WO2008018190A1

WO2008018190A1 - Fat-derived neural crest cells

Info

Publication number: WO2008018190A1
Application number: PCT/JP2007/051643
Authority: WO
Inventors: Kotaro Yoshimura; Takashi Nagase; Daisuke Matsumoto; Tomokuni Shigeura
Original assignee: Biomaster, Inc.
Priority date: 2006-08-09
Filing date: 2007-01-31
Publication date: 2008-02-14

Abstract

The invention provides neural crest cells applicable to regenerative medicine in a treatment of a nervous system disease, disorder or state by a simple method. In particular, the invention provides fat-derived neural crest cells. The invention also provides neural crest cells induced from fat-derived stem cells. The invention also provides a method for preparing the neural crest cells. The method includes the steps of: A) obtaining progenitor cells from fat; and B) subjecting the stem cells to conditions to induce neural crest cells. The invention also provides a composition and a mixture for cell transplantation containing the neural crest cells, a method for cell transplantation and use of the neural crest cells for preparing a pharmaceutical.

Description

Specification

Fat-derived neural crest cells

Technical field

[0001] 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.

Background art

[0002] Regenerative medicine, centered on the use of stem cells, has made significant progress in recent years.

Various 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.

[0003] However, regenerative medicine has not yet been routinely applied to many patients with organ or tissue dysfunction. To date, it has only been applied to such patients with limited use of assistive systems in medical devices as well as organ transplants! /. These therapies have problems such as donor shortage, rejection, infection, and lifetime. In particular, lack of donors is a serious problem.

[0004] 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.

[0005] The presence of stem cells in fat has been a major factor. 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. (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.

[0006] As stem cell research has progressed, a new concept of cell differentiation across the germ layer boundary has been discovered. MSCs and adipose-derived stem cells can be differentiated into derivatives of the nervous system (ie, ectoderm), which is essentially a mesoderm system (Non-patent Documents 4 and 5). Recently, it has been reported that stem cells having nervous system characteristics can be isolated from mesoderm tissue (eg, dermis and heart) (Non-Patent Documents 6 to 8). In these reports, 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.

[0007] 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 (Non-patent Document 9). However, 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.

[0008] A group of Rang, KS, et al. Has found that adipose tissue stromal cells derived from primates (in non-patent documents 10 and 12, akakasaru is used. In non-patent document 11, the animals used are “ It seems that it is only described as being a “non-human” primate.) By culturing in a Neurobasal medium ™ medium developed for the maintenance of neurons-an attempt was made to form eurospheres (Non-patent documents 10 to 12). However, according to Rang KS et al., The produced -Eurosphere expresses NeuN, MAP2, NF160, etc., and thus aggregates of those differentiated into neural cells (ie, differentiated cells) are formed. Seems to be

[0009] 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.

[0010] As described above, 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.

[0011] Regarding this, it can be said that there are evidences in many documents such as Patent Documents 1 to 6 and Non-Patent Documents 4, 5, and 13-22. These documents describe that adipose-derived stem cells appear to have the ability to be divided into nerves. However, 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.

[0012] Thus, it seems that the possibility of nerve differentiation from fat, particularly fat-derived stem cell force, is quite high. On the other hand, from fat, particularly from fat-derived stem cells, the pluripotency of the nervous system. Inducing cells has been considered difficult.

[0013] However, since fat, in particular, 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

4; Aug 15; 117 (Pt 18): 4289-99

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

Sep; 54 (3): 132-41

Non-Patent Document 22: Parker AM, Katz AJ. Expert. Opin. Biol. Ther. 2006 Jun; 6 (6): 567-78

Disclosure of the invention

Problems to be solved by the invention

[0014] Thus, there is a growing demand in the art for a controlled and simple method for preparing neural stem cells and neural progenitor cells that can be used for the treatment of nervous system diseases, disorders or conditions. 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.

Means for solving the problem

[0015] As a result of the present inventors diligently examining the above problems, the present invention was partially completed by unexpectedly preparing a neural crest cell derived from a fat or a fat-derived stem cell. Accordingly, the present invention provides the following.

(Item 1)

Fat-derived neural crest cells.

