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WO2008018190A1 - Cellules de la crête neurale dérivées de tissu adipeux - Google Patents

Cellules de la crête neurale dérivées de tissu adipeux Download PDF

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Publication number
WO2008018190A1
WO2008018190A1 PCT/JP2007/051643 JP2007051643W WO2008018190A1 WO 2008018190 A1 WO2008018190 A1 WO 2008018190A1 JP 2007051643 W JP2007051643 W JP 2007051643W WO 2008018190 A1 WO2008018190 A1 WO 2008018190A1
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WIPO (PCT)
Prior art keywords
cells
cell
neural crest
derived
stem cells
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PCT/JP2007/051643
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English (en)
Japanese (ja)
Inventor
Kotaro Yoshimura
Takashi Nagase
Daisuke Matsumoto
Tomokuni Shigeura
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Biomaster, Inc.
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Publication of WO2008018190A1 publication Critical patent/WO2008018190A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0623—Stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
    • C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
    • C12N2506/1384—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from adipose-derived stem cells [ADSC], from adipose stromal stem cells

Definitions

  • the present invention relates to the field of cell sorting. More particularly, the present invention relates to a fat-derived or fat-derived stem cell-derived neural crest cell, a method for producing the same, and a transplantation therapy using the same.
  • tissue stem cells that were previously thought to be absent have been discovered and identified from various tissues. Thus, disease treatment by regenerative medicine is attracting attention.
  • a fertilized egg is divided into three germ layers of endoderm, mesoderm and ectoderm after gastrulation, and cells derived from ectoderm mainly exist in the brain, and include neural stem cells and the like.
  • Cells derived from mesoderm are mainly present in the bone marrow, and include vascular stem cells, hematopoietic stem cells, mesenchymal stem cells, and the like.
  • Endoderm-derived cells are mainly present in the pharynx, lungs, liver, spleen, intestine, etc., and include spleen stem cells, liver stem cells, and the like.
  • Adipose-derived stem cells are originally subtypes of mesenchymal stem cells (MSCs) that have also isolated liposuction aspirate powers that can be separated into mesenchymal tissues such as bone, cartilage and adipose tissue.
  • MSCs mesenchymal stem cells
  • Non-Patent Document 1 At present, adipose-derived stem cells are considered as one of the most promising adult stem cells for regenerative medicine (Non-Patent Documents 2 to 3). This is because fat-derived stromal cells can be safely recovered by liposuction and good yields can be expected. Ruka.
  • Non-patent Documents 4 and 5 derivatives of the nervous system
  • mesoderm tissue eg, dermis and heart
  • Non-Patent Documents 6 to 8 stem cells with neural characteristics have been collected by the neurosphere method.
  • the neurosphere method was developed as a method for culturing isolated spheres of neural stem cells derived from embryos and human brain.
  • Non-patent Document 9 It has been reported that neural stem cells are also present in the central nervous system of adults, which has long been thought to regenerate the central nervous system of adult mammals.
  • the number is small and it requires a great deal of labor to isolate, and it is difficult to actually use it in regenerative medicine such as nervous system diseases. Therefore, obtaining pluripotent cells of the nervous system from the nervous system is not practical. Therefore, studies have been conducted on the division of the nervous system using fat.
  • Neural crest cells also called neural crest cells, have the ability to migrate and have multipotency into differentiated cells of the nervous system (eg dorsal root ganglion cells, autonomic ganglion cells, adrenal medulla chromaffinity) A cell, a Schwann cell, and a cell having a differentiation potential into a coat pigment cell). Therefore, neural crest cells can be said to be broad stem cells rather than differentiated cells. Nerve crest cells are identified as a group of cells (fourth germ layer) that originates from the limbal limbus (nerve crest) in the early fetal period and migrates widely in the embryo and divides into a very wide and specific repertoire. It was done. At present Neural crest cells have been used to treat facial malformations.
  • the eurosphere produced by the group of Kang, KS, etc. can be said to be an aggregate of differentiated cells, and this -eurosphere has multipotency to differentiate into cells of the nervous system. No cells are present, and even neural crest cells are expected to be completely absent from this -Eurosphere. Therefore, when adipose-derived stem cells are used, differentiation is remarkably induced and it is considered that induction into pluripotent cells of the nervous system, such as neural crest cells, does not occur.
  • Patent Documents 1 to 6 describe that adipose-derived stem cells appear to have the ability to be divided into nerves.
  • these documents do not substantially describe the induction of adipose-derived stem cells into pluripotent cells of the nervous system such as neural stem cells, neural progenitor cells, and neurospheres, and have the ability to differentiate directly into nerves. Whether or not it is being studied. Therefore, these documents do not describe anything that can be induced from fat to neural pluripotent cells such as neural stem cells, neural crest cells, etc. In other words, since it has been attempted to differentiate into differentiated cells), it is considered that it cannot be induced into pluripotent cells of the nervous system in this field.
  • adipose-derived stem cells are abundant in supply, if adipose-derived, in particular, adipose-derived stem cells can be used to produce pluripotent cells of the nervous system, It can be said that it will bring about a revolutionary change in the treatment of diseases and disorders of the trans-system, and the demand for such methods will increase.
  • Patent Document 1 Reprinted WO2003Z008592
  • Patent Document 2 Japanese Translation of Special Publication 2005-502352
  • Patent Document 3 Japanese Translation of Special Publication 2002-537849
  • Patent Document 4 US Patent Application Publication No. 2001Z0033834
  • Patent Document 5 Special Table 2003-523767
  • Patent Document 6 Special Table 2005-502712
  • Non-patent literature l Zuk PA, Zhu M, Mizuno H, et al. Tissue Eng. 2001; 7: 2 11-228
  • Non-Patent Document 2 Zuk PA, Zhu M, Ashjian P, et al. Mol. Biol. Cell 2002; 1 3: 4279-4295
  • Non-Patent Document 3 Yoshimura K, Shigeura T, Matsumoto D, et al. J. Cell. Phisiol. 2006; 208: 64-76
  • Non-Patent Document 4 Ashjian PH, Elbarbary AS, Edmonds B, et al. Plast. Recon nstr. Surg. 2003; 111: 1922-1931
  • Non-Patent Document 5 Kokai LE, Rubin JP, Marra KG. Plast. Reconstr. Surg. 20 05; 116: 1453-1460
  • Non-Patent Document 6 Toma JG, Akhavan M, Fernandes KJ, et al. Nat. Cell. Bio 1.2001; 3: 778-784
  • Non-Patent Document 7 Fernandes KJ, McKenzie IA, Mill P, et al. Nat. Cell. Biol. 2004; 6: 1082-1093
  • Non-Patent Document 8 Tomita Y, Matsumura K, Wakamatsu Y, et al. J. Cell. Biol. 2005; 170: 1135-1146
  • Non-Patent Document 9 Enoyuki Okano, Experimental Medicine, No. 20, No. 9, 2002; 1276-1279
  • Non-Patent Document 10 Kang SK, Putnam LA, Ylostalo J, et al. J. Cell. Sci. 200
  • Non-patent literature ll Kang SK, Putnam L, Dufour J, et al. Stem Cells 2004; 22: 1356-1372
  • Non-Patent Document 12 Bunnell BA, Ylostalo J, Kang SK.Biochem.Biophys.Res.Commun.2006; May 12; 343 (3): 762-71
  • Non-Patent Document 13 Safford KM, Hicok KC, Safford SD, et al. Biochem. Biophys. Res. Commun. 2002; Jun 7; 294 (2): 371-9
  • Non-Patent Document 14 Tholpady SS, Katz AJ, Ogle RC. Anat. Rec. A Discov. Mol. Cell. Evol. Biol. 2003; May; 272 (1): 398-402
  • Non-Patent Document 15 Kang SK, Lee DH, Bae YC, et al. Exp. Neurol. 2003; 0 ct; 183 (2): 355-66
  • Non-patent document 16 Hiroshi Mizuno J. Nippon Med. Sch. 2003; Oct; 70 (5): 428-31
  • Non-patent document 17 Saf ford KM, Safford SD, Gimble JM, et al. Exp. Neurol. 2004; Jun; 187 (2): 319-28
  • Non-Patent Document 18 Yang LY, Liu XM, Sun B, et al. Chin. Med. J. (Engl) .2 004; Mar; 117 (3): 425—9
  • Non-Patent Document 19 Safford KM, Rice HE. Curr. Drug. Targets. 2005; Feb 6 (1): 57-62
  • Non-Patent Document 20 Fujimura J, Ogawa R, Mizuno H, et al. Biochem. Biophys. Res. Commun. 2005; Jul 22; 333 (1): 116-21
  • Non-Patent Document 21 Strem BM, Hicok KC, Zhu M, et al. Keio J. Med. 2005
  • Non-Patent Document 22 Parker AM, Katz AJ. Expert. Opin. Biol. Ther. 2006 Jun; 6 (6): 567-78
  • an object of the present invention is to meet such a demand. Furthermore, an object of the present invention is to provide a surgical method capable of obtaining a desired treatment effect and a material or a medicine used therefor. An object of the present invention is to induce neural pluripotent cells using abundant fat as a raw material. More specifically, an object is to induce neural crest cells from fat.
  • the present invention provides the following.
  • Fat-derived neural crest cells Fat-derived neural crest cells.
  • the neural crest cell according to item 1 which is derived from an adipose-derived stem cell.
  • the neural crest cell according to item 1 wherein the neural crest cell expresses at least one neural stem cell marker.
  • neural stem cell marker is selected from the group consisting of Nestin, Musashi-1, CD133, notch1, Hesl, Mashl, Neurogenin, Pax6, CD15 and PDGFR.
  • Item 7 The neural crest cell according to item 6, wherein the neural stem cell marker includes Nestin and Musashi-1.
  • the neural crest cell according to item 1 wherein the neural crest cell expresses at least one neural crest cell marker.
  • the neural crest cell marker is selected from the group consisting of CRABP1, AP2, Slug, SoxlO, Snail, Twist, Pax3, Pax7, HNK1, p75NTR, TRP2, Wntl, PO, and tPA.
  • the neural crest cell according to 1.
  • the neural crest cell according to item 1 wherein the neural crest cell expresses Nestin and Musashi-1, and the expression level of Leptin is lower than the expression level of adipose-derived progenitor cells.
  • Item 2 The neural crest cell according to Item 1, wherein the fat is derived from a mammal.
  • Item 13 The neural crest cell according to Item 12, wherein the fat is derived from a human.
  • a method for preparing neural crest cells comprising the following steps:
  • Item 15 The method according to Item 14, wherein the anterior stem cell is an adipose-derived stem or stromal cell.
  • Item 15 The method according to Item 14, wherein the step of obtaining the stem cell comprises collagenase treatment and centrifugation treatment.
  • Item 15 The method according to Item 14, wherein the stem cell is selected from the group consisting of PLA cell and LAF cell force.
  • step B) the adipose-derived progenitor cells are seeded at a density of about 1 ⁇ 10 4 cells Zml to about 1 ⁇ 10 6 cells Zml.
  • Item 19 The method according to Item 18, wherein in the step B), the adipose-derived stem cells are seeded at a density of about 1 ⁇ 10 5 cells Zml.
  • Item 15 The method according to Item 14, wherein in the step B), the adipose-derived stem cells are cultured in a medium containing at least epidermal growth factor (EGF).
  • EGF epidermal growth factor
  • Item 15 The method according to Item 14, wherein in step B), the adipose-derived stem cells are cultured in a medium containing epidermal growth factor, basic fibroblast growth factor, nerve induction factor, penicillin, and streptomycin as auxiliary components. .
  • the nerve-inducing factor is piotin, L-carcin, corticosterone, ethanolamine, D (+)-galactose, dartathione (reduced form), linoleic acid, linolenic acid, progesterone, retulacetate, selenium, 28.
  • the item according to item 21, comprising triodothyronine (T3), DL-a-tocopherol, DL-a-tocopherol acetate, albumin (ushi), catalase, insulin, superoxide dismutase, and transferrin.
  • Item 15 The method according to Item 14, wherein in the step B), a medium used for culturing the stem cells contains glutamic acid and aspartic acid.
  • the medium used for culturing the stem cells is an eagle basic medium (BME), a minimum essential medium (MEM), a Dulbecco's modified Eagle medium (DMEM), a HAMF12 medium, or a mixture thereof.
  • BME eagle basic medium
  • MEM minimum essential medium
  • DMEM Dulbecco's modified Eagle medium
  • HAMF12 a HAMF12 medium
  • Item 15 The method according to Item 14, which is a medium.
  • Item 15 The method according to Item 14, wherein the medium used for culturing the stem cells in Step B) is Dulbecco's Modified Eagle Medium (DMEM) ZF12 (1: 1).
  • DMEM Dulbecco's Modified Eagle Medium
  • step B) includes replacing the medium with a new medium on days 2 to 6.
  • step B) includes replacing the medium with a new medium on the 4th to 5th days.
  • step B) includes subculture on the 6th to the 10th day.
  • Item 15 The method according to Item 14, wherein the fat is derived from a mammal.
  • the adipose-derived stem cells are human-derived
  • the adipose-derived stem cells are seeded at a density of about 1 ⁇ 10 5 cells Zml;
  • the adipose-derived stem cells are cultured in a medium containing epidermal growth factor, basic fibroblast growth factor, nerve induction factor, penicillin and streptomycin as auxiliary components, wherein the nerve induction factor is piotin, L— Carcin, corticosterone, ethanolamine, D (+) galactose, dartathione (reduced form), linoleic acid, linolenic acid, progesterone, retino-acetate, selenium, tolydothyrone (T3), DL—a Tokov Contains erol, DL-a-tocopherol acetate, albumin (usi), catalase, insulin, superoxide dismutase, transferrin, and
  • Item 15 The method according to Item 14, wherein the medium is Dulbecco's Modified Eagle Medium (DMEM) ZF12 (1: 1).
  • DMEM Dulbecco's Modified Eagle Medium
  • Item 15 The method according to Item 14, further comprising the step of confirming whether the stem cell expresses a neural stem cell marker.
  • the neural stem cell marker is selected from the group consisting of Nestin, Musashi-1, CD133, notch 1, Hesl, Mashl, Neurogenin, Pax6, CD15 and PDGFR The method according to 32.
  • Item 15 The method according to Item 14, further comprising the step of confirming whether the stem cells express a neural crest cell marker.
  • Item 15 The method according to Item 14, wherein the cell expresses at least one neural crest cell marker.
  • the neural crest cell marker is selected from the group consisting of CRABP1, AP2, Slug, SoxlO, Snail, Twist, Pax 3, Pax7, HNK1, p75NTR, TRP2, Wntl, PO, and tPA force. .
  • Item 15 The method according to Item 14, further comprising the step of confirming the level of expression of the adipocyte marker by the stem cell.
  • Item 15 The method according to Item 14, wherein the neural crest cell becomes a fat-derived neural crest cell precursor containing neural crest cells and adipose-derived stem cells by the step B).
  • the method according to item 14 further comprising the step of confirming whether the stem cell expresses a neural stem cell marker and a neural crest cell marker, and confirming the level of expression of the adipocyte marker by the stem cell.
  • Item 44 The method according to Item 41, wherein the neural stem cell marker is Nestin and Musashi-1, and the fat cell marker is Leptin.
  • a composition for cell transplantation for the treatment of a nervous system disease, disorder or condition comprising the cell according to item 1.
  • composition according to item 45 wherein the nervous system disease, disorder or condition is a disease, disorder or condition caused by a defect in differentiated cells of the nervous system.
  • composition according to item 45 wherein the fat is derived from an individual having an allogeneic relationship with an individual subject to the nervous system disease, disorder or condition.
  • composition according to item 45 wherein the fat is derived from an individual having a heterogeneous relationship with the subject of the nervous system disease, disorder or condition.
  • composition according to item 45 wherein the fat is derived from an individual having an allogeneic relationship with an individual subject to the nervous system disease, disorder or condition.
  • a method for cell transplantation for the treatment of a nervous system disease, disorder or condition comprising the step of administering the cell according to item 1.
  • Said nervous system disease, disorder or condition is a disease caused by a loss of differentiated cells of the nervous system, 51.
  • the method of item 50, wherein the method is a disorder or condition.
  • the nervous system disease, disorder or condition is a disease, disorder or condition caused by a defect in differentiated cells of the nervous system.
  • An adipose-derived neural crest cell mixture comprising neural crest cells and adipose-derived stem cells.
  • regenerative treatment can be performed using a neural crest cell derived from fat and a neural crest cell derived from fat precursor cell.
  • a neural crest cell can be further prepared by recovering a fat progenitor cell and subjecting the progenitor cell to conditions for isolating the neural crest cell.
  • the present invention also facilitates treatment of nervous system diseases, disorders or conditions by transplanting neural crest cells. These treatments provide a simple and efficient treatment method in regenerative medicine because few side effects are expected and their sources are abundant.
  • neurospheres containing neural crest cells could be provided at a remarkably high rate. Since fat can be used as a raw material, an effect that a large amount of neural crest cell mixture can be provided is also provided.
  • Fig. 1 shows culture in a neurosphere culture medium, Day 5 (Day 5: A), Day 6 (Day 6 (Day 2).
  • Figure 2 shows neural stem cell markers Nestin (A) and Musashi— 1 (B), and The results of quantitative real-time RT-PCR analysis of gene expression of the adipogenic marker Leptin (C) are shown (control: undifferentiated adipose-derived stem cells, sphere: Nyuro sphere). Atsey was conducted in triplicate. Standard error is indicated by an error bar.
  • FIG. 3 shows neural crest-like migration of GFP-transformed adipose-derived stem cells transplanted into mouse embryos cultured in vivo.
  • A A photograph of a mouse embryo that was cultured for 40 hours on day 8 embryos.
  • B, C Fluorescent photographs of embryos. GFP positive-Eurosphere cells were arranged in a row (arrow). This suggests that neurosphere cells are migrating along the second arch. The bar is 500 ⁇ m.
  • cell used herein is defined as the broadest meaning used in the field, and is a structural unit of a tissue of a multicellular organism and is enclosed in a membrane structure that isolates the living body from the outside world. Rarely, an organism with self-regenerative ability and genetic information and its expression mechanism. Any cell can be targeted in the method of the present invention.
  • the number of “cells” used in the present invention can be counted through an optical microscope. When counting through an optical microscope, count by counting the number of nuclei. The tissue is sliced into tissue slices and stained with hematoxylin-eosin (HE) to separate extracellular matrix (eg, elastin or collagen) and cell-derived nuclei.
  • HE hematoxylin-eosin
  • the cells used in the present specification may be naturally occurring cells or artificially modified cells (eg, fusion cells, genetically modified cells, etc.).
  • Examples of cell sources include: It can be a single cell culture, or a cell mixture such as cells from a normally grown transgenic animal embryo, blood, or body tissue (eg, adipose tissue), or a normally grown cell line However, it is not limited to them. Further, such a supply source can be used as a cell as it is.
  • the fat cells (fat eels or adipocytes) used in the present invention and their counterparts may be any organism (for example, larvae, jellyfishes) as long as the following organisms have adipocytes or their counterparts.
  • Cartilaginous fish, teleosts, amphibians, reptiles, birds, mammals, etc. Preferably, mammals (e.g. single pores, marsupials, rodents, wings, wings, carnivores, carnivores, long noses, odd-hoofed animals, even-hoofed animals, rodents, It may be derived from scales, sea cattle, cetaceans, primates, rodents, maggots, etc.). In one embodiment, cells derived from primates (eg, chimpanzee, dihonosa, human), particularly cells derived from humans, are used, but are not limited thereto.
  • stem cell refers to a precursor cell of a differentiated cell (pr ecursor or progenitor), which is monopotency, multipotency. Refers to a cell having totipotency.
  • stem cell can be used interchangeably with “progenitor cell”.
  • Stem cells can be differentiated in response to specific stimuli. Stem cells are usually able to regenerate the tissue when it is damaged.
  • Stem cells as used herein can be, but are not limited to, embryonic stem (ES) cells or tissue stem cells (including tissue stem cells, tissue-specific stem cells or somatic stem cells) or other progenitor cells. Not.
  • an artificially produced cell can also be a stem cell as long as it has the above-mentioned ability.
  • 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 are being used in regenerative medicine. Unlike embryonic stem cells, tissue stem cells are cells that have a relatively limited degree of separation, exist in the tissue, and have an undifferentiated intracellular structure. Tissue stem cells are poor in organelles with a high nuclear Z cytoplasm ratio. Tissue stem cells are generally individuals with pluripotency and slow cell cycle Maintains proliferative capacity over the life of the body. As used herein, stem cells can preferably be embryonic stem cells, although tissue stem cells can also be used depending on the circumstances.
  • stem cell may refer to a tissue containing at least a certain amount of stem cells or progenitor cells. Therefore, the stem cells can be stem cells (eg, adipose-derived stem cells used in the following examples) that have been treated with collagenase and collected adipose tissue strength, but are not limited thereto.
  • stem cells eg, adipose-derived stem cells used in the following examples
  • tissue stem cells When classified according to the site from which the cells are derived, 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.
  • tissue stem cells of the extinct system 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.
  • mesenchymal stem cell refers to a stem cell found in the mesenchyme.
  • the term “mesenchymal stem cell” may be abbreviated herein as “MSC”.
  • mesenchyme is defined by a population of free cells with stellate or irregular projections that fill the gaps between epithelial tissues, which are observed at various stages of development of multicellular animals, and the associated cytoplasm. This refers to the organization that is formed.
  • 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 to cultivate or proliferate bone marrow cells collected from patients, have chondrocytes! /, To differentiate into osteoblasts, and, for example, bones such as alveolar bone, arthropathy, It is used as a reconstruction material for cartilage or joints, and its demand is great. In addition, since mesenchymal stem cells can be separated into blood cells and lymphoid cells, the demand for them is increasing.
  • the term "adipose-derived stem cell” refers to a stem cell obtained by liposuction and also other progenitor cells (eg, peripheral blood or vascular stromal cells (vascular- stromal cell) (stem cell derived from preadipocyte). Adipose-derived stem cells are derived from adipose tissue or from any multipotent precursor cell) or monopotent precursor cell population obtained by a liposuction procedure. means.
  • Adipose-derived vascular stromal cells (adipose—derived interstitial cells, adipose—derived stromal cells), adipose-derived stem cells, adipose-derived progenitor cells, adipose stem cells , Endothelial progenitor cells, hematopoietic stem cells, and the like.
  • stem cell separation methods are known, and are described, for example, in Non-Patent Document 1, Patent Documents 1 and 2. The matters described in these documents are hereby incorporated by reference in particular in the present specification.
  • adipose-derived stem cells refers to all adipose tissue-derived stem cells including adipose tissue-derived stem cells obtained by these known separation methods.
  • progenitor cell includes unipotent undifferentiated cells as well as pluripotent undifferentiated cells.
  • stem cell includes progenitor cells.
  • Aspirated material collected by liposuction is separated into two layers in a suction bottle.
  • the upper layer of aspirate is composed of floating fat (lipoaspimte) and the lower layer is composed of liquid (liquid-aspirate or liposcution-aspirate fluid) forces.
  • PVA processed lipo aspirate cell
  • LAF cells liquid—aspirate
  • eel is a liposcution—aspirate fluid cell)), which is a progenitor cell derived from the liquid part of the aspirate.
  • Adipose-derived stem cells include PLA cells and LAF cells.
  • the term "somatic cell” refers to all cells that are cells other than germ cells, such as eggs and sperm, and that do not deliver the DNA to the next generation. Somatic cells usually have power or loss of limited pluripotency. Somatic cells as used herein may be naturally occurring or genetically modified as long as the intended treatment can be achieved.
  • neural stem cell refers to a cell that can differentiate into the nervous system and has both self-renewal ability and pluripotency. Neural stem cells It can be identified by confirming cell markers, self-renewal ability, and pluripotency of vesicles.
  • the "self-renewal ability" of a neural stem cell refers to the ability to repeat division and generate the same cell population as self).
  • the “self-renewal ability” of a neural stem cell can be confirmed by, for example, the neurosphere method.
  • the neurosphere method is the method originally reported by Reynolds et al., J Neurosci 1992; 12: 4565-4574 and Science 1992; 255: 17 07-1710.
  • the neurosphere method is one of the most frequently used methods for isolating neural stem cells from the embryonic or adult central nervous system.
  • neural stem cells are cultured in a floating state in the presence of epidermal growth factor (E GF) and basic fibroblast growth factor (basic-FGF, b-FGF) as growth factors.
  • E GF epidermal growth factor
  • basic-FGF basic fibroblast growth factor
  • Single cell force-forming the eurosphere, dissociating the formed eurosphere, seeding the dissociated cell in a new medium, and the ability of the dissociated cell to form the eurosphere again
  • a method including a step of confirming whether or not. In this method, a dissociated cell forms a neurosphere again when it forms a eurosphere.
  • pluripotency refers to the nervous system of nerve cells and glial cells (astrocytes, oligodendrocytes, etc.). The ability to divide into multiple cells.
  • the pluripotency of neural stem cells is, for example, that when neurospheres are placed under the condition of dividing them into cells of the nervous system, they enter the nervous system of neurons and glial cells (astrocytes, oligodendrocytes, etc.). If it is determined that the cell is pluripotent.
  • pluripotency refers to having the ability to differentiate into cells of multiple nervous systems, but is not limited thereto.
  • Examples of conditions for separating cells of the nervous system include, for example, a medium for separating the nervous system (for example, DMEMZF 12:49 ml in a nerve induction medium (50 ml), a nerve inducer (for example, B27 (GIBCO)) : Lml, which may contain antibiotics) Powers including, but not limited to, 1 week of culture (culture with half the medium every 2 days).
  • a glial induction medium 50 ml of medium, DMEMZF12: 45 ml, FBS: 5 ml, may contain antibiotics
  • the neurotrophic factor that can be used herein may be any as long as it has an action of assisting or promoting nerve induction.
  • the neuroinductive factor is piotin, L-carthine, corticosterone, ethanolamine, D (+)-galactose, dartathione (reduced form), linoleic acid, linolenic acid, progesterone, retort acetate , Selenium, tolyothyrone (T3), DL-a-tocopherol, DL-a-tocopherol acetate, albumin (usi), catalase, insulin, superoxide dismutase, transferrin.
  • Examples of neural pluripotent cells include neural stem cells, neural crest cells, neural progenitor cells, and the like.
  • Neural stem cells can also be identified using only neural stem cell markers.
  • a "neural stem cell marker” refers to a neural stem cell that assists in identifying a neural stem cell by its localization or expression. .
  • its localization or expression for example, Nestin (NM006617 (t)), Musashi-1 (NM002442 (human)), CD133 (NM0006017 (human)), notchl (NM017617 (human))) Hes l ( NM005524 (human)) ⁇ Mashl (NM004316 (human)) ⁇ Neurogenin (N M006161 (human)), Pax6 (NM001604 (human)), CD15 (NM002033 (human)), PDG FR (NM002609 (human))
  • neural stem cell markers include, for example, Nestin (N M006617 (Hin)), Musashi-1 (NM002442 (Hin))) , CD133 (NM0006017 (human)), notch 1 (NM017617 (human)), Hesl (NM
  • neural stem cell markers can be performed using methods known in the art (eg, RT-PCR, Northern blot, Western blot, immunostaining, FACS, etc.). Here, whether it is expressed or not can be appropriately selected by RT-PCR, Northern blot, Western blot, immunostaining, FACS, etc.
  • “neural crest cell (NCC)” has at least one characteristic selected from the group consisting of a unique migratory ability and the expression of a cell marker of at least one neural crest cell. Cell.
  • neural crest cells are identified by confirming characteristics of both unique migration ability and expression of cell markers of at least one neural crest cell.
  • CRABP1 NM004378 (human)
  • a P2 NM002097 (chicken)
  • Slug NM003068 (chicken)
  • SoxlO NM006941 (chicken)
  • Snail NM005985)
  • Twist NM000474 (chicken)
  • Pax3 NM000438 (chicken)
  • Pax7 NM002584 (human)
  • HNKl NM004854 (human)
  • p75NTR NM0025 07 (human)
  • TRP2 NM006267 (human)
  • Wntl NM005430 (human)
  • PO NM002 723 (human)
  • tPA NM000930 (human)
  • neural stem cell markers are Nestin (NM006617 (Human)), Musashi-1 (NM002442 (Human)), CD133 (NM0006017 (Human)), Notch 1 (NM017617 (Human)), Hesl (NM005524 (Human)) ), Mashl (NM00 4316 (chicken)), Neurogenin (NM006161 (chicken)), Pax6 (NM001604 (chicken)), CD 15 (NM002033 (human)), PDGFR (NM002609 (human)), etc.
  • neural stem cell markers that can be used include Nestin and Musashi-1.
  • Neural crest cells originate from the edge of the neural tube (nerve crest) in the early fetal period and migrate widely in the embryo, and are divided into an extremely wide and specific repertoire (fourth germ layer). ) was identified. Neural crest cells can differentiate into, for example, dorsal root ganglion cells, autonomic ganglion cells, adrenal medulla chromaffin cells, Schwann cells, and outer pigment cells. Neural crest cells are also called neural crest cells. Neural crest cells have self-renewal and pluripotency and are very similar to neural stem cells.
  • the neural crest cell of the present invention may have a characteristic that expression of an adipocyte marker is lower than that of a fat-derived stem cell.
  • the adipocyte marker used may be any as long as it can determine adipocytes, but is preferably not limited to the force with which Leptin (NM000230 (human)) can be used. Accordingly, it is preferable that Leptin expression is lower than that of adipose-derived stem cells.
  • the neural crest cells of the present invention can be characterized by expressing Nestin and Musashi-1 and expressing Leptin, which is an adipocyte marker, lower than the expression level of adipose-derived stem cells.
  • a "neural crest cell marker” is a neural crest cell that has V, and its localization or expression assists in identifying the neural crest cell.
  • its localization or expression eg, CRABP1 (NM004378 (human)), AP2 (N M002097 (human)), Slug (NM003068 (human)), SoxlO (NM006941 (human)), Snail (NM005985 (Human)), Twist (NM000474 (Human)), Pax3 (NM000438 (Human)), Pa x7 (NM002584 (Human)), HNKl (NM004854 (Human)), p75NTR (NM002507 (Human)), TRP2 (NM006267 (human)), Wntl (NM005430 (human)), PO (NM002723 (human);), tPA (NM000930 (human)), which can be identified as neural crest cells.
  • CRABP1 NM004378 (human)
  • cell markers for crest cells include CRABP1 (NM004 378 (Hin)), AP2 (NM002097 (Hin)), Slug (NM003068 (Hin)), SoxlO (NM0 06941 (Hin)), Snail ( NM005985 (chicken)), Twist (NM000474 (chicken)), Pax3 (N M000438 (chicken)), Pax7 (NM002584 (chicken)), HNKl (NM004854 (chicken)), p75 NTR (NM002507 (human) )), TR P2 (NM006267 (human)), Wntl (NM005430 (human)), PO (NM002723 (human)), tPA (NM000930 (human)) and the like can be mentioned, but not limited thereto.
  • CRABP1 NM004 378 (Hin)
  • AP2 NM002097 (Hin)
  • Slug NM003068 (Hin)
  • Determination by these neural crest cell marker, neural stem cell marker or adipocyte marker is a method known in the art (for example, RT-PCR, Northern blot, Western blot, immunostaining, FACS, etc.) Can be used. Here, whether it is expressed or not can be appropriately selected by RT-PCR, Northern blot, Western blot, immunostaining, FACS and the like.
  • Leptin NM000230 Han
  • embryo or “embryonic cell” refers to the state of an early era of development in a multicellular organism.
  • the “head region” of the embryo refers to the region that migrates to the hindbrain, in particular, the second arch, that is, around Lombomea 3 and 4.
  • the term “divided cell” refers to a cell with a special function and morphology (eg, muscle cell, nerve cell, etc.), and unlike stem cells, There is little or no performance.
  • Differentiated cells include, for example, epidermal cells, spleen Stromal cells, knee duct cells, hepatocytes, blood cells, cardiomyocytes, skeletal muscle cells, osteoblasts, skeletal myoblasts, neurons, vascular endothelial cells, pigment cells, smooth muscle cells, fat cells, bone cells, soft Examples include bone cells.
  • sperm cells may be in the form of a population or tissue! /.
  • the stem cell origin can be classified into ectoderm, mesoderm and endoderm.
  • Stem cells derived from ectoderm are mainly present in the brain and include neural stem cells.
  • Stem cells derived from mesoderm are mainly present in bone marrow, and include vascular stem cells and their differentiated cells, hematopoietic stem cells and their differentiated cells, and mesenchymal stem cells and their differentiation cells.
  • Endoderm-derived stem cells exist mainly in organs, and include hepatic stem cells and their differentiation cells, spleen stem cells and their differentiation cells.
  • somatic cells may be derived from any germ layer.
  • mesenchymal cells can be used as the somatic cells.
  • adipocyte refers to interstitial tissues, or forms adipose tissue as a population along a capillary run as one of the loose connective tissues.
  • a cell containing a large amount of fat include yellow fat cells and brown fat cells, and any of these can be used equivalently in this specification.
  • Intracellular fat can be easily detected by Sudan I or osmium tetroxide.
  • the term "desired site” refers to any site in a subject for which treatment is desired. In the present invention, it is understood that such a desired site can be selected from any organ or tissue in the subject.
  • tissue refers to a population of cells having substantially the same function and Z or morphology in a multicellular organism. “Organization” is usually
  • stem cell of the present invention is used.
  • tissue When regenerating a tissue by V, cell populations having two or more different origins can constitute one tissue.
  • tissue constitutes part of an organ. Animal tissues are classified into epithelial tissues, connective tissues, muscle tissues, and nerve tissues based on morphological, functional, or developmental basis. In plant tissue, meristem and permanent tissue depend on the stage of development of the cells that make up the tissue. Various classifications are made, such as dividing into single tissue and composite tissue according to the type of constituent cells. As used herein, any tissue can be intended as a subject to be treated.
  • an organ when an organ is a target, such an organ may be any organ, and the tissue or cell targeted by the present invention may be derived from any organ or organ of an organism.
  • the term “organ” refers to a structure in which a function of an individual organism is localized and operates in a specific part of the individual, and that part is morphologically independent. .
  • an organ In multicellular organisms (eg animals, plants), an organ consists of several tissues with a specific spatial arrangement, and each tissue also has a number of cellular forces. Such organs include those 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, spleen, brain, peripheral limbs, retina, and the like. Is not limited to them.
  • any organ may be a target, but preferably a mesenchymal tissue (for example, fat, bone, ligament, etc.) may be a target. It is not limited to that.
  • condition for inducing neural crest cells refers to time, medium, temperature, humidity, or a combination thereof, etc. that allow neural crest cells to be induced.
  • a condition seems to maintain adipose-derived stem cells or neural stem cells alone. It is understood that this can overlap with other conditions. Therefore, such conditions can be changed as appropriate. Also, once such favorable conditions have been established, such conditions can subsequently be used to induce similar neural crest cells.
  • sufficient conditions for inducing such neural crest cells can be used in vitro, in vivo, or ex vivo.
  • the conditions in the transplanted body part are applied as they are.
  • the stem cells and the differentiated cells may be immediately transplanted and mixed and cultured in an in-vitro environment in an in vivo environment. Autotransplantation can also be called ex vivo transplantation.
  • a “condition for inducing neural crest cells” the trunk cells that have been separated from the conventional tissue strength of the nervous system are used.
  • a condition known as a condition for dividing the cell into -eurosphere can be mentioned. It has been found for the first time in the present invention that such “conditions for separating the stem cells of the nervous system into the eurospheres” are effective as conditions for inducing adipose-derived stem cells into neural crest cells. Effect.
  • condition for separating the stem cells separated from the nervous system into -eurospheres are the conditions described in the examples (cultivation in "neurosphere culture medium”). In addition to the above, those having the following conditions can be mentioned.
  • epidermal growth factor e.g. epidermal growth factor, basic fibroblast growth factor, B27 supplement (GIBCO)>
  • BME Eagle Basal Medium
  • MEM Minimum Essential Medium
  • DMEM Dulbecco's Modified Eagle Medium
  • HAMF12 medium there is! /, or their mixed medium
  • the cell density condition is the condition of Kang KS et al.
  • DMEMZF12 (1: 1) is used, and it has been found that it is more advantageous to include glutamic acid, aspartic acid, and iron sulfate.
  • the present invention includes an excitatory amino acid, glutamate or aspartate, so the environment is completely different from the Neurobasal medium TM used to maintain differentiated neurons. It's different.
  • excitable amino acids is the Although it is thought to have an adverse effect such as wasting, on the other hand, it is considered to have a positive effect on the induction from fat into pluripotent cells of the nervous system (for example, neural crest cells).
  • in vivo refers to the interior of an organism. In a particular context, “in vivo” refers to the location (eg, desired site! / Where desired) where the tissue or organ of interest is to be placed.
  • in vitro refers to the removal or release of a portion of a living organism "ex vivo" (eg, in a test tube) for various research purposes. Let's see the state. A term that contrasts with in vivo.
  • ex vivo refers to the extraction of a target cell for gene transfer from a subject and the introduction of a therapeutic gene into the cell in vitro, and then again. When returning to the same subject, a series of operations is called ex vivo.
  • autograft tissue, cell, organ, etc.
  • tissue, cell, organ, etc. refers to a graft (tissue, cell, organ, etc.) from another genetically identical individual (eg, monozygotic twin) in a broad sense.
  • self expression is used interchangeably when derived from a subject.
  • the expression not derived from a subject has the same meaning as not being self (ie, non-self).
  • allogeneic grafts, tissues, cells, organs, etc.
  • allogeneic ones grafts, tissues, cells, organs, etc.
  • immune response in the transplanted individual (recipient).
  • examples of such include, but are not limited to, those derived from the parent (grafts, tissues, cells, organs, etc.).
  • heterologous refers to a transplant (graft, tissue, cell, organ, etc.) transplanted from a heterogeneous individual.
  • a human-derived one graft, tissue, cell, organ, etc.
  • tissue, cell, organ, etc. graft, tissue, cell, organ, etc.
  • the conditions for isolating such neural stem cells are independently about 1 X 10 4 cells. Zml to about 1 X 10 6 cells Zml. Density (eg, about 1 X 10 5 cells Zml), addition of growth factors (eg, EGF, bFGF), nerve inducers (eg, piotin, L-carcin, corticosterone, ethanolamine, D ( +) —Galactose, dartathione (reduced form), linolenoreic acid, linolenic acid, progesterone, retinoreacetate, selenium, triodothyronine (T3), DL—a—tocopherol, DL-a—tocopherol acetate, Addition of albumin (us), catalase, insulin, superoxide dismutase, transferrin), penicillin and streptomycin [], penicillin!
  • growth factors eg, EGF, bFGF
  • nerve inducers
  • the trophic factor may be any as long as it has the effect of assisting or promoting nerve induction
  • the nerve inducer may be piotin, L-carcin, corti Costerone, ethanolamine, D (+) galactose, dartathione (reduced form), linoleic acid, linolenic acid, progesterone, retino-acetate, selenium, tolydothyrone (T3), DL—a Tocopheronole, DL- a Tocopherol acetate, albumin (usi), catalase, insulin, superoxide dismutase, transferrin
  • the nerve inducer may be B27 supplement (GIBCO).
  • the above conditions may be employed to maintain neural stem cells, neural crest cells, and the like, but are not limited thereto.
  • any culture medium containing a factor that promotes induction from adipose-derived stem cells to neural crest cells can be used.
  • Incubation in a medium supplemented with lOOUZml penicillin and 100 gZ ml streptomycin includes, but is not limited to, 37 ° C, oxygen 20%, carbon dioxide 5% and 80% or more. Can be used in humidity.
  • the term "factor that promotes induction into neural crest cells” or “nerve crest cell induction promoting factor” refers to a factor that promotes induction into neural crest cells (eg, As long as it is a chemical substance, temperature, etc.).
  • factors include various environmental factors. Examples of such factors include temperature, humidity, pH, salt concentration, nutrition, metal, gas, organic solvent, pressure, chemical Examples include, but are not limited to, substances (eg, steroids, antibiotics, etc.) or any combination thereof.
  • EGF epidermal growth factor
  • BMP-4 NM001202 (human)
  • Noelinl NM01 4279 (human)
  • condition for separating neuronal tissue from separated stem cells into neurospheres are effective as conditions for inducing adipose-derived stem cells to neural crest cells.
  • the “factor that promotes induction into neural crest cells” or “factor that promotes neural crest cell induction” is essentially based on “conditions for differentiating stem cells from the nervous system to differentiate into eurospheres”. Can be determined.
  • the term "adipose-derived neural crest cell mixture” refers to a mixture comprising the neural crest cells of the present invention and adipose stem cells.
  • the mixture of the present invention may be in any form as long as it contains neural crest cells and fat-derived hepatocytes.
  • the mixture of the present invention may further comprise neural stem cells.
  • the term "transplant” refers to the transfer of a cell, mixture, composition, medicament, etc. of the present invention into the body alone or in combination with other therapeutic agents.
  • the present invention relates to the following method and mode of introduction to the treatment site (for example, bone). And the amount introduced can be used: a method of directly injecting the pharmaceutical agent of the present invention into an injured site, suturing after insertion, and inserting.
  • the combination of adipose-derived stem cells of the invention and differentiated cells can be administered, for example, either 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 “self” or “self” refers to an individual, or an individual derived from the individual or a part thereof (eg, cell, thread and fabric, Organ). As used herein, the term “self” or “self” can broadly include grafts from other genetically identical individuals (eg, identical twins).
  • homologous refers to another individual force that is homogenous but genetically different (eg, cell, Tissue, organ, etc.). Because allogeneic individuals are genetically different, allogeneic individuals can elicit an immune response in the transplanted individual (recipient). Examples of such cells include, but are not limited to, parent-derived cells.
  • heterologous refers to something that is transplanted from a heterologous individual.
  • a transplant from a pig is a xenotransplant.
  • the term "recipient” refers to an individual that receives cells to be transplanted and the like, and is also referred to as "host”.
  • an individual that provides cells to be transplanted is called a “donor”.
  • the recipient and donor can be the same or different.
  • the cells used in the present invention may be derived from the same line (derived from the self (autologous)), from the same lineage (derived from another individual (other house)), or from a different species. Self-derived cells are preferred because of possible rejection, but may be from allogeneic if rejection is not a problem [0073]
  • "nervous disease, disorder or condition” refers to any disease, disorder or abnormal condition involving cells of the nervous system.
  • diagnosis, prevention, treatment or prognostic effective amount is medically effective in diagnosis, prevention, treatment (or therapy) or prognosis, respectively. This is the amount that can be recognized. Such an amount can be determined by a person skilled in the art using techniques well known in the art, taking into account various parameters.
  • the animal targeted by the present invention is an animal having adipocytes, any animal (for example, metal eels, shark eels, cartilaginous fish, teleosts, amphibians, reptiles, birds, mammals, etc.) ).
  • animals are mammals (eg, single pores, marsupials, rodents, wings, wings, carnivores, carnivores, long noses, odd hoofed animals, even hoofs, even Hoofs, rodents, scales, sea cattle, cetaceans, primates, rodents, maggots).
  • Exemplary subjects include, but are not limited to, animals such as rabbits, pigs, horses, chickens, cats and dogs. More preferably, a primate (for example, a chimpanzee, a second monkey, a human, etc.) is used. Most preferably, a human is used.
  • such medicament may further comprise a pharmaceutically acceptable carrier and the like.
  • a pharmaceutically acceptable carrier known in the art can be used.
  • Suitable formulation materials or pharmaceutically acceptable carriers include antioxidants, preservatives, colorants, flavors, diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffering agents. Including, but not limited to, delivery vehicles and Z 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 at least one physiologically acceptable carrier, excipient or diluent.
  • a suitable vehicle may be an injection solution, a physiological solution, or an artificial cerebrospinal fluid that is supplemented with other substances commonly used in compositions for parenteral delivery. Is possible.
  • Acceptable carriers, excipients or stabilizers used herein are preferably non-toxic to the recipient, and preferably the dosage and concentration used.
  • Inactive preferably, for example, phosphate, citrate, or other organic acids; ascorbic acid, OC tocopherol; low molecular weight polypeptides; proteins (eg, serum albumin, gelatin or immunoglobulins) Hydrophilic polymers (eg polyvinylpyrrolidone); amino acids (eg glycine, glutamine, asparagine, arginine or lysine); monosaccharides, disaccharides and other carbohydrates (glucose, mannose or dextrin); chelating agents ( Eg, EDTA); sugar alcohols (eg, mantol or sorbitol); salt-forming counterions (eg, sodium); and Z or non-ionic surfactants (eg, Tween, pluronic or Polyethylene glycol (PEG)) I can get lost.
  • Inactive preferably, for example
  • Exemplary 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 a Tris buffer having a pH of about 7.0 to 8.5 or an acetate buffer having a pH of about 4.0 to 5.5, which further includes sorbitol or a suitable replacement thereof. Can contain things.
  • a general method for preparing the pharmaceutical composition of the present invention is shown below. It should be noted that veterinary pharmaceutical compositions, quasi-drugs, marine pharmaceutical compositions, food compositions, baboon cosmetic compositions, and the like can also be produced by known preparation methods.
  • the cells of the present invention are blended with a pharmaceutically acceptable carrier as needed, and are administered parenterally, for example, as liquid preparations such as injections, suspensions, solutions and sprays. be able to.
  • pharmaceutically acceptable carriers include excipients, lubricants, binders, disintegrants, disintegration inhibitors, absorption enhancers, absorbents, wetting agents, solvents, solubilizers, suspending agents. , Isotonic agents, buffering agents, soothing agents and the like.
  • formulation additives such as preservatives, antioxidants, colorants, sweeteners and the like can be used as necessary.
  • substances other than the polynucleotide, polypeptide and the like of the present invention can be added to the composition of the present invention.
  • parenteral routes of administration include, but are not limited to, intravenous, intramuscular, subcutaneous, intradermal, mucosal, intrarectal, intravaginal, topical, and dermal.
  • the medicament used in the present invention does not contain pyrogens It can be in the form of a pharmaceutically acceptable aqueous solution. It is within the skill of the artisan to consider pH, isotonicity, stability, etc. for the preparation of such pharmaceutically acceptable compositions.
  • Preferable examples of the solvent in the liquid preparation include an injection solution, alcohol, propylene dallicol, macrogol, sesame oil, corn oil and the like.
  • solubilizer in the liquid preparation include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, and ken Examples include, but are not limited to, sodium acid.
  • suspending agent in the liquid preparation include, for example, stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, salted benzethonium, glyceryl monostearate, etc.
  • Surfactants such as polyvinyl alcohol, polypyrrole pyrrolidone, sodium carboxymethylcellulose, methinoresenorelose, hydroxymethinoresenorelose, hydroxyethinoresenorelose, hydroxyethylcellulose, hydroxypropylcellulose, etc. Functional polymers, etc.
  • Preferable examples of the isotonic agent in the liquid preparation include, but are not limited to, sodium chloride salt, glycerin, D-manntol and the like.
  • buffering agent in the liquid preparation include, but are not limited to, phosphate, acetate, carbonate, citrate and the like.
  • the soothing agent in the liquid preparation include, but are not limited to, benzyl alcohol, benzalkonium chloride and pro-in hydrochloride.
  • Preferable examples of the preservative in the liquid preparation include, but are not limited to, paraoxybenzoates, chlorobutanol, benzyl alcohol, 2-phenylethyl alcohol, dehydroacetic acid, sorbic acid and the like. .
  • antioxidant in the liquid preparation include, but are not limited to, sulfite, ascorbic acid, a tocopherol and cysteine.
  • solutions and suspensions are sterilized and blood or other It is preferably isotonic with the solvent at the injection site for these purposes.
  • these are sterilized by filtration using a bacteria retention filter or the like, blending with a bactericide, or irradiation.
  • the pharmaceutical composition of the present invention may contain a coloring agent, 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, the target disease (type, severity, etc.), the patient's age, weight, sex, medical history, cell morphology or type, etc. In view of this, 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 those skilled in the art. Examples of the frequency include administration once a few months every day (for example, once a week, once a month). It is preferable to administer once a week-once a month with the progress of the test.
  • the amount to be administered can be determined by estimating the amount required by the site to be treated.
  • the term "instruction” refers to a method for administering or diagnosing the medicament of the present invention, a person who administers the medicament of the present invention, or the like. It is written for a person, a person to be diagnosed, or a person to be diagnosed (eg, doctor, patient, etc.). This instruction describes a word indicating an appropriate method for administering the diagnostic agent or medicine of the present invention. These instructions are 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). It will be clearly stated that it has been approved. Instructions are so-called package inserts, and are not normally limited to paper-delivered forces, such as electronic media (eg, home pages (websites) provided on the Internet, emails, etc. Etc.) can also be provided.
  • the end of treatment according to the method of the present invention may be determined by the results of standard laboratory tests using commercially available accessories or instrumentation or diseases associated with the intended treatment (eg, For example, it can be supported by the disappearance of clinical symptoms characteristic of bone disease, heart disease, nerve disease, etc., or recovery of the cosmetic state (eg, visual recovery, etc.). Treatment can be resumed by a recurrence of a disease (eg, neurological disease) associated with a lack of differentiated cells or a cosmetic condition injury.
  • a disease eg, neurological disease
  • the present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more ingredients of a pharmaceutical composition.
  • a notice of the form prescribed by the government that regulates the manufacture, use or sale of a medicinal product or biological product may optionally be attached to such containers, and this notice may be made, used or administered for human administration. Represents approval by a government agency regarding sales.
  • the kit can include an infusion device.
  • Toxicity studies such as cells of the present invention can be performed using a model.
  • toxicity studies can be performed in appropriate animal models such as: (1) compounds are administered to mice (untreated control mice should also be used); (2) each One mouse in the treatment group also obtains blood samples periodically via the tail vein; and (3) The samples are analyzed for cells of the nervous system and the like. Comparison of the results for each dosing regimen with controls shows whether toxicity is present.
  • the neural crest cells of the present invention can be used for various purposes, for example, treatment of the nervous system.
  • forces including treatment of any disease, disorder or abnormal condition involving cells of the nervous system are not limited to these.
  • the present invention provides a method for preparing neural crest cells.
  • neural crest cells can be provided in a certain amount or more.
  • This method comprises the steps of A) obtaining stem cells from fat; and B) subjecting the stem cells to conditions for inducing neural crest cells.
  • stem cells derived from adipose are disclosed in International Publication No. OOZ53795, No. 03Z 022988, No. 01Z62901, Zuk, PA et al., Tissue Engineering, Vol. 7, 211-228, 2001, And Zuk, PA, et al., Molecular Biology of the Cell Vol., 13, 4279-4295, 2002, etc., or modifications thereof, and the fat portion of aspirate (lipoaspirate) by liposuction Force can also be separated. Specifically, for example, (1) suctioned fat is thoroughly washed with physiological saline using a 1-liter separatory funnel; (2) suction fat is in the upper layer, and physiological saline is in the lower layer.
  • the above sample can be filtered by suction using a 100 m mesh mesh; and (8) The resulting filtrate can be separated by centrifuging at 1200 g for 5 minutes.
  • adipose-derived stem cells can be isolated from, for example, a liquid part (liquid aspirate) of aspirate by liposuction as follows: (1) The liquid part of aspirate by liposuction is prepared. (2) The liquid portion is centrifuged to obtain a cell fraction; (3) The cell fraction is subjected to density gradient centrifugation, and the cell separation is performed based on specific gravity. And (4) the cells are recovered from a cell layer having a specific gravity lower than that of red blood cells.
  • the liquid portion of the aspirate can be prepared using saline or Ringer's infusion. Centrifugation can be performed at a speed of about 800 Xg or less, or about 400 Xg or more.
  • Density gradient centrifugation is performed at a rate of about 370 X g to l, 100 X g. Density gradient centrifugation is carried out using a solvent having a specific gravity (20 ° C.) of about 1.0 76 gZml to 1.07 gZml.
  • the solvent used in density gradient centrifugation can be Ficoll TM, Percoll TM or sucrose.
  • the specific gravity of the recovered cell layer can range from about 1.050 to 1.075.
  • the cell layer can be collected using a pipette.
  • the adipose-derived stem cells used in the present invention are selected from the group consisting of CD13, CD29, CD34, CD36, CD44, CD49d, CD54, CD58, CD71, CD73, CD90, CD105, CD106, CD 151, and SH3. Can express at least one (preferably two, three,... N) proteins. More preferably, the adipose-derived stem cells used in the present invention express all of CD13, CD29, CD34, CD36, CD44, CD49d, CD54, CD58, CD71, CD73, CD90, CD105, CD106, CD151, and SH3. It is possible.
  • CD antigens can be determined using a method known in the art (for example, an immunological technique using an antibody). Here, whether it is expressed or not can be appropriately selected by an immunological method or the like.
  • the adipose-derived stem cells used in the present invention are CD3, CD4, CD14, CD15, CD16, CD19, CD33, CD38, CD56, CD61, CD62e, CD62p, CD69, CD104, CD135, and CD144. Does not express at least one (especially CD56).
  • a CD antigen is a marker for differentiated cells, and since it can be an indicator of stem cells that it is not expressed, it is not limited thereto.
  • the adipose-derived stem cells used in the present invention are CD3, CD4, CD14, CD15, CD16, CD19, CD33, CD38, CD56, CD61, CD62e, CD62p, CD69, CD104, CD1 35 , And CD144 !, no deviations expressed! It may be advantageous to be a cell.
  • cells that express CD49d and do not express CD56 may be conveniently selected as adipose-derived stem cells.
  • the adipose-derived stem cells used in the present invention can be derived from aspirated fat.
  • aspirated fat Conventionally, sucked fat has been discarded, but in the present invention, neural stem cells (preferably neural crest cells) can be isolated from fat-derived stem cells.
  • neural stem cells preferably neural crest cells
  • aspiration fat may be, for example, the liquid part or the fat part of the aspirate from liposuction! /.
  • isolated ones may be used. Force-purified ones or completely purified ones may be used.
  • the adipose stem cells used in the present invention also have a portion of fat (for example, fat in the abdomen, chest, hips, thigh, upper arm, face, etc.) that is unnecessary for modern humans. Can be prepared from adipocytes.
  • the abdomen, buttocks, etc. are preferred. This is because the abdomen, buttocks, etc. are sites where fat tends to accumulate and are often desired to be removed.
  • a neural cell is prepared by subjecting a stem cell obtained from fat to a condition known as a condition for separating a stem cell separated from the tissue power of a conventional nervous system into -eurosphere.
  • Such conditions include, independently, a density of about 1 ⁇ 10 4 cells / ml to about 1 ⁇ 10 6 cells / ml (eg, about 1 ⁇ 10 5 cells / ml), a growth factor (eg, EGF) BFGF), nerve inducer (eg, piotin, L-carcin, corticosterone, ethanolamine, D (+)-galatose, glutathione (reduced form), linoleic acid, linolenic acid, progesterone, (Including tuluacetate, selenium, tolydothyrone (T3), DL-a-tocopherol, DL-a-tocopherol acetate, albumin, catalase, insulin,
  • T3 a density of about 1
  • the power to use uncoated dishes can also be selected.
  • the neurotrophic factor that can be used herein may be any as long as it has an effect of assisting or promoting nerve induction.
  • the nerve-inducing factor is piotin, L-carthine, corticosterone, ethanolamine, D (+)-galactose, dartathione (reduced form), linoleic acid, linolenic acid, progesterone, Contains leturyl acetate, selenium, triodothyronine (T3), DL-a-tocopherol, DL-a-tocopherol acetate, albumin (usi), catalase, insulin, superoxide dismutase, transferrin.
  • the neural inducer can be B27 supplement (GIBCO).
  • neural stem cells eg, neural crest cells
  • neural stem cells eg, neural crest cells
  • any culture medium containing a factor that promotes induction from adipose-derived stem cells to neural crest cells can be used.
  • examples of such medium include DMEM / HAMF12 (1: 1) and human recombinant EGF (20 ng Zml, PeproTech, human thread and basic-FGF (20 ng / ml, Kaken Pharmaceutical, Japan), 2% B27 supplement.
  • EGF important for induction.
  • the method may further comprise the step of ascertaining whether the stem cell is expressing a neural crest cell marker, and optionally a neural stem cell marker.
  • the neural crest cells express at least one neural crest cell marker.
  • the neural crest cell may further express at least one neural stem cell marker.
  • cell markers of neural crest cells include, for example, CRABP1 (NM004378 (human)), AP2 (NM00 2097 (Human)), Slug (NM003068 (Human)), SoxlO (NM006941 (Human)), Snail (NM 005985 (Human)), Twist (NM000474 (Human)), Pax3 (NM000438 (Human)), Pax7 (N M002584 (chicken)), HNKl (NM004854 (chicken)), p75NTR (NM002507 (chicken)), TRP2 (NM006267 (human)), Wntl (NM005430 (human)), PO (NM002723 (human)) ), TPA (NM000930 (human)) and the like, but are not limited thereto.
  • CRABP1 NM004378 (human)
  • AP2 NM00 2097 (Human)
  • Slug NM003068 (Human)
  • SoxlO
  • neural stem cell markers include Nestin (NM006617 (human)), Musashi—1 (NM002 442 (blue)), CD133 (NM0006017 (blue)), notchl (NM017617 (blue)), Hesl (NM005524 ( ⁇ )), Mashl (NM004316 ( ⁇ )), Neurogenin (NM006161 ( ⁇ )), Pax6 (NM001604 ( ⁇ )), CD15 (NM002033 ( ⁇ )), PDGFR (NM0026 09 (human)) Although it is mentioned, it is not limited to these.
  • neural stem cell markers include Nestin and Musashi-l.
  • the method may further comprise the step of confirming the level of expression of the adipocyte marker by the stem cell.
  • the adipocyte marker used in the present method may be any as long as it can determine adipocytes, but preferably Leptin (NM000230 (human)) can be used. It is not limited to. Thus, confirmation of the level of adipocyte markers can be confirmed by a decrease in Leptin (NM000230 (human)).
  • the stem cells that have also obtained fat power become fat-derived neural crest cell precursors including neural crest cells and adipose-derived stem cells by the step of subjecting them to conditions for inducing neural crest cells. Can do.
  • the method wherein the stem cell expresses a neural crest cell marker or neural stem cell marker! may further comprise the step of confirming whether or not the stem cell is confirmed, and the step of confirming the level of expression of the fat cell marker by the stem cell.
  • cell markers of neural crest cells include, for example, CRABP1 (NM004378 (Hin)), AP2 (NM002097 (Hin)), Slug (NM003068 (Hin)), SoxlO (NM0069 41 (Hin) )), Snail (NM005985 (Hui)), Twist (NM000474 (H2)), Pax3 (NM00 0438 (H2)), Pax7 (NM002584 (H2)), HNKl (NM004854 (H2)), p75NTR (NM002507 (human)), TRP2 (NM006267 (human)), Wntl (NM005430 (human)), P0 (NM002723 (human)), tPA (NM000930 (human)), and the like.
  • CRABP1 NM004378 (Hin)
  • AP2 NM002097 (Hin)
  • Slug NM003068 (Hin)
  • SoxlO NM0069 41 (Hin)
  • neural stem cell markers include Nestin (NM006617 (tl)), Mu sashi-1 (NM002442 (chicken)), CD133 (NM0006017 (chicken)), notchl (NM017 617 (chicken)), Hesl (NM005524 ( ⁇ )), Mashl (NM004316 (()), Neurogenin (NM006161 ( ⁇ )), Pax6 (NM001604 ( ⁇ )), CD15 (NM002033 ( ⁇ )), PDGFR (NM002609 (human)) But is not limited to these!
  • neural stem cell markers include Nestin and Musashi-l.
  • the neural crest cells of the present invention may be characterized by expressing Nestin and Musashi-1 and expressing Leptin, which is an adipocyte marker, lower than the expression level of adipose-derived stem cells.
  • the determination using these neural crest cell marker, neural stem cell marker, or adipocyte marker is performed using a method known in the art (eg, RT-PCR, Northern blot, Western blot, immunostaining, FACS, etc.). Can be done. Here, whether it is expressed or not can be appropriately selected by RT-PCR, Northern blot, Western blot, immunostaining, FACS and the like.
  • a method known in the art eg, RT-PCR, Northern blot, Western blot, immunostaining, FACS, etc.
  • the method may further comprise the step of determining whether the stem cell has the ability to migrate. This migratory ability can be confirmed by transplanting into the head region of the embryonic cell and then observing that it migrates to the frontal nasal protuberance, maxillary protuberance, and arch mesenchyme.
  • a neural crest cell derived from fat is provided.
  • Such neural crest cells can be obtained by the above-described method of the present invention.
  • the fat can be a fat-derived stem cell, but it will be understood that the adipose tissue itself may be used.
  • the neural crest cells of the present invention may also have migration ability. This migration ability can be confirmed by transplanting the cells of interest into the head region of the embryo and then observing that they migrate to the frontal nasal protuberance, maxillary protuberance, and arch mesenchyme.
  • the neural crest cells of the present invention also have neural crest cell markers (for example, CRABP1 (NM004 378 (Hin)), AP2 (NM002097 (Hin)), Slug (NM003068 (Hin)), SoxlO (NM0).
  • CRABP1 NM004 378 (Hin)
  • AP2 NM002097 (Hin)
  • Slug NM003068 (Hin)
  • SoxlO NM0
  • the neural crest cells of the present invention further comprise neural stem cell markers (for example, Nestin (NM0066 17 (Hin)), Musashi-1 (NM002442 (Hin)), CD133 (NM0006017 (Hin)), not chl (NM017617 (Human)), Hesl (NM005524 (Human)), Mashl (NM004316 (Human)), Neurogenin (NM006161 (Human)), Pax6 (NM001604 (Human)), CD15 (NM00 2033 (Human)) PDGFR (NM002609 (human)) can be expressed, preferably the neural crest cells of the present invention can be identified by identifying cells that express Nestin and Musashi-1.
  • neural stem cell markers for example, Nestin (NM0066 17 (Hin)), Musashi-1 (NM002442 (Hin)), CD133 (NM0006017 (Hin)), not chl (NM017617 (Human)), Hesl (NM005524 (Human)), Mashl (
  • the present invention it is confirmed whether or not it has at least one characteristic selected from the group consisting of unique migration ability and expression of a cell marker of at least one neural crest cell. It can be identified as a neural crest cell derived from fat. Preferably, neural crest cells of the present invention are identified by confirming both the unique migration ability and the expression of cell markers of at least one neural crest cell. This unique migratory ability can be confirmed by transplanting into the embryonic head region and then observing that it migrates to the frontal nasal protuberance, maxillary protuberance, and meridian mesenchyme.
  • Leptin NM000230 (human) expression is lower than that of a fat-derived stem cell.
  • the cell of the present invention is characterized in that it expresses Nestin and Musashi-1, and the expression level of Leptin is lower than that of an adipose-derived stem cell.
  • the present invention provides a fat-derived neural crest cell mixture comprising neural crest cells and adipose-derived stem cells.
  • the neural crest cells contained in the fat-derived neural crest cell mixture of the present invention are in any form as described in the “adipose-derived neural crest cells” and “method for preparing neural crest cells” in the present specification. possible.
  • the adipose-derived stem cells contained in the adipose-derived neural crest cell mixture of the present invention can be in any form as described in “Adipose-derived stem cells” in the present specification.
  • the mixture of the present invention Any form is acceptable as long as it includes vesicles and adipose-derived hepatocytes.
  • the mixture of the present invention may also contain neural stem cells.
  • the present invention provides a composition for cell transplantation for the treatment of nervous system diseases, disorders or conditions, comprising adipose-derived or adipose-derived stem cell-derived neural crest cell.
  • the nervous system disease, disorder or condition can be, but is not limited to, a disease, disorder or condition resulting from, for example, a loss of differentiated cells of the nervous system.
  • the composition can be used for any purpose as long as it is intended to treat or prevent a nervous system disease, disorder or condition.
  • the neural crest cells in the composition used may be in any form as described herein in “Fat-derived neural crest cells” and “Methods for preparing neural crest cells”. .
  • the neural crest cells can be independently xenogeneic, allogeneic or syngeneic with respect to the host to be transplanted. Preferably, they are allogeneic or syngeneic, more preferably syngeneic, but not limited thereto. Without being bound by theory, it is possible to suppress immune rejection if it is syngeneic. However, if rejection is predicted, a step of avoiding rejection may be further included. Procedures to avoid rejection are well known in the art, for example, from the New Surgery System, Section 12, Heart / Lung Transplantation from technical and ethical arrangements to implementation (Revised 3rd Edition), See Nakayama Shoten.
  • Immunosuppressants that prevent rejection include, for example, “Cyclosporine” (Sunday Miyun Z Neoral), “Tacrolimus” (Prograf), “Azathioprine” (Imlan), “Steroid Hormone” (predonin, methylpredonin) ), And “T-cell antibodies” ( ⁇ KT3, ATG, etc.), and the method used in many centers around the world as a preventive immunosuppressive therapy is a combination of three drugs “cyclosporine, azathioprine and steroid hormones”. is there.
  • the immunosuppressive agent is desirably administered at the same time as the agent of the present invention, but it is not absolutely necessary. Therefore, as long as the immunosuppressive effect is achieved, the immunosuppressive agent can be administered before or after the regeneration therapy / treatment of the present invention.
  • Such a composition may be provided as a medicament.
  • Such medicines are used for nervous system diseases, disorders It is used to treat or prevent a harm or condition (eg, a disease, disorder or condition resulting from a loss of neuronal dividing cells).
  • the medicament of the present invention may contain a pharmaceutically acceptable carrier in addition to such a composition.
  • a carrier e.g, a pharmaceutically acceptable carrier
  • any carrier described herein can be selected and used by one of ordinary skill in the art.
  • the invention provides a method for cell transplantation for the treatment of a nervous system disease, disorder or condition (eg, a disease, disorder or condition resulting from a loss of differentiated cells of the nervous system). To do.
  • This method includes the step of administering a neural crest cell derived from a fat or a fat-derived stem cell.
  • the neural crest cells used for transplantation can be in any form as described herein in “Adipose-derived neural crest cells” and “Methods for preparing neural crest cells”.
  • the neural crest cells can be administered by any method known in the art.
  • the neural crest cells can be injected using a syringe, a catheter, a tube, and the like, but are not limited thereto.
  • exemplary dosage forms include, but are not limited to, local injection (subcutaneous injection, intramuscular or intramuscular injection), intravenous injection, intraarterial injection, or tissue administration.
  • the present invention provides the use of neural crest cells for the preparation of a medicament for treating or preventing a nervous system disease, disorder or condition.
  • the neural crest cells used for the preparation of the medicament are in any form as described herein in “Adipose-derived neural crest cells” and “Method of preparing neural crest cells”. obtain [0131]
  • the present invention will be described based on examples. However, the following examples are provided for illustrative purposes only. Accordingly, the scope of the present invention is limited only by the appended claims, which are not limited to the above-described embodiment and the following examples.
  • adipose-derived stem cells were prepared from sucked fat from humans who gave consent to this experiment. Specifically, the aspirated fat was thoroughly washed with physiological saline using a 1-liter separatory funnel. After confirming that the aspirated fat was sufficiently separated in the upper layer and the physiological saline was sufficiently separated in the lower layer, the lower layer was discarded, and this was repeated until the physiological saline became almost transparent with the naked eye. In this example, it was performed five times.
  • the aspirated fat was added in an amount equal to 10 ml of 0.075% collagenase ZPBS, and incubated at 37 ° C for 30 minutes with good agitation. To this sample, the same amount of 10% serum-added DMEM was added and centrifuged at 1200 ⁇ g for 10 minutes.
  • the precipitated cells (mostly red blood cells) were transferred to several 50 ml polypropylene tubes and centrifuged (400 X g, 5 minutes).
  • Ficoll registered trademark
  • the cell solution was separated into 4 layers. From the top (A layer) Cell-free layer (Transparent), (B layer) Mononuclear cell layer (light red), (C layer) Ficoll layer (transparent), (D layer) Red blood cell layer (dark red) Adherent cell groups including vesicles were included in B and C layers. After the A layer was aspirated, the B layer and about 3 ml of the C layer were collected as a cell suspension and transferred to a 50 ml tube.
  • Rough cell specific gravity is determined by mixing density gradient centrifuge separation media such as Percoll TM , Readygrad TM, etc. in saline-sodium solution or sucrose solution, and collecting collected cells and density-one marker beads ( Add density marker beads) to the mixture, centrifuge, and check by checking the force of cells in which of the 5-10 layers divided by the beads (the layer containing the cells indicates the specific gravity of the cells) It is possible.
  • density gradient centrifuge separation media such as Percoll TM , Readygrad TM, etc. in saline-sodium solution or sucrose solution
  • Example 2 The stem cells collected in Example 2 were characterized using FACS according to the following procedure:
  • SM staining medium
  • a gZml labeled antibody (phycoerythrin (PE), allophycocyanin (APC) and Z or fluorescein isothiocyanate (FITC) was used for labeling) was added.
  • PE phytoerythrin
  • APC allophycocyanin
  • FITC fluorescein isothiocyanate
  • FACS Vantage (Becton Dickinson) was used. Using the antibody label as an indicator, the expression of various CD proteins in the isolated stem cells was analyzed. As a result, as shown in Table 4, stem cells derived from the liquid portion of the aspirate from fat suction were found to express CD90 and CD49d.
  • the isolated stem cells were subcultured twice in DMEM medium! Passaging was done at 80% confluence. Use the same procedure as above to incubate cells after the second passage. Analysis was performed. The results are shown in Table 4 below.
  • stem cells prepared by liquid partial force of aspirate by liposuction contain mesenchymal stem cells, but differ from the adipose-derived stem cell group prepared by conventional methods, and CD31, 34 positive cells are Included. Therefore, it can be understood that the stem cells prepared by the method of the present invention are a group of cells that can easily differentiate into vascular endothelium (angiogenesis) with high efficiency. Furthermore, since the CD expression used as an index in the present specification has been confirmed after being subcultured twice, the stem cell of the present invention shows its phenotype after about two subcultures. It is understood that there is little change.
  • stem cells were collected from the liquid portion of the aspirate obtained by multiple subject force liposuction and characterized. The results are shown below.
  • Numbers indicate the percentage of stem cells expressing each protein in the cell population.
  • the collected stem cells are most of the population of CD13, CD29, CD34, CD36, CD44, CD49d, CD54, CD58, CD71, CD73, CD90, CD105, CD106 Positive for CD151 and SH3. Therefore, the adipose-derived stem cells of the present invention are selected from the group consisting of CD13, CD29, CD34, CD36, CD44, CD49d, CD54, CD58, CD71, CD73, CD90, CD105, CD106, CD151, and SH3 force. Both are cells that express one protein.
  • One feature of the adipose-derived stem cells used in the present invention is that they are stem cells that express CD106. Also, for CD31, CD45, CD117, and CD146, some of the stem cell populations were positive and some were negative.
  • the stem cell population was negative for CD3, CD4, CD14, CD15, CD16, CD19, CD33, CD38, CD56, CD61, CD62e, CD62p, CD69, CD104, CD135, and CD144. Therefore, the adipose-derived stem cells of the present invention do not express at least one of CD3, CD4, CD14, CD15, CD16, CD19, CD33, CD38, CD56, CD61, CD62e, CD62p, CD69, CD104, CD135, and CD144 It is a cell.
  • this stem cell population was cultured in a differentiation-inducing medium, expression of organ-specific proteins such as bone, cartilage, and fat was observed in 2 to 3 weeks.
  • This stem cell population unlike human fibroblast-derived cultured fibroblasts, did not express CD56, which is expressed in many fibroblast cells.
  • CD105 expressed by this population of stem cells was not normally seen in fibroblasts.
  • CD49d expressed by this stem cell population was not normally seen in bone marrow-derived mesenchymal stem cells.
  • CD31, CD34, CD36, CD45, CD106, and CD117 tended to disappear when the culture period was prolonged. Therefore, when subculture is continued, CD106 expression observed before subculture may not be observed.
  • Example 5 Isolation of human adipose-derived stem cells and cell culture of neurospheres
  • Adipose-derived stem cells were also isolated from human aspirated fat as reported by Yoshimura K et al., J Cell Physiol 2006; 208: 64-76.
  • suctioned fat was digested on a shaker for 30 minutes at 37 ° C with 0.075% collagenase in PBS. Mature adipocytes and connective yarns and weaves were removed by centrifugation. Blood cells again The fat-derived stem cell pellet was obtained by treatment with an erythrocyte lysis buffer.
  • the fluid partial force of liposuction aspirate can also be achieved by treating fat-derived stem cells with erythrocyte lysis buffer and density gradient centrifugation using FicolKGE Healthcare Bio-sciences, Piscataway, NJ). Isolated. It was confirmed that the obtained cells were adipose-derived stem cells by subjecting the cells to a method for inducing differentiation into fat, cartilage, and bone.
  • a method slightly modified from the method described in Kanemura Y et al., J Neurosci Res 2002; 69: 869-879 was used.
  • Freshly isolated adipose-derived stem cells are plated in 10 cm uncoated dishes at a density of 2 x 10 7 cells (approximately 1 x 10 5 cells Zml) and in a 5% CO environment under humidity. Underneath at 37 ° C, Neuros Hue
  • Neurosphere culture medium consists of human recombinant EGF (20ng / ml, PeproTech), human thread and basic-FGF (20ngZ ml, Kaken Pharmaceutic al, Japan), 2% B27 supplement (GIBCO), lOOUZml penicillin and 100 ⁇ gZml DMEMZHAMF12 (1: 1) as a basal medium supplemented with streptomycin. Half of the medium was replaced with fresh medium on days 4-5 and passage was performed on day 8.
  • adipose-derived stem cells When adipose-derived stem cells are cultured in a neurosphere culture medium (without serum, and containing EGF and basic FGF), the floating adipose-derived stem cells form small clumps on the third day of culture of the newly prepared adipose-derived stem cells.
  • aggregation of o neuro Sufuea like cells were observed in bright et force on day 5 (see FIG. 1A.) 0 the number and size of the Sufuea, due within 2 days (See Figures 1B and 1C.) Passaged on the 8th day. During this passage, the spheres were dissociated and resuspended in fresh media. On day 7 after passage, a new globular cell mass formed (data not shown.) O This suggests the neurosphere's ability to self-replicate.
  • Adipose-derived stem cells were treated with M199 medium and 10% fetal bovine serum. : Pre-cultured in a standard medium containing FBS). Next, total mRNA was extracted from Neurospora derived from the first passage of adipose-derived stem cells using an RNeasy-mini kit (Qiagen, Hilden Germany). This preculture was necessary to reduce blood cell contamination. Control mRNA was also extracted from undissolved adipose-derived stem cells cultured in M199 and 10% FBS.
  • RT-PCR was used to examine the expression of the neural stem cell markers Nestin and Musashi-1 genes and the adipogenic marker Leptin.
  • the Nestin and Musashi-1 gene expression was cultured in neurosphere culture media! /, Well upregulated in neurospheres compared to control adipose-derived stem cells (see Figure 2A and B). O This result indicates that neurospheres have characteristics of neural progenitor cells Is suggested. In contrast, Leptin expression was dramatically reduced in neurospheres (see Figure 2C). 0 This result indicates that neurospheres have reduced adipogenic capacity.
  • adipose-derived stem cell force-eurosphere was obtained.
  • This-you The cell growth of the rothsphere is remarkably rapid and can be achieved with a variety of other organs (eg, dermis and heart)-faster than the eurosphere.
  • This remarkably fast cell growth suggests the availability of adipose-derived stem cells as a source of neural progenitor cells in regenerative medicine.
  • the cells constituting the neurosphere expressed Nestin and Musaxhi-l, which are the genes of neural stem cells. This is thought to reflect the tendency of neurospheres to differentiate into neural progenitor cells. This finding is further supported by reduced expression of Leptin, a marker for adipogenesis and maturation.
  • Eurospheres derived from human adipose-derived stem cells were isolated from the Sendai virus vector (as described in Li HO et al., J Virol 2000; 74: 6 564-6565 and Inoue M et al., J Virol 2003; 77: 3238-3246).
  • DNAVEC corp. Tsukuba, Japan was used to transfect with green fluorescent protein (GFP).
  • GFP green fluorescent protein
  • the original vector SeVZ lacks the F gene encoding the fusion protein required for entry of the ribonucleotide complex into infected cells, so this vector is neither infectious nor pathogenic.
  • the modified SevZ A F vector has additional mutations that reduce cytotoxicity.
  • a modified vector was used. Neurospheres were incubated for 1 hour in a medium containing modified SeVZ (multiplicity of infection of 250) carrying the GFP gene, and then rinsed with phosphate buffered saline.
  • the eurospheres induced by the present invention expressed the neural stem cell marker genes Ne stin and Musashi-1 (see Example 6), and had the same migration ability as neural crest cells. Therefore, it can be said that this -Eurosphere contains neural crest cells.
  • the human adipose-derived stem cells obtained by this example were able to produce -eurosphere in about one week. It was observed to form. Furthermore, it was confirmed that the neurosphere formed from the human adipose-derived stem cells of the present invention is a cell mixture containing neural crest cells, adipose-derived stem cells, and neural stem cells. The cell mixture of the present invention was also able to form eurospheres in about one week even after repeated passages.
  • the cell mixture of the present invention proliferates at a remarkably fast rate. It was confirmed that it has the ability to form a and includes neural crest cells.
  • the neural crest cells derived from fat or adipose-derived stem cells of the present invention are considered to be applicable to regenerative medicine for treating nervous system diseases, disorders or conditions.
  • Example 8 Isolation of mouse adipose-derived stem cells and cell culture of neurospheres
  • Mouse adipose-derived stem cells are isolated using the same method as in Example 5 except that mice are used instead of humans as specimens.
  • the mouse adipose-derived stem cells are characterized by the same method as in Example 3.
  • mouse adipose-derived stem cells are cultured in a neurosphere culture medium. Half of the medium is replaced with fresh medium on days 4-5 and passage is performed on day 8.
  • Adipose-derived stem cells were treated with neurosphere culture medium (serum-free, EGF and basic
  • Example 9 Quantitative real-time RT—PCR (reverse transcriptase polymerase chain reaction)
  • PCR reverse transcriptase polymerase chain reaction
  • Example 10 Mouse whole embryo culture and mouse-eurosphere-like cell transplantation
  • Example 8 Mouse whole embryo culture and mouse-eurosphere-like cell transplantation
  • Mouse GFP positive cells transplanted into mouse embryos are clearly observed in some of the transplanted embryos and appear to survive.
  • This GFP positive cell can be confirmed to be incorporated into the craniofacial region of the embryo and the heart and trunk.
  • the transplanted cells are arranged in a row along the second arch, which can be confirmed to be very similar to the turn of the neural crest cells that migrate in the second arch. This result suggests an interesting possibility that the cells of murine adipose-derived stem cells exhibit neuronal crest-like properties in mouse embryos.
  • the mouse adipose-derived stem cells obtained in this example form the spheres in about one week.
  • the neurosphere formed with the mouse adipose-derived stem cell force of the present invention is a cell mixture containing neural crest cells, adipose-derived stem cells and neural stem cells.
  • the cell mixture of the present invention can also form a microsphere in about 1 week even after repeated passages.
  • the cell mixture of the present invention has the ability to proliferate at a remarkably fast rate to form a rosphae and contains neural crest cells.
  • the neural crest cells in which the mouse adipose-derived stem cell force of the present invention is induced can also be used for transplantation into mouse embryos of the same species.
  • DMEMZHAMF12 (1: 1) as the medium for neurosphere culture
  • BME MEM DMEM or HAMF12 medium to examine the effect of the medium on cell culture of rosophane.
  • the other materials and methods are the same as in Example 5.
  • Adipose-derived stem cells were treated with neurosphere culture medium (serum-free, EGF and basic
  • Example 7 (Mouse whole embryo culture and transplantation of eurosphere-like cells) [0183] The same method as in Example 7 is used, except that the eurosphere cultured in this example is used.
  • GFP positive cells transplanted into mouse embryos are clearly observed in some of the transplanted embryos and appear to survive. It can be confirmed that the GFP positive cells are incorporated into the craniofacial region of the embryo and the heart and trunk. In particular, the transplanted cells are arranged in a row along the second arch, which can be confirmed to be very similar to the pattern of neural crest cells that migrate in the second arch. This result suggests that the neurosphere cells induced by adipose-derived stem cell force have a neural crest cell-like property, which is interesting and possible.
  • Example 5 the human adipose-derived stem cells isolated in the above are passaged 5 times in DMEM medium to prepare and culture eurosphere.
  • Example 7 The same method as in Example 7 is used except that the spheres of this example are used.
  • Mouse GFP positive cells transplanted into mouse embryos are clearly observed in some of the transplanted embryos and appear to survive.
  • This GFP positive cell can be confirmed to be incorporated into the craniofacial region of the embryo and the heart and trunk.
  • the transplanted cells are arranged in a row along the second arch, which can be confirmed to be very similar to the turn of the neural crest cells that migrate in the second arch. This result suggests an interesting possibility that the cells of murine adipose-derived stem cells exhibit neuronal crest-like properties in mouse embryos.
  • adipose-derived stem cells can be used even if they are cultured and expanded and maintained after acquisition. Furthermore, it is confirmed that neurospheres can be prepared from adipose-derived stem cells cultured and proliferated, and neural crest cells can be prepared.
  • CRABP1 (NM004378 (human)), AP2 (NM002097 (human)), Slug (NM003068 (human)), SoxlO (NM006941 (human)), Snail (NM005985 (human)), Twist (NM0004 74) (Human)), Pax3 (NM000438 (human)), Pax7 (NM002584 (human)), HNKl (NMO 04854 (human)), p75NTR (NM002507 (human)), TRP2 (NM006267 (human)), Wntl (NM005430 ( (Human)), PO (NM002723 (Human)), tPA (NM000930 (Human)) gene expression is significantly up-regulated in neurospheres compared to control adipose-derived stem cells not cultured in neurosphere culture medium Make sure. This result indicates that neurospheres express neural crest cell markers.
  • neurospheres express neural crest cell markers by specific staining methods, immunohistochemical methods, in situ hybridization methods, and Western blotting methods for each marker.
  • the present invention proves that fat-derived or fat-derived stem cell-derived neural crest cells that can be obtained by a simple method can be applied to regenerative medicine. Therefore, the industrial use of the present invention is found in the pharmaceutical industry.

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  • Virology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Ophthalmology & Optometry (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
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Abstract

La présente invention concerne des cellules de la crête neurale pouvant être appliquées à la médecine régénérative dans le traitement d'une maladie, d'un trouble ou d'un état du système nerveux par un procédé simple. En particulier, la présente invention concerne des cellules de la crête neurale dérivées de tissu adipeux. La présente invention concerne également des cellules de la crête neurale obtenues à partir de cellules souches dérivées de tissu adipeux. La présente invention concerne également un procédé destiné à préparer les cellules de la crête neurale. Le procédé comprend les étapes consistant : A) à obtenir des cellules progéniteurs à partir de tissu adipeux; et B) à soumettre les cellules souches aux conditions nécessaires pour induire des cellules de la crête neurale. La présente invention concerne également une composition et un mélange pour transplantation cellulaire contenant les cellules de la crête neurale, un procédé de transplantation cellulaire et l'utilisation des cellules de la crête neurale pour préparer un produit pharmaceutique.
PCT/JP2007/051643 2006-08-09 2007-01-31 Cellules de la crête neurale dérivées de tissu adipeux WO2008018190A1 (fr)

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JP2013063088A (ja) * 2012-12-14 2013-04-11 Nippon Institute For Biological Science 脂肪組織間質細胞の神経細胞への分化誘導方法
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US11959100B2 (en) 2017-11-30 2024-04-16 Kyoto University Method for culture of cells

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WO2008150001A1 (fr) * 2007-06-08 2008-12-11 Biomaster, Inc. Amas adipeux
JP5388297B2 (ja) * 2007-06-08 2014-01-15 株式会社バイオマスター アディポクラスター
JP2011520434A (ja) * 2008-05-07 2011-07-21 ボーン セラピューティクス エス．アー． 新規の間葉系幹細胞及び骨形成性細胞
US9371515B2 (en) 2008-05-07 2016-06-21 Bone Therapeutics S.A. Mesenchymal stem cells and bone-forming cells
JP2017099386A (ja) * 2008-05-07 2017-06-08 ボーン セラピューティクス エス．アー． 新規の間葉系幹細胞及び骨形成性細胞
US20110110900A1 (en) * 2008-06-13 2011-05-12 Philip Stephens Novel adult progenitor cell
US11560546B2 (en) 2009-02-17 2023-01-24 Memorial Sloan Kettering Cancer Center Methods for neural conversion of human embryonic stem cells
US10287546B2 (en) 2009-02-17 2019-05-14 Memorial Sloan Kettering Cancer Center Kits for neural conversion of human embryonic stem cells
US10260041B2 (en) 2009-02-17 2019-04-16 Memorial Sloan Kettering Cancer Center Methods for neural conversion of human embryonic stem cells
JP5700301B2 (ja) * 2009-06-03 2015-04-15 国立大学法人大阪大学 多能性幹細胞からの神経堤細胞群の分化誘導方法
WO2010150650A1 (fr) * 2009-06-23 2010-12-29 学校法人日本大学 Nouveau procédé pour maintenir des cellules souches dans un état indifférencié
JP2011004607A (ja) * 2009-06-23 2011-01-13 Nihon Univ 幹細胞の未分化状態を維持する新規方法
US9453198B2 (en) 2010-05-25 2016-09-27 Memorial Sloan Kettering Cancer Center Method of nociceptor differentiation of human embryonic stem cells and uses thereof
JP2013532961A (ja) * 2010-05-25 2013-08-22 メモリアル スローン−ケタリング キャンサー センター ヒト胚性幹細胞の侵害受容器分化のための方法およびその使用
US10711243B2 (en) 2011-11-04 2020-07-14 Memorial Sloan-Kettering Cancer Center Midbrain dopamine (DA) neurons for engraftment
US10280398B2 (en) 2011-11-04 2019-05-07 Memorial Sloan-Kettering Cancer Center Midbrain dopamine (DA) neurons for engraftment
US11970712B2 (en) 2011-11-04 2024-04-30 Memorial Sloan-Kettering Cancer Center Midbrain dopamine (DA) neurons for engraftment
JP2013063088A (ja) * 2012-12-14 2013-04-11 Nippon Institute For Biological Science 脂肪組織間質細胞の神経細胞への分化誘導方法
US10662411B2 (en) 2013-01-31 2020-05-26 Ajinomoto Co., Inc. Culture method for stable proliferation of pluripotent stem cell while maintaining undifferentiated state
EP2952579A4 (fr) * 2013-01-31 2016-07-20 Ajinomoto Kk Méthode de culture pour une prolifération stable indifférenciée de cellules souches pluripotentes
US10745669B2 (en) 2013-01-31 2020-08-18 Ajinomoto Co., Ltd. Culture method for stable proliferation of pluripotent stem cell while maintaining undifferentiated state
US11649431B2 (en) 2013-04-26 2023-05-16 Memorial Sloan-Kettering Cancer Center Cortical interneurons and other neuronal cells produced by the directed differentiation of pluripotent and multipotent cells
CN103751845A (zh) * 2014-01-30 2014-04-30 中国人民解放军海军总医院 一种用于移植修复周围神经缺损的组织工程生物材料
US11959100B2 (en) 2017-11-30 2024-04-16 Kyoto University Method for culture of cells
CN111304167A (zh) * 2018-12-12 2020-06-19 上海泉眼生物科技有限公司 人源脂肪干细胞来源的神经元前体细胞及其制备方法和应用
CN111304167B (zh) * 2018-12-12 2024-03-26 上海泉眼生物科技有限公司 人源脂肪干细胞来源的神经元前体细胞及其制备方法和应用

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