の脂肪部分力 だけではなぐ液体部分力 も単離され得ることが強く示された。 [0171] The strength of CD34 expression decreased with the passage of culture time. 10-20% of cells maintained CD34 expression even after 10 weeks of culture in PLA Itoda and LAF cells (Figure 5). CD34 + cells were sorted, about half of the force cells cultured under the same culture protocol Power cultures were found to be negative for CD34 after 2 weeks (Figure 3b). This suggests that some CD34 + cells have lost CD34 expression in culture and others have grown without losing CD34 expression. CD34 expression in adherent PLA cells and adherent LAF cells was confirmed up to 20 weeks (passage 19) and 10 weeks (passage 9), respectively. In addition, the maintenance of the expression of mesenchymal markers (CD13, CD29, CD44, CD73, and CD90) and other markers (CD49d, CD59, CD105, and CD151) in adherent PLA cells and adherent LAF cells, respectively, at 20 weeks and Observed up to 10 weeks. No significant change in surface expression was observed between adherent and adherent LAF cells throughout the culture period after the first 2 weeks. Taken together, it is suggested that adherent PLA cells and adherent LAF cells can be expanded by the culture method of the present invention without losing surface markers associated with sternness. These facts reveal that adherent and adherent LAF cells have very similar surface marker expression profiles throughout the culture period after the first 1-2 weeks, and both cell populations are called ASCs. Suggest to get. Therefore, it was strongly shown that ASC can isolate not only the fat partial force of lipoaspirate but also liquid partial force.
[0172] (実施例 2 :長期培養の効果の確認実験） [Example 2: Confirmation experiment of long-term culture effect]
上記実施例に準じて幹細胞の単離を行い、 20週間 PLA細胞および LAF細胞の 長期培養による影響を調べた。図 6および図 7は 20週間までの、それぞれ CD34陽 性細胞比率および CD105陽性細胞比率を示すグラフを示す。示されるように、予想 外にも、 CD34陽性細胞が 10— 20%程度 20週間経っても維持されていることが確 認された。 CD105陽性細胞については、 50%程度維持されていることが判明した。 CD34は一般的には DMEMで培養され、：!〜 2週間で CD34は完全にネガティブに なることが多く報告されている（Mizuno, J Nippon Med Sch 2003; 70 (5) 428-431)) 0ま た、最初から CD34 (_)と報告している論文も存在する（Mizuno J Nippon Med Sch 2 003; 70 (5) 428-431))。したがって、この細胞は、長期培養後でも未分化性を保って レ、ると結論付けた。 Stem cells were isolated according to the above example, and the effects of long-term culture of PLA cells and LAF cells for 20 weeks were examined. Figures 6 and 7 show graphs showing the ratio of CD34 positive cells and CD105 positive cells up to 20 weeks, respectively. As shown, it was unexpectedly confirmed that CD34 positive cells were maintained even after 20 weeks, about 10-20%. It was found that about 50% of CD105 positive cells were maintained. CD34 is generally cultured in DMEM, and it has been reported that CD34 becomes completely negative in 2 weeks (Mizuno, J Nippon Med Sch 2003; 70 (5) 428-431)) 0 There is also a paper that has been reported as CD34 (_) from the beginning (Mizuno J Nippon Med Sch 2 003; 70 (5) 428-431)). Therefore, it was concluded that the cells remained undifferentiated even after long-term culture.
[0173] したがって、本発明において、 CD34の挙動に関して、従来知られていた間葉系幹 細胞とは異なる特徴を有することが明らかとなった。 [0174] (実施例 3 :コンフルェントに近い条件での増殖のアツセィ） [0173] Therefore, in the present invention, it has been clarified that the behavior of CD34 has characteristics different from those of conventionally known mesenchymal stem cells. [0174] (Example 3: Growth at a condition close to confluence)
本実施例において、実施例 1および 2で調製した細胞を分化誘導条件に曝すと、種 々の分化細胞への分化誘導を生じることを示す。  In this example, it is shown that when the cells prepared in Examples 1 and 2 are exposed to differentiation-inducing conditions, differentiation induction into various differentiated cells occurs.
[0175] 使用されたプロトコールは、以下の通りである： [0175] The protocol used is as follows:
1)骨への分化  1) Differentiation into bone
収集された細胞を、 DMEM培地において 1. 8 X 107細胞 Z皿の濃度にて、 60m m皿上で培養する。 10日間の培養後、この細胞はコンフルェント近くになり、その時 点で、その現在の培地と、分化誘導培地 (組成:骨生成性培地の例は、 10% FBS および 5%馬血清を含む DMEMに、 1 μ Μデキサメタゾン、 50 μ Μァスコルべート _ 2_ホスフェート、 10mM /3—グリセ口ホスフェート、および 1% ABAMを添加 する培地であり得る）とを交換し、 22日間培養する。この細胞を、 von Kossa染色を 用いて観察する。 The collected cells are cultured in DMEM medium at a concentration of 1.8 × 10 7 cells Z dish on a 60 mm dish. After 10 days of culture, the cells are close to confluence, at which point their current medium and differentiation induction medium (composition: an example of osteogenic medium is DMEM containing 10% FBS and 5% horse serum). , 1 μΜ dexamethasone, 50 μΜ ascorbate _ 2_phosphate, 10 mM / 3-glyceose phosphate, and 1% ABAM), and incubate for 22 days. The cells are observed using von Kossa staining.
[0176] von Kossa染色：  [0176] von Kossa staining:
培養培地を捨て、 目的のサンプルをリン酸緩衝化生理食塩水（PBS)で洗浄する。 この洗浄するサンプルを、 4%パラホルムアルデヒドで 10分間固定する。このサンプ ルを、 milliQ水で洗浄する。このサンプルを、喑所で 20分間、 2. 5%硝酸銀と接触さ せる。このサンプルを、 milliQ水で洗浄する。このサンプルを、蛍光灯の下に 15分間 放置する。このサンプルを、 0. 5%ヒドロキノンと 2分間インキュベーションし、続いて 5 %チォ硫酸ナトリウムと 2分間インキュベーションする。次いで、このサンプルを、 milli Q水で洗浄する。このサンプルを、ヌクレアファーストレッド（Nuclear First Red)で 2分間染色する。次いで、このサンプルを、 milliQ水で洗浄する。このサンプルを、マ ゥントクイック（大道産業、 日本）と共にマウントし、顕微鏡で観察する。  Discard the culture medium and wash the target sample with phosphate buffered saline (PBS). Fix the sample to be washed with 4% paraformaldehyde for 10 minutes. Wash this sample with milliQ water. This sample is contacted with 2.5% silver nitrate for 20 minutes at a certain place. The sample is washed with milliQ water. Leave this sample under fluorescent light for 15 minutes. The sample is incubated for 2 minutes with 0.5% hydroquinone followed by 2 minutes with 5% sodium thiosulfate. The sample is then washed with milliQ water. This sample is stained for 2 minutes with Nuclear First Red. The sample is then washed with milliQ water. Mount this sample with Mount Quick (Daido Sangyo, Japan) and observe with a microscope.
[0177] 2)軟骨への分化  [0177] 2) Differentiation into cartilage
収集された細胞を、 DMEM培地において 1. 8 X 107細胞 Z皿の濃度にて、 60m m皿上で培養する。 10日間の培養後、この細胞はコンフルェント近くになり、その時 点で、その現在の培地と、分化誘導培地 (組成:軟骨生成性培地の例は、 1% FBS を含む DMEMに、 6. 25 z g/mlインスリン、 6. 25 μ gZmlトランスフェリン、 10ng /ml TGF j3 1、 50nMァスコルべート一 2_ホスフェート、および 1% ABAMを添 加する培地であり得る）とを交換し、 22日間培養した。この細胞を、アルシアンブルー 染色を用いて観察する。 The collected cells are cultured in DMEM medium at a concentration of 1.8 × 10 7 cells Z dish on a 60 mm dish. After 10 days of culture, the cells are close to confluence, at which point their current medium and differentiation induction medium (composition: an example of chondrogenic medium is 6.25 zg in DMEM containing 1% FBS. / ml insulin, 6.25 μg Zml transferrin, 10 ng / ml TGF j3 1, 50 nM ascorbate 2_phosphate, and 1% ABAM The medium to be added may be replaced) and cultured for 22 days. The cells are observed using Alcian blue staining.
[0178] アルシアンブルー染色：  [0178] Alcian blue staining:
目的のサンプルから培地を捨てる。このサンプルを、 4%パラホルムアルデヒドで 10 分間固定する。このサンプルを、 milliQ水で洗浄する。このサンプルを、 3%ァセテ ートで 3分間洗浄する。このサンプルを、アルシアンブルー染色溶液 (組成:脂肪生 成培地の例は、 10%FBSを含む DMEMに、 0. 5mM IBMX、 1 μ Mデキサメタゾ ン、 10 μ Μインスリン、 200 μ Μインドメタシン、および 1%ΑΜΑΜを添加した培地で あり得る）に、 30分間浸す。このサンプルを、 3%アセテートで 3分間洗浄する。このサ ンプノレを、 milliQ水で洗浄する。このサンプルを、ヌクレアファーストレッドで 2分間染 色する。次いで、このサンプルを、 milliQ水で洗浄する。次いで、このサンプルを、マ ゥントクイック（大道産業、 日本）と共にマウントし、顕微鏡で観察する。  Discard the medium from the desired sample. The sample is fixed with 4% paraformaldehyde for 10 minutes. The sample is washed with milliQ water. Wash the sample with 3% acetate for 3 minutes. This sample was added to an Alcian Blue staining solution (composition: example of adipogenic medium, DMEM containing 10% FBS, 0.5 mM IBMX, 1 μM dexamethasone, 10 μΜ insulin, 200 μΜ indomethacin, and Immerse in medium supplemented with 1% koji for 30 minutes. The sample is washed with 3% acetate for 3 minutes. Wash the sample with milliQ water. The sample is dyed with Nuclea Fast Red for 2 minutes. The sample is then washed with milliQ water. The sample is then mounted with MountQuick (Daido Sangyo, Japan) and observed with a microscope.
[0179] 3)脂肪への分化  [0179] 3) Differentiation into fat
収集された細胞を、 DMEM培地において 1. 8 X 107細胞/皿の濃度にて、 60m m皿上で培養した。 10日間の培養後、この細胞はコンフルェント近くになり、その時 点で、その現在の培地と、分化誘導培地 (組成:脂肪生成培地の例は、 10% FBS を含む DMEMに、 0· 5mM IBMX、 1 μ Μデキサメタゾン、 10 μ Μインスリン、 200 /i Mインドメタシン、および 1 % AMAMを添加した培地であり得る）とを交換し、 12 日間培養する。この細胞を、オイルレッド O染色を用いて観察する。 The collected cells were cultured in DMEM medium at a concentration of 1.8 × 10 7 cells / dish on a 60 mm dish. After 10 days of culture, the cells are close to confluence, at which point their current medium and differentiation induction medium (composition: an example of adipogenic medium is DMEM containing 10% FBS, 0.5 mM IBMX, Medium) supplemented with 1 μΜ dexamethasone, 10 μΜ insulin, 200 / i M indomethacin, and 1% AMAM) and cultured for 12 days. The cells are observed using oil red O staining.
[0180] オイルレッド〇染色：  [0180] Oil red 〇 dyeing:
培養培地を捨て、 目的のサンプルをリン酸緩衝化生理食塩水（PBS)で洗浄した。 この洗浄したサンプノレを、 4%パラホルムアルデヒド（PFA)で 10分間固定する。この サンプルを、 milliQ水で洗浄する。このサンプルを、 60%イソプロピルアルコールに 1分間浸す。このサンプルを、 milliQ水で洗浄する。このサンプルを、へマトキシリン に 10分間浸す。このサンプルを、 milliQ水で洗浄する。次いで、このサンプルを、炭 酸リチウムに数秒間浸し、次いで milliQ水で洗浄する。このサンプルを、マウントクイ ック（大道産業、 日本）と共にマウントし、顕微鏡で観察する。  The culture medium was discarded, and the target sample was washed with phosphate buffered saline (PBS). Fix the washed sampnore with 4% paraformaldehyde (PFA) for 10 minutes. Wash this sample with milliQ water. Immerse this sample in 60% isopropyl alcohol for 1 minute. The sample is washed with milliQ water. Immerse this sample in hematoxylin for 10 minutes. The sample is washed with milliQ water. The sample is then immersed in lithium carbonate for a few seconds and then washed with milliQ water. Mount this sample with Mount Quick (Daido Sangyo, Japan) and observe with a microscope.
[0181] 1)オイルレッド〇染色溶液を、以下のように調製する： オイルレッド〇 0· 3gを、 100mlの 99%イソプロピルアルコールに溶解する。使用 に際して、この溶液を 6 : 4の割合で1111111(3水と混合し、そのまま 30分間攪拌する。こ の混合溶液を、使用前に濾過する。 [0181] 1) Prepare an oil red dyeing solution as follows: Dissolve 0-3 g of oil red in 100 ml of 99% isopropyl alcohol. At the time of use, this solution is mixed with 1111111 (3 water in a ratio of 6: 4 and stirred for 30 minutes as it is. The mixed solution is filtered before use.
[0182] 2)炭酸リチウム溶液 [0182] 2) Lithium carbonate solution
3mlの炭酸リチウム飽和溶液（100ml当たり 1. 54gの炭酸リチウム）を、 100mlの m illiQ水に加える。  Add 3 ml of saturated lithium carbonate solution (1.54 g of lithium carbonate per 100 ml) to 100 ml of milliQ water.
[0183] このような実験を通じて、実施例 1または実施例 2において調製された幹細胞は、良 質な多能性を示すことが理解される。  [0183] Through such experiments, it is understood that the stem cells prepared in Example 1 or Example 2 exhibit good pluripotency.
[0184] (実施例 4 :分化培地での分化） [Example 4: Differentiation in differentiation medium]
本実施例において、分化培地を使用する場合、種々の分化細胞への分化を誘導 することを示す。  In this example, it is shown that when a differentiation medium is used, differentiation into various differentiated cells is induced.
[0185] 使用されたプロトコールは、以下の通りである： [0185] The protocol used is as follows:
1)骨への分化  1) Differentiation into bone
収集された細胞を、分化誘導培地 (組成:骨生成性培地の例は、 10% FBSおよ び 5%ゥマ血清を含む DMEMに、 1 μ Μデキサメタゾン、 50 μ Μァスコルべ一トー 2 ホスフェート、 10mM βーグリセ口ホスフェート、および 1% ΑΒΑΜを添加した 培地であり得る）において 1. 8 X 107細胞/皿の濃度にて、 60mm皿上に播種する。 Collected cells are differentiated into differentiation-inducing medium (composition: example of osteogenic medium is DMEM containing 10% FBS and 5% horse serum, 1 μΜ dexamethasone, 50 μΜascorbate 2 phosphate In a medium supplemented with 10 mM β-glycose mouth phosphate, and 1%）) 1. Seed on a 60 mm dish at a concentration of 8 × 10 7 cells / dish.
22日間培養後、このサンプルを von Kossa染色で評価した。 von Kossa染色は、 上記実施例で説明したように行う。  After 22 days of culture, the samples were evaluated by von Kossa staining. von Kossa staining is performed as described in the above examples.
2)脂肪への分化  2) Differentiation into fat
収集された細胞を、分化誘導培地 (組成:脂肪生成培地の例は、 10% FBSを含 む DMEMに、 0. 5mM IBMX、 1 μ Μデキサメタゾン、 10 μ Μインスリン、 200 μ Μインドメタシン、および 1 % ΑΜΑΜを添加した培地であり得る）において 1. 8 X 1 07細胞/皿の濃度にて、 60mm皿上に播種する。 25日間培養後、このサンプルを、 オイルレッド〇染色を用いて評価する。オイルレッド O染色は、上記実施例 17で説明 したように行う。 Collected cells are differentiated into differentiation-inducing medium (composition: adipogenic medium is an example of DMEM containing 10% FBS, 0.5 mM IBMX, 1 μΜ dexamethasone, 10 μΜ insulin, 200 μΜ indomethacin, and 1 Seed on a 60 mm dish at a concentration of 8 X 10 7 cells / dish. After culturing for 25 days, the sample is evaluated using Oil Red Staining. Oil red O staining is performed as described in Example 17 above.
[0186] 以上の実験を通じて、実施例 1または実施例 2において調製された幹細胞は、コン フルェントに近い状態への培養なしで分化誘導培地を用いて、良質な多能性を示す ことが理解される。 [0186] Through the above experiments, the stem cells prepared in Example 1 or Example 2 exhibit high-quality pluripotency using a differentiation-inducing medium without culturing to a state close to confluence. It is understood.
[0187] 特定の好ましい実施形態が本明細書中で説明されたが、添付の特許請求の範囲 以外に、このような実施形態が本発明の範囲を制限すると解釈されることを意図しな レ、。本明細書中で引用された全ての特許、公開特許出願および刊行物は、あたかも 本明細書中にその全体が示されたかのように、参考として援用される。本発明の範囲 および精神を逸脱せずに、種々の改変が当業者に明らかであり、そして当業者により 容易になされ得る。従って、本明細書に添付の特許請求の範囲は、本明細書の記載 により制限されることを意図されず、むしろ特許請求の範囲は広く解釈されることが意 図される。  [0187] Although certain preferred embodiments have been described herein, it is not intended that such embodiments be construed as limiting the scope of the invention, other than the appended claims. ,. All patents, published patent applications and publications cited herein are incorporated by reference as if set forth in full herein. Various modifications will be apparent to and can be readily made by those skilled in the art without departing from the scope and spirit of the invention. Accordingly, the claims appended hereto are not intended to be limited by the description herein, but rather the claims are intended to be construed broadly.
産業上の利用可能性  Industrial applicability
[0188] 本発明は、臨床的に有用な性質を維持した ASCを大量に供給し得ることから、医 療分野 (特に、脂肪再生、骨再生、心筋再生、血管新生、難治性潰瘍など)等への 適用に有用である。本発明は、簡便な方法で取得できる脂肪吸引物由来の幹細胞 、再生医療に応用できることを証明した。従って、当業者は、医薬品業界などにお ける本発明の産業上の利用可能性を容易に見出す。 [0188] Since the present invention can supply a large amount of ASC maintaining clinically useful properties, the medical field (particularly, fat regeneration, bone regeneration, myocardial regeneration, angiogenesis, refractory ulcer, etc.), etc. Useful for application. The present invention proved that lipoaspirate-derived stem cells that can be obtained by a simple method can be applied to regenerative medicine. Accordingly, those skilled in the art easily find the industrial applicability of the present invention in the pharmaceutical industry and the like.

Claims

請求の範囲 The scope of the claims
単離された多能性幹細胞であって、 CD34、 CD105、またはこれらの両方の細胞 表面マーカーを発現し得る、細胞。  An isolated pluripotent stem cell, which can express a cell surface marker of CD34, CD105, or both.
培養された多能性幹細胞であって、 CD34、 CD105、またはこれらの両方の細胞 表面マーカーを発現し得る、細胞。  A cultured pluripotent stem cell that can express a cell surface marker of CD34, CD105, or both.
培養された脂肪由来幹細胞であって、 CD34、 CD105、またはこれらの両方の細 胞表面マーカーを発現し得る、細胞。  A cultured adipose-derived stem cell that can express a cell surface marker of CD34, CD105, or both.
少なくとも 2週間培養された、請求項 2または 3に記載の細胞。  The cell according to claim 2 or 3, which has been cultured for at least 2 weeks.
少なくとも 10週間培養された、請求項 2または 3に記載の細胞。  The cell according to claim 2 or 3, which has been cultured for at least 10 weeks.
少なくとも 20週間培養された、請求項 2または 3に記載の細胞。  The cell according to claim 2 or 3, which has been cultured for at least 20 weeks.
新たに単離された脂肪由来幹細胞を培養する工程を包含する、 CD34+および/ または CD105+脂肪由来幹細胞の調製方法。 A method for preparing CD34 + and / or CD105 + adipose-derived stem cells, comprising culturing newly isolated adipose-derived stem cells.
前記脂肪由来幹細胞を少なくとも 2週間培養する、請求項 7に記載の方法。 前記脂肪由来幹細胞を少なくとも 10週間培養する、請求項 7に記載の方法。 前記脂肪由来幹細胞を少なくとも 20週間培養する、請求項 7に記載の方法。 前記培養が、  8. The method of claim 7, wherein the adipose-derived stem cells are cultured for at least 2 weeks. 8. The method of claim 7, wherein the adipose-derived stem cells are cultured for at least 10 weeks. The method according to claim 7, wherein the adipose-derived stem cells are cultured for at least 20 weeks. The culture is
5〜20% ゥシ胎仔血清  5-20% urine fetal serum
50〜400IUまたは 50〜400mg/ml 抗生物質、  50-400 IU or 50-400 mg / ml antibiotic,
l^lO z g/ml へパリン、および  l ^ lO z g / ml heparin, and
0. 5〜： 10ng/ml 酸性線維芽細胞増殖因子  0.5 ~: 10ng / ml acidic fibroblast growth factor
を含有する培地を用いて行われる、請求項 7〜： 10のいずれかに記載の方法。 前記培養の基本培地が、 M_ 199培地である、請求項 11に記載の方法。 The method according to claim 7, wherein the method is carried out using a medium containing 12. The method according to claim 11, wherein the basic culture medium is M_199 medium.
脂肪由来幹細胞を調製する方法であって、  A method for preparing adipose-derived stem cells, comprising:
ヒトドナー由来の脂肪吸引物を得る工程、および  Obtaining a lipoaspirate from a human donor, and
前記脂肪吸引物から、脂肪由来幹細胞を単離する工程、  Isolating adipose-derived stem cells from the lipoaspirate,
前記単離した脂肪由来幹細胞を培養する工程  Culturing the isolated adipose-derived stem cells
を包含し、 Including
前記培養が、 5〜20% ゥシ胎仔血清 Said culture is 5-20% urine fetal serum
50〜400IUまたは 50〜400mg/ml 抗生物質、  50-400 IU or 50-400 mg / ml antibiotic,
1〜： 10 /i g/ml へパリン、および  1 to: 10 / i g / ml heparin, and
0. 5〜： 10ng/ml 酸性線維芽細胞増殖因子  0.5 ~: 10ng / ml acidic fibroblast growth factor
を含有する培地を用いて行われる、  Carried out using a medium containing
方法。  Method.
[14] 前記培養のための基本培地が、 M_ 199培地である、請求項 13に記載の方法。  14. The method according to claim 13, wherein the basal medium for culturing is M_199 medium.
[15] ゼラチンで被覆した基板上で接着培養を行う、請求項 13または 14に記載の方法。 15. The method according to claim 13 or 14, wherein adhesion culture is performed on a substrate coated with gelatin.
[16] 前記培養を少なくとも 2週間行う、請求項 13〜： 15のいずれかに記載の方法。 [16] The method according to any one of [13] to [15], wherein the culture is performed for at least 2 weeks.
[17] 培地交換を少なくとも週 2回、細胞継代を少なくとも週 1回行う、請求項 16に記載の 方法。 17. The method according to claim 16, wherein medium exchange is performed at least twice a week and cell passage is performed at least once a week.
[18] 請求項 7〜： 17のいずれかに記載の方法によって調製された脂肪由来幹細胞であ つて、 CD34、 CD105、またはこれらの両方の細胞表面マーカーを発現する、細胞。  [18] A fat-derived stem cell prepared by the method according to any one of claims 7 to 17, wherein the cell expresses a cell surface marker of CD34, CD105, or both of them.
[19] 請求項:!〜 6のいずれか、または請求項 18に記載の細胞を含有する、再生医療用 材料。  [19] A regenerative medical material comprising the cell according to any one of claims:! To 6 or claim 18.
[20] 請求項:!〜 6のいずれか、または請求項 18に記載の細胞を使用する工程を包含す る、再生医療用材料の製造方法。  [20] A method for producing a material for regenerative medicine, comprising a step of using the cell according to any one of claims 6 to 18 or claim 18.
[21] 請求項:!〜 6のいずれか、または請求項 18に記載の細胞を含有する、ヒトもしくは動 物の疾患の治療、またはヒトもしくは動物の細胞、組織、もしくは臓器の再生に使用 するための薬剤。 [21] Claim: Used for treatment of human or animal diseases or regeneration of human, animal cells, tissues, or organs, comprising the cells according to any one of! To 6 or claim 18. Drugs for.
[22] 請求項:!〜 6のいずれか、または請求項 18に記載の細胞を被験体に投与する工程 を包含する、ヒトもしくは動物の疾患を治療するため、またはヒトもしくは動物の細胞、 組織、もしくは臓器を再生するための方法。  [22] A method for treating a human or animal disease, or a human or animal cell, tissue, comprising the step of administering to the subject a cell according to any one of! To 6 or claim 18 Or a method for regenerating an organ.
[23] 多能性幹細胞を含む組成物であって、該組成物は、 CD34、 CD105,またはこれ らの両方の細胞表面マーカーを発現し得る多能性幹細胞を少なくとも 10%含む、組 成物。 [23] A composition comprising pluripotent stem cells, the composition comprising at least 10% pluripotent stem cells capable of expressing CD34, CD105, or both cell surface markers. .
[24] 前記組成物は、 CD34、 CD105、またはこれらの両方の細胞表面マーカーを発現し 得る多能性幹細胞を少なくとも 50%含む、請求項 23に記載の組成物。 [25] 前記組成物は、 CD105の細胞表面マーカーを発現し得る多能性幹細胞を少なくと も 50%含む、請求項 23に記載の組成物。 [24] The composition of claim 23, wherein the composition comprises at least 50% pluripotent stem cells capable of expressing CD34, CD105, or both cell surface markers. [25] The composition of claim 23, wherein the composition comprises at least 50% of pluripotent stem cells capable of expressing a cell surface marker of CD105.
[26] 前記組成物は、 CD34の細胞表面マーカーを発現し得る多能性幹細胞を少なくとも[26] The composition comprises at least pluripotent stem cells capable of expressing a cell surface marker of CD34.
10%含む、請求項 23に記載の組成物。 24. The composition of claim 23, comprising 10%.
[27] CD34+および/または CD105+脂肪由来幹細胞を含む組成物を調製する方法 であって、 [27] A method for preparing a composition comprising CD34 + and / or CD105 + adipose-derived stem cells,
A)新たに単離された脂肪由来幹細胞を培養する工程を  A) A step of culturing newly isolated adipose-derived stem cells
包含する、方法。  The method of inclusion.
[28] 前記脂肪由来幹細胞を少なくとも 2週間培養する、請求項 27に記載の方法。  [28] The method of claim 27, wherein the adipose-derived stem cells are cultured for at least 2 weeks.
[29] 前記脂肪由来幹細胞を:!〜 2週間培養する、請求項 27に記載の方法。 [29] The method according to claim 27, wherein the adipose-derived stem cells are cultured for:! To 2 weeks.
[30] 前記脂肪由来幹細胞を少なくとも 10週間培養する、請求項 27に記載の方法。 [30] The method of claim 27, wherein the adipose-derived stem cells are cultured for at least 10 weeks.
[31] 前記脂肪由来幹細胞を少なくとも 20週間培養する、請求項 27に記載の方法。 [31] The method of claim 27, wherein the adipose-derived stem cells are cultured for at least 20 weeks.
[32] 前記培養が、 [32]
5〜20% ゥシ胎仔血清  5-20% urine fetal serum
50〜400IUまたは 50〜400mg/ml 抗生物質、  50-400 IU or 50-400 mg / ml antibiotic,
1〜： ίθ /i g/ml へパリン、および  1 to: ίθ / i g / ml heparin, and
0. 5〜： 10ng/ml 酸性線維芽細胞増殖因子  0.5 ~: 10ng / ml acidic fibroblast growth factor
を含有する培地を用いて行われる、  Carried out using a medium containing
請求項 27〜31のいずれか 1項に記載の方法。  32. A method according to any one of claims 27 to 31.
[33] 前記脂肪由来幹細胞は、 [33] The adipose-derived stem cells are
ヒトドナー由来の脂肪吸引物を得る工程、および  Obtaining a lipoaspirate from a human donor, and
前記脂肪吸引物から、脂肪由来幹細胞を単離する工程  Isolating fat-derived stem cells from the lipoaspirate
によって、単離される、請求項 27に記載の方法。  28. The method of claim 27, isolated by.
[34] 前記脂肪吸引物は、液体部分を含む、請求項 33に記載の方法。 [34] The method of claim 33, wherein the lipoaspirate comprises a liquid portion.
[35] 前記脂肪吸引物を得る工程は、液体部分を分離する工程をさらに包含する、請求項 33に記載の方法。 35. The method of claim 33, wherein obtaining the lipoaspirate further comprises separating a liquid portion.
PCT/JP2006/311882 2005-06-13 2006-06-13 Long-term culture proliferation method for fat-derived stem cell WO2006134951A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2007521313A JPWO2006134951A1 (en) 2005-06-13 2006-06-13 Long-term growth method for adipose-derived stem cells

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2005171886 2005-06-13
JP2005-171886 2005-06-13

Publications (1)

Publication Number Publication Date
WO2006134951A1 true WO2006134951A1 (en) 2006-12-21

Family

ID=37532309

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2006/311882 WO2006134951A1 (en) 2005-06-13 2006-06-13 Long-term culture proliferation method for fat-derived stem cell

Country Status (2)

Country Link
JP (1) JPWO2006134951A1 (en)
WO (1) WO2006134951A1 (en)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008018450A1 (en) * 2006-08-08 2008-02-14 National University Corporation Nagoya University Cell preparation containing multipotential stem cells originating in fat tissue
WO2008147057A1 (en) 2007-05-25 2008-12-04 Rnl Bio Co., Ltd Composition for treating ischemic limb disease comprising stem cells derived from adipose tissue
EP2166084A1 (en) * 2007-06-14 2010-03-24 Foundation for Biomedical Research and Innovation Multipotent progenitor cell derived from adipose tissue
JP2011520434A (en) * 2008-05-07 2011-07-21 ボーン セラピューティクス エス．アー． Novel mesenchymal stem cells and osteogenic cells
JP2014076048A (en) * 2012-09-20 2014-05-01 Yuichiro Onishi Generation and isolation method and production method of olfactory sphere cells, and production method of demyelinating disease treating agent and peripheral nerve axon regeneration promoting agent using the olfactory sphere cells
CN111471651A (en) * 2020-04-24 2020-07-31 中国人民解放军第四军医大学 Serum-free medium for human adipose-derived stem cells and preparation method thereof
JP2020143008A (en) * 2019-03-05 2020-09-10 達也 楠堂 Novel brown adipocyte differentiation inducing agent

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005035738A1 (en) * 2003-10-07 2005-04-21 Biomaster Inc. Cell differentiation of adipose-derived precursor cells
WO2005042730A2 (en) * 2003-11-04 2005-05-12 Biomaster, Inc. Method and system for preparing stem cells from fat tissue

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005035738A1 (en) * 2003-10-07 2005-04-21 Biomaster Inc. Cell differentiation of adipose-derived precursor cells
WO2005042730A2 (en) * 2003-11-04 2005-05-12 Biomaster, Inc. Method and system for preparing stem cells from fat tissue

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
BOQUEST A.C. ET AL.: "Isolation and transcription profiling of purified uncultured human stromal stem cells: alteration of gene expression after in vitro cell culture", MOL. BIOL. CELL, vol. 16, no. 3, March 2005 (2005-03-01), pages 1131 - 1141, XP003005683 *
MATSUMOTO D. ET AL.: "Kansaibo source toshite no Shibo Soshiki", ORGAN BIOLOGY, vol. 12, no. 2, 10 August 2005 (2005-08-10), pages 167 - 173 *
MATSUMOTO D. ET AL.: "Liposuction Aspirate Fluid karano Saibo no Saishu Oyobi characterization", DAI 13 KAI JAPAN SOCIETY OF PLASTIC AND RECONSTRUCTIVE SURGERY KISO GAKUJUTSU SHUKAI, 2004, pages 60 *
MATSUMOTO D. ET AL.: "Shibo Soshiki Chu no Kansaibo ni Tsuite", JAPAN SOCIETY OF AESTHETIC, vol. 27, no. 1, 25 March 2005 (2005-03-25), pages 62 *
RODRIGUEZ A.M. ET AL.: "Transplantation of a multipotent cell population from human adipose tissue induces dystrophin expression in the immunocompetent mdx mouse", J. EXP. MED., vol. 201, no. 9, 2 May 2005 (2005-05-02), pages 1397 - 1405, XP003005684 *
ZUK P.A. ET AL.: "Human adipose tissue is a source of multipotent stem cells", MOL. BIOL. CELL, vol. 13, no. 12, 2002, pages 4279 - 4295, XP002249630 *

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008018450A1 (en) * 2006-08-08 2008-02-14 National University Corporation Nagoya University Cell preparation containing multipotential stem cells originating in fat tissue
JP2010528107A (en) * 2007-05-25 2010-08-19 アールエヌエル バイオ カンパニー リミテッド Composition for treating ischemic limb disease comprising adipose tissue-derived stem cells
WO2008147057A1 (en) 2007-05-25 2008-12-04 Rnl Bio Co., Ltd Composition for treating ischemic limb disease comprising stem cells derived from adipose tissue
EP2162141A1 (en) * 2007-05-25 2010-03-17 RNL Bio Co., Ltd. Composition for treating ischemic limb disease comprising stem cells derived from adipose tissue
EP2162141A4 (en) * 2007-05-25 2011-01-26 Rnl Bio Co Ltd Composition for treating ischemic limb disease comprising stem cells derived from adipose tissue
EP2166084A1 (en) * 2007-06-14 2010-03-24 Foundation for Biomedical Research and Innovation Multipotent progenitor cell derived from adipose tissue
EP2166084A4 (en) * 2007-06-14 2010-06-23 Found Biomedical Res & Innov Multipotent progenitor cell derived from adipose tissue
US9845458B2 (en) 2007-06-14 2017-12-19 Akifumi Matsuyama Multipotent progenitor cell derived from adipose tissue
JP2011520434A (en) * 2008-05-07 2011-07-21 ボーン セラピューティクス エス．アー． Novel mesenchymal stem cells and osteogenic cells
US9371515B2 (en) 2008-05-07 2016-06-21 Bone Therapeutics S.A. Mesenchymal stem cells and bone-forming cells
JP2017099386A (en) * 2008-05-07 2017-06-08 ボーン セラピューティクス エス．アー． Novel mesenchymal stem cells and bone-forming cells
JP2014076048A (en) * 2012-09-20 2014-05-01 Yuichiro Onishi Generation and isolation method and production method of olfactory sphere cells, and production method of demyelinating disease treating agent and peripheral nerve axon regeneration promoting agent using the olfactory sphere cells
JP2020143008A (en) * 2019-03-05 2020-09-10 達也 楠堂 Novel brown adipocyte differentiation inducing agent
CN111471651A (en) * 2020-04-24 2020-07-31 中国人民解放军第四军医大学 Serum-free medium for human adipose-derived stem cells and preparation method thereof

Also Published As

Publication number Publication date
JPWO2006134951A1 (en) 2009-01-08

Similar Documents

Publication Publication Date Title
EP1692275B1 (en) Method and system for preparing stem cells from fat tissue
US9074190B2 (en) Cell differentiation of adipose-derived precursor cells
Kuhbier et al. Isolation, characterization, differentiation, and application of adipose-derived stem cells
EP2739292B1 (en) Means for liver regeneration
US20100330047A1 (en) Mesenchymal Stem Cells Grown Under Hypoxic Conditions: Compositions, Methods and Uses Therefor
US20150118748A1 (en) High-concentration stem cell production method
EP3019599B1 (en) Method for isolating stromal vascular fraction
DE102017002458B4 (en) STRUCTURAL STEM CELLS FROM ADIPOSIVE FABRICS FOR USE IN THE TREATMENT OF TREATMENT-RESISTANT COMPLEX PERIANAL FISTLES AT MORBUS CROHN
WO2006134951A1 (en) Long-term culture proliferation method for fat-derived stem cell
WO2014046417A1 (en) Method for preparing mesenchymal stem cell aggregates
JP5388297B2 (en) Adipo cluster
WO2014201986A1 (en) Monoclonal mesenchymal stem cell generation method and use thereof
WO2019237812A1 (en) Adipose tissue digestive juice and method for rapidly obtaining stromal vascular fraction cells
US20230097931A1 (en) Method for treating chronic graft versus host disease
JP2023517641A (en) Method II for treating inflammatory bowel disease
Okura et al. Adipose tissue-derived multi-lineage progenitor cells as a promising tool for in situ stem cell therapy
WO2023217130A1 (en) Biological formulation containing skeletal muscle precursor-like cells, and preparation method and application therefor
Xie et al. Individual Heterogeneity Screened Umbilical Cord Derived Mesenchymal Stem Cells With High Treg Promotion Significantly Recovered the Mouse Liver Fibrosis
AU2021234104A1 (en) Method for treating inflammatory bowel disease I
CN117802039A (en) Serum-free mesenchymal stem cell three-dimensional culture medium and application thereof
KR20100069376A (en) Chondrogenic differentiations of adipose tissue-derived mesenchymal stem cells using growth factors

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 2007521313

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: 06766664

Country of ref document: EP

Kind code of ref document: A1