(Item 2)

2. The neural crest cell according to item 1, which is derived from an adipose-derived stem cell.

(Item 3)

2. The neural crest cell according to item 1, wherein the neural crest cell has a migration ability.

(Item 4)

4. The neural crest according to item 3, wherein the migratory ability is confirmed by transplanting to the head region of the embryo and then observing the migratory ability to the frontal nasal protuberance, maxillary protuberance, or mesenteric mesenchyme. cell.

(Item 5)

2. The neural crest cell according to item 1, wherein the neural crest cell expresses at least one neural stem cell marker.

(Item 6)

6. The neural crest cell according to item 5, wherein the 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)

Item 7. The neural crest cell according to item 6, wherein the neural stem cell marker includes Nestin and Musashi-1.

(Item 8)

2. The neural crest cell according to item 1, wherein the neural crest cell expresses at least one neural crest cell marker.

(Item 9) 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.

(Item 10) Item 2. The neural crest cell according to item 1, wherein the neural crest cell has an expression level of Leptin lower than that of an adipose-derived stem cell.

(Item 11)

2. 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 12)

Item 2. The neural crest cell according to Item 1, wherein the fat is derived from a mammal.

(Item 13)

Item 13. The neural crest cell according to Item 12, wherein the fat is derived from a human.

(Item 14)

A method for preparing neural crest cells comprising the following steps:

A) the step of obtaining fat stem cells as well

B) Step of subjecting the stem cells to conditions for inducing neural crest cells

Including the method.

(Item 15)

Item 15. The method according to Item 14, wherein the anterior stem cell is an adipose-derived stem or stromal cell.

(Item 16)

Item 15. The method according to Item 14, wherein the step of obtaining the stem cell comprises collagenase treatment and centrifugation treatment.

(Item 17)

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.

(Item 18)

15. The method of item 14, wherein in 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) 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 20)

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).

(Item 21)

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. .

(Item 22)

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. Method. (Item 23)

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.

(Item 24)

In step B), 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. Item 15. The method according to Item 14, which is a medium. (Item 25)

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).

(Item 26)

15. The method according to item 14, wherein the step B) includes replacing the medium with a new medium on days 2 to 6. (Item 27)

15. The method according to item 14, wherein the step B) includes replacing the medium with a new medium on the 4th to 5th days.

(Item 28)

The method according to Item 14, wherein the step B) includes subculture on the 6th to the 10th day.

(Item 29)

Item 15. The method according to Item 14, wherein the fat is derived from a mammal.

(Item 30)

30. The method of item 29, wherein the fat is derived from a human.

(Item 31)

In the step B), 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).

(Item 32)

Item 15. The method according to Item 14, further comprising the step of confirming whether the stem cell expresses a neural stem cell marker.

(Item 33)

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 34)

34. The method of item 33, wherein the neural stem cell marker comprises Nestin and Musashi-1.

(Item 35)

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 36)

Item 15. The method according to Item 14, wherein the cell expresses at least one neural crest cell marker.

(Item 37)

37. The method of item 36, wherein 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 38)

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 39)

The method according to Item 38, wherein the confirmation of the level of the adipocyte marker is confirmation of a decrease in Leptin.

(Item 40)

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).

(Item 41)

15. 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 42) Item 15. The method according to Item 14, further comprising the step of confirming whether the stem cell has a migration ability.

(Item 43)

43. The method according to Item 42, wherein the migration ability is confirmed by transplanting to a head region of an embryo and observing the subsequent migration ability.

(Item 44)

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.

(Item 45)

A composition for cell transplantation for the treatment of a nervous system disease, disorder or condition, comprising the cell according to item 1.

(Item 46)

46. The 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.

(Item 47)

46. The 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.

(Item 48)

46. A 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.

(Item 49)

(Item 50)

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.

(Item 51)

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.

(Item 52)

Use of the cell according to item 1 for the preparation of a medicament for treating or preventing a nervous system disease, disorder or condition.

(Item 53)

53. Use according to item 52, 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.

(Item 54)

An adipose-derived neural crest cell mixture comprising neural crest cells and adipose-derived stem cells.

(Item 55)

55. The fat-derived neural crest cell mixture according to item 54, further comprising neural stem cells.

[0017] Accordingly, these and other advantages of the present invention will be apparent upon reading the following detailed description with reference to the accompanying drawings.

The invention's effect

[0018] According to the present invention, regenerative treatment can be performed using a neural crest cell derived from fat and a neural crest cell derived from fat precursor cell. In the present invention, 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. Furthermore, 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. According to the present invention, 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.

Brief Description of Drawings

[0019] [Fig. 1] Fig. 1 shows culture in a neurosphere culture medium, Day 5 (Day 5: A), Day 6 (Day

6: Photo (X200) showing the formation of eurosphere of adipose-derived stem cells on day 7 (Day 7: C).

[Figure 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 μm.

BEST MODE FOR CARRYING OUT THE INVENTION

[0020] Hereinafter, the present invention will be described. Throughout this specification, it should be understood that singular articles (eg, “a”, “an”, “the”, etc. in English) also include the plural concept unless otherwise stated. It should be. In addition, it should be understood that the terms used in this specification are used in the meanings usually used in the art unless otherwise specified. In case of conflict, the present specification, including definitions, will control.

[0021] (Definition of terms)

Listed below are definitions of terms particularly used in the present specification.

[0022] The term "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. This tissue section can be examined with an optical microscope, and the number of nuclei per specific area (for example, 200 m × 200 m) can be estimated and counted. 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.

[0023] 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.

[0024] As used herein, the term "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. As used herein, “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. Also, an artificially produced cell (eg, a fused cell, a reprogrammed cell, etc. used herein) 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.

[0025] As used herein, the term "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.

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. Examples of tissue stem cells of the extinct system include knee (common) stem cells and liver stem cells. Examples of myeloid tissue stem cells include hematopoietic stem cells and mesenchymal stem cells. Neural tissue stem cells include neural stem cells and retinal stem cells.

[0027] As used herein, the term "mesenchymal stem cell" refers to a stem cell found in the mesenchyme. The term “mesenchymal stem cell” may be abbreviated herein as “MSC”. Here, 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.

[0028] As used herein, 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. As these 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. Some of such 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. As used herein, the term “adipose-derived stem cells” refers to all adipose tissue-derived stem cells including adipose tissue-derived stem cells obtained by these known separation methods. As used herein, the term “progenitor cell” includes unipotent undifferentiated cells as well as pluripotent undifferentiated cells. As used herein, the term “stem cell” includes progenitor cells.

[0029] 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. As used herein, the term “PLA (processed lipo aspirate cell)” refers to a progenitor cell obtained from the aspirated fat of the aspirate by liposuction. As used herein, the term “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.

[0030] As used herein, 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.

[0031] As used herein, "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.

[0032] As used herein, 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. As an example of the neurosphere method, 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. 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 There is 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.

[0033] As used herein, with respect to nervous system cells, "pluripotency" or "multipotency" 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. Preferably, 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). As the conditions for separating cells into glial cells, for example, a glial induction medium (50 ml of medium, DMEMZF12: 45 ml, FBS: 5 ml, may contain antibiotics) is cultured for 1 week (half volume every 2 days). Replace the medium.) It is not limited to. The neurotrophic factor that can be used herein may be any as long as it has an action of assisting or promoting nerve induction. In one embodiment, 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.

[0034] Neural stem cells can also be identified using only neural stem cell markers.

[0035] As used herein, a "neural stem cell marker" refers to a neural stem cell that assists in identifying a neural stem cell by its localization or expression. . Preferably, its localization or expression (for example, Nestin (NM006617 (t)), Musashi-1 (NM002442 (human)), CD133 (NM0006017 (human)), notchl (NM017617 (human))) Hes l ( NM005524 (human)) ゝ Mashl (NM004316 (human)) ゝ Neurogenin (N M006161 (human)), Pax6 (NM001604 (human)), CD15 (NM002033 (human)), PDG FR (NM002609 (human)) In this way, neural stem cell markers include, for example, Nestin (N M006617 (Hin)), Musashi-1 (NM002442 (Hin))) , CD133 (NM0006017 (human)), notch 1 (NM017617 (human)), Hesl (NM005524 (human)), Mashl (NM004 316 (human)), Neurogenin (NM006161 (human)), Pax6 (NM001604 (human))卜)), CD15 (NM002033 (human)), PDGFR (NM002609 (human)) and the like, but are not limited thereto.

[0036] Determination with these 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. [0037] In the present specification, "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. Preferably, 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. This unique migratory ability can be confirmed by transplanting into the head region of the embryo and then observing that it migrates to the frontal nasal protuberance, maxillary protuberance, and arch mesenchyme. As cell markers of neural crest cells, for example, CRABP1 (NM004378 (human)), A P2 (NM002097 (chicken)), Slug (NM003068 (chicken)), SoxlO (NM006941 (chicken)), Snail (NM005985) (Chicken)), 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)), and the like, but are not limited thereto. This neural crest cell can also be confirmed by observing that at least one neural stem cell marker is expressed. For example, 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. However, it is not limited to these. Preferably, neural stem cell markers that can be used include Nestin and Musashi-1.

[0038] 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.

[0039] Preferably, in an embodiment, 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. This method! 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.

[0040] As used herein, 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. Uh. Preferably, 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. Examples of 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.

[0041] 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.

[0042] The neural crest cell marker, neural stem cell marker, and adipocyte marker that can be used in the present invention are listed below. [0043] [Table 1]

(Table 1. Neural crest cell markers)

[0044] [Table 2] (Table 2. Neural stem cell markers)

Neural stem cell marker accession number

Nesti n NM006617

Musashi— 1 NM002442

CD133 NM0006017 (Hui) notch! NM017617 (Hige)

Hes1 NM005524

Mashl NM004316

Neur ogenin NM006161

Pax6 NM001604 (Hui)

CD15 NM002033

PDGFR NM002609 (Hui)

13]

(Table 3. Adipocyte marker)

Adipocyte marker accession number

Leptin NM000230 (Han) As used herein, “embryo” or “embryonic cell” refers to the state of an early era of development in a multicellular organism. As used herein, 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.

As used herein, 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. When used in the present invention, sperm cells may be in the form of a population or tissue! /.

[0047] 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. As used herein, somatic cells may be derived from any germ layer. Preferably, mesenchymal cells can be used as the somatic cells.

[0048] As used herein, the term "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. Adipocytes 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.

[0049] As used herein, 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.

[0050] As used herein, the term "tissue" refers to a population of cells having substantially the same function and Z or morphology in a multicellular organism. “Organization” is usually

However, cell populations having the same origin, but cell populations having different origins, may have the same function and Z or morphology. Therefore, the stem cell of the present invention is used.

When regenerating a tissue by V, cell populations having two or more different origins can constitute one tissue. Usually, 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.

[0051] In the present invention, 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. Good. As used herein, 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. . 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. In one embodiment, 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. In the present specification, when an organ is used as a target, 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.

[0052] As used herein, the term "conditions for inducing neural crest cells" refers to time, medium, temperature, humidity, or a combination thereof, etc. that allow neural crest cells to be induced. In the present invention, it has been found for the first time to prepare neural crest cells from adipose-derived stem cells. However, in view of the contents of this specification, such 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. In the present invention, sufficient conditions for inducing such neural crest cells can be used in vitro, in vivo, or ex vivo. In vivo, the conditions in the transplanted body part are applied as they are. In the present invention, after the stem cells and the differentiated cells are mixed, they 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. In the present invention, as 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.

[0053] In the present specification, the "conditions 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.

[0054] a) about 1 X 10 ⁴ cells Zml to about 1 X 10 ⁶ cells Zml

b) Presence of epidermal growth factor (EGF) <e.g. epidermal growth factor, basic fibroblast growth factor, B27 supplement (GIBCO)>

c) Eagle Basal Medium (BME), Minimum Essential Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM) or HAMF12 medium, there is! /, or their mixed medium

d) Replace the medium with a new one on the 2nd to 6th day (eg 4th to 5th day).

e) 6 ~: Passing on the LO day (eg, day 8),

And combinations of these.

[0055] This condition can be prepared by those skilled in the art with reference to Kanemura Y, Mori H, Kobayashi S, et al. J. Neurosci. Res. 2002; 69: 869-879.

[0056] As used herein, in the "conditions for differentiating stem cells of the nervous system to differentiate into neurospheres", in a preferred embodiment, a) the cell density condition is the condition of Kang KS et al. Patent Documents 10 to 12; 10-20 cells (density of Zcm ² )) and is nectar. C) Medium conditions were developed by Rang KS et al. For the maintenance of nerve cells, with the exception of glutamic acid, aspartic acid, and iron sulfate. On the other hand, in the present invention, in a particularly preferred embodiment, DMEMZF12 (1: 1) is used, and it has been found that it is more advantageous to include glutamic acid, aspartic acid, and iron sulfate. In this preferred embodiment, in particular, the present invention includes an excitatory amino acid, glutamate or aspartate, so the environment is completely different from the Neurobasal medium ™ used to maintain differentiated neurons. It's different. Although not wishing to be bound by theory, the presence of 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).

As used herein, the term “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.

[0057] As used herein, the term "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.

[0058] As used herein, the term " _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.

In this specification, the term “autograft (tissue, cell, organ, etc.)” refers to a graft (tissue, cell, organ, etc.) from another genetically identical individual (eg, monozygotic twin) in a broad sense. Can also be included. As used herein, such self expression is used interchangeably when derived from a subject. Thus, as used herein, the expression not derived from a subject has the same meaning as not being self (ie, non-self).

[0059] In the present specification, "same species (allogeneic) (grafts, tissues, cells, organs, etc.)" refers to those that are transplanted to other individual forces that are genetically different even if they are the same species. Tissue, cell, organ, etc.). Because of genetic differences, allogeneic ones (grafts, tissues, cells, organs, etc.) can elicit an immune response in the transplanted individual (recipient). Examples of such (grafts, tissues, cells, organs, etc.) include, but are not limited to, those derived from the parent (grafts, tissues, cells, organs, etc.).

As used herein, the term “heterologous (graft, tissue, cell, organ, etc.)” refers to a transplant (graft, tissue, cell, organ, etc.) transplanted from a heterogeneous individual. Thus, for example, when a mouse is a recipient, a human-derived one (graft, tissue, cell, organ, etc.) is said to be heterogeneous (tissue, cell, organ, etc.). In the present invention, it should be noted in a sense that it can be used in different species and has demonstrated good transplantation results.

[0061] 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! ], Addition of streptomycin, media selection (BME, MEM, DMEM or HAMF12 media, or mixed media thereof (eg, DMEMZHAMF12 (1: 1)), once every 2-6 days (eg, 4-5 days) Medium change, passage once every 6-10 days (eg 8 days), temperature 20 ° C-40 ° C (eg 37 ° C), carbon dioxide 5%, humidity above 80% ( For example, 100%), and the use of uncoated dishes, such as 1 x 10 ⁵ cell Zml density, EGF addition, bFGF addition, neurotrophic factor (eg B27 supplement (GIBCO)) ), Penicillin and streptomycin, DMEMZHAMF12 (1: 1), medium change once every 4-5 days, passage once every 8 days, 37 ° C, carbon dioxide 5%, 80% It is the condition that the culture is performed with the above humidity and the use of an uncoated dish. The trophic factor may be any as long as it has the effect of assisting or promoting nerve induction, In one embodiment, 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 In one embodiment, the nerve inducer may be B27 supplement (GIBCO).

[0062] The above conditions may be employed to maintain neural stem cells, neural crest cells, and the like, but are not limited thereto.

[0063] In order to induce neural crest cells from adipose-derived stem cells, any culture medium containing a factor that promotes induction from adipose-derived stem cells to neural crest cells can be used. like that As the medium, for example, 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 (GIBCO) 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.

[0064] As used herein, 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.). Examples of such 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. Typical examples of such factors include epidermal growth factor (EGF), growth factors such as basic fibroblast growth factor (basic—FGF, b—FGF) (especially EGF), Examples of biofactors include BMP-4 (NM001202 (human)), Noelinl (NM01 4279 (human)), but are not limited to these.

[0065] In the present invention, it has been found for the first time that "conditions 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.

[0066] As used herein, 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.

[0067] As used herein, 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. Say. 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. This includes the presentation that the combined drugs are administered together as a therapeutic mixture, and the procedure in which the combined drugs are administered separately but at the same time (for example, a protein enhancer) is also included. Including. “Combination” administration further includes the separate administration of one of the compounds or agents given first, followed by the second.

[0068] As used herein, 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).

[0069] As used herein, the term "homologous" (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.

[0070] As used herein, the term "heterologous" refers to something that is transplanted from a heterologous individual. Thus, for example, if a human is the recipient, a transplant from a pig is a xenotransplant.

[0071] As used herein, the term "recipient" (recipient) refers to an individual that receives cells to be transplanted and the like, and is also referred to as "host". In contrast, an individual that provides cells to be transplanted is called a “donor”. The recipient and donor can be the same or different.

[0072] 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] As used herein, "nervous disease, disorder or condition" refers to any disease, disorder or abnormal condition involving cells of the nervous system.

[0074] As used herein, the term "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.

[0075] As long as 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.) ). Preferably, such 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.

[0076] When the present invention is used as a medicament, such medicament may further comprise a pharmaceutically acceptable carrier and the like. In the medicament of the present invention, any pharmaceutically acceptable carrier known in the art can be used.

[0077] 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. Typically, the medicament of the present invention is administered in the form of a composition comprising the cells of the present invention and other active ingredients together with at least one physiologically acceptable carrier, excipient or diluent. For example, 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.

[0078] 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.

[0079] Exemplary suitable carriers include neutral buffered saline or saline mixed with serum albumin. Preferably, the product is formulated as a lyophilizer using a suitable excipient (eg, sucrose). Other standard carriers, diluents and excipients may be included as desired. Other exemplary compositions include 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.

[0080] 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.

[0081] 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. Examples of 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. In addition, formulation additives such as preservatives, antioxidants, colorants, sweeteners and the like can be used as necessary. In addition, substances other than the polynucleotide, polypeptide and the like of the present invention can be added to the composition of the present invention. Examples of parenteral routes of administration include, but are not limited to, intravenous, intramuscular, subcutaneous, intradermal, mucosal, intrarectal, intravaginal, topical, and dermal. When administered systemically, 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.

[0082] Preferable examples of the solvent in the liquid preparation include an injection solution, alcohol, propylene dallicol, macrogol, sesame oil, corn oil and the like.

[0083] Preferable examples of the 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.

[0084] Preferable examples of the 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.

[0085] 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.

[0086] Preferable examples of the buffering agent in the liquid preparation include, but are not limited to, phosphate, acetate, carbonate, citrate and the like.

[0087] Preferable examples of the soothing agent in the liquid preparation include, but are not limited to, benzyl alcohol, benzalkonium chloride and pro-in hydrochloride.

[0088] 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. .

[0089] Preferable examples of the antioxidant in the liquid preparation include, but are not limited to, sulfite, ascorbic acid, a tocopherol and cysteine.

[0090] When prepared as an injection, solutions and suspensions are sterilized and blood or other It is preferably isotonic with the solvent at the injection site for these purposes. Usually, these are sterilized by filtration using a bacteria retention filter or the like, blending with a bactericide, or irradiation. Furthermore, after these treatments, solidify by freeze-drying or other methods, and add sterile water or sterile diluent for injection (lidocaine hydrochloride aqueous solution, physiological saline, aqueous glucose solution, ethanol or a mixture of these) immediately before use. May be.

[0091] Further, 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.

[0092] 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.

[0093] As used herein, 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.

[0094] 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.

[0095] 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.

[0096] Toxicity studies such as cells of the present invention can be performed using a model. For example, 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.

[0097] Further studies can be performed by sacrificing animals at the end of each toxicology study (preferably ii, American Veterinary Medical Association guidelines Report of the American Veterinary Medical Assoc. Panel on Eutha nasia, (1993) J. Am. Vet. Med. Assoc. 202: 229—249. Then, representative animals from each treatment group can be tested by global examination for direct evidence of metastasis, abnormal illness or toxicity. Overall abnormalities in the tissue are described and the tissue is examined histologically. Drugs that cause abnormalities in the nervous system or cause weight loss or blood component reduction are not preferred, as are compounds that have adverse effects on major organs. In general, the greater the adverse effect, the less preferred the compound.

[0098] (Use of neural crest cells)

The neural crest cells of the present invention can be used for various purposes, for example, treatment of the nervous system. For example, forces including treatment of any disease, disorder or abnormal condition involving cells of the nervous system are not limited to these. (Description of Preferred Embodiment)

Hereinafter, preferred embodiments of the present invention will be described. The embodiments provided below are provided for a better understanding of the present invention, and the scope of the present invention should not be limited to the following description. Therefore, it is obvious that those skilled in the art can make appropriate modifications within the scope of the present invention in consideration of the description in the present specification.

[0099] (Method for preparing neural crest cells)

In one aspect, the present invention provides a method for preparing neural crest cells. By this method, 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.

[0100] Here, 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. Make sure it is well separated and discard the lower layer. Physiological diet with the naked eye Repeat this until the saline is almost clear; (3) Hold 0.075% collagenase ZPBS in the same amount as the aspirated fat and incubate for 30 minutes with good agitation at 37 ° C. (4) Add an equal volume of 10% serum-added DMEM to the above sample; (5) Centrifuge the above sample at 1200 g for 10 minutes; (6) Add 0.16M NH 4 C1ZPBS to the resulting pellet. Suspended and suspended for 10 minutes at room temperature

Four

(7) 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. Here, it is within the technical scope of those skilled in the art to scale up or scale down the above protocol according to the preparation amount.

[0101] On the other hand, 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 ™, Percoll ™ 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.

[0102] 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.

[0103] These 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.

[0104] Preferably, 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). Such 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. Therefore, in a preferred embodiment, 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. [0105] In another embodiment, cells that express CD49d and do not express CD56 may be conveniently selected as adipose-derived stem cells.

[0106] The adipose-derived stem cells used in the present invention can be derived from 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. Thus, it has become clear that it can be used as a source of stem cells that can actually be used to treat neurological diseases, disorders or conditions. Thus, such aspiration fat may be, for example, the liquid part or the fat part of the aspirate from liposuction! /.

[0107] As the adipose-derived stem cells used in the present invention, isolated ones may be used. Force-purified ones or completely purified ones may be used.

[0108] In a preferred embodiment, 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.

[0109] In this method, 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. be able to. 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, superoxide dismutase, transferrin), Add at least one selected from the group that also has penicillin and streptomycin activity, select medium (BME, MEM, DMEM or HAMF12 medium, or a mixed medium thereof) For example, DMEM / HAMF12 (1: 1)), medium change every 2-6 days (eg 4-5 days), passage once every 6-10 days (eg 8 days), 20 ° C-40 ° C temperature (e.g. 37 ° C), carbon dioxide 5%, humidity above 80% (e.g. 100%), and The power to use uncoated dishes can also be selected. For example, density of 1 × 10 ⁵ cells / ml, addition of EGF, addition of bFGF, addition of neurotrophic factors (eg B27 supplement (GIBCO)), addition of penicillin and streptomycin, DMEM / HAMF1 2 (1: 1 ), Medium change once every 4-5 days, passage once every 8 days, 37 ° C, carbon dioxide gas 5%, humidity of 80% or higher, use uncoated dish It can be the condition. The neurotrophic factor that can be used herein may be any as long as it has an effect of assisting or promoting nerve induction. In one embodiment, 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. In one embodiment, the neural inducer can be B27 supplement (GIBCO).

[0110] The above conditions can be employed to maintain neural stem cells (eg, neural crest cells), but are not limited thereto.

[0111] In order to induce neural crest cells from adipose-derived stem 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. (GIBCO), lOOUZml penicillin, and culture in a medium supplemented with 100 gZ ml streptomycin, including but not limited to: 37 ° C, 20% oxygen, 5% carbon dioxide and 80% In the present invention, it was found that EGF is important for induction.

[0112] In another embodiment, 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. For example, 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. For example, neural stem cell markers include Nestin (NM006617 (human)), Musashi—1 (NM002 442 (blue)), CD133 (NM0006017 (blue)), notchl (NM017617 (blue)), Hesl (NM005524 (が)), Mashl (NM004316 (卜)), Neurogenin (NM006161 (ヒ)), Pax6 (NM001604 (卜)), CD15 (NM002033 (卜)), PDGFR (NM0026 09 (human)) Although it is mentioned, it is not limited to these. Preferably, neural stem cell markers include Nestin and Musashi-l.

[0113] In another embodiment, 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)). In one embodiment, 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.

[0114] In another embodiment, the method wherein the stem cell expresses a neural crest cell marker or neural stem cell marker! The method 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. Preferably, for example, 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. It is not limited. For example, neural stem cell markers include Nestin (NM006617 (tl)), Mu sashi-1 (NM002442 (chicken)), CD133 (NM0006017 (chicken)), notchl (NM017 617 (chicken)), Hesl (NM005524 (卜)), Mashl (NM004316 (()), Neurogenin (NM006161 (卜)), Pax6 (NM001604 (卜)), CD15 (NM002033 (卜)), PDGFR (NM002609 (human)) But is not limited to these! Preferably, neural stem cell markers include Nestin and Musashi-l. In this case, 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.

[0115] 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.

[0116] In another embodiment, 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.

[0117] (Adipose-derived neural crest cells)

In the present invention, 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. Preferably, the fat can be a fat-derived stem cell, but it will be understood that the adipose tissue itself may be used.

[0118] 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.

[0119] 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). 06941 (Hui)), Snail (NM005985 (H 卜)), Twist (NM000474 (H 卜)), Pax3 (N M000438 (H 卜)), Pax7 (NM002584 (H 卜)), HNKl (NM004854 (H 卜) )), P75 NTR (NM002507 (human)), TRP2 (NM006267 (human)), Wntl (NM005430 (human)), PO (NM002723 (human)) tPA (NM000930 (human))) can be expressed.

[0120] 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.

[0121] In 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.

[0122] In one embodiment, in the cell of the present invention, Leptin (NM000230 (human)) expression is lower than that of a fat-derived stem cell.

[0123] In a preferred embodiment of the present invention, 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.

[0124] (mixture)

In another aspect, 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.

[0125] (Composition)

In another aspect, 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”. .

[0126] Here, 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. Examples of such methods include, but are not limited to, methods such as the use of immunosuppressants and steroids. Immunosuppressants that prevent rejection include, for example, “Cyclosporine” (Sunday Miyun Z Neoral), “Tacrolimus” (Prograf), “Azathioprine” (Imlan), “Steroid Hormone” (predonin, methylpredonin) ), And “T-cell antibodies” (ΟKT3, 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.

[0127] 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. As such a carrier

Depending on its application, any carrier described herein can be selected and used by one of ordinary skill in the art.

[0128] (Methods of cell transplantation for the treatment of nervous system diseases, disorders or conditions)

In another aspect, 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. Here, 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”.

[0129] The neural crest cells can be administered by any method known in the art. For example, the neural crest cells can be injected using a syringe, a catheter, a tube, and the like, but are not limited thereto. Preferably, exemplary dosage forms include, but are not limited to, local injection (subcutaneous injection, intramuscular or intramuscular injection), intravenous injection, intraarterial injection, or tissue administration. For example, as an advantage compared with the prior art, for example, (1) it is necessary to create a regenerated tissue ex vivo (ex vivo production); 2) Larger tissue regeneration is possible more reliably; (3) It can be produced by simple and short-time treatment; (4) Needle insertion without the need for surgery to open organs such as skin It is possible to administer (transplant) cells and tissues by, but not limited to.

[0130] (Use)

In another aspect, 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. Here, 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] Hereinafter, 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.

Example

[0132] Reagents used in the following examples were obtained from Wako Pure Chemicals and Sigma force unless otherwise specified. Animal breeding was created by the National Society for Medical Researchg (Principles of Laboratory Animal CareJ and the Institute of Laboratory Animal Resource S, National Institute of Healths, published `` Guide for the Care and Use of Laboratory AnimalsJ ( NIH Public Ratio, No. 86-23, 1985, revised), and conducted in accordance with the spirit of animal welfare.

(Example 1: Preparation of adipose-derived stem cells using collagenase)

In this example, first, 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.

[0134] 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.

[0135] Add 0.16M NH 4 ClZPBS to the pellet obtained by centrifugation and suspend it at room temperature.

Four

Incubated for 10 minutes. This sample was subjected to suction filtration using a mesh (Whatman) having a diameter of 100 μm. The obtained filtrate was centrifuged at 1200 × g for 5 minutes. This cell preparation is also called PLA. Stem cells are confirmed using cell markers (eg, CD4, CD13, CD34, CD36, CD49d, CD71, CD90, CD105, CD117, CD151, etc.).

[0136] (Example 2: Preparation of cell suspension of liquid partial force of aspirate by liposuction)

Use one of the following two methods for the liquid part of the aspirate by liposuction. Thus, a stem cell suspension was prepared. Either of the following two methods is different from the conventional method in that no treatment with an enzyme such as collagenase is required, and therefore no enzyme such as collagenase is mixed.

[0137] (I) Preparation Method 1

1) The liquid part (usually about 2-4 liters) of the aspirate by liposuction was centrifuged at 400 X g for 10 minutes.

2) The supernatant was discarded. However, since the precipitated cells tend to float, they were carefully aspirated using an aspirator so as not to damage the cells.

3) The precipitated cells (mostly red blood cells) were transferred to several 50 ml polypropylene tubes and centrifuged (400 X g, 5 minutes).

4) The supernatant was aspirated and the precipitated cells were collected so that the total volume was 15-20 ml. When many matrix components were included, the matrix components were filtered out with a 100 / zm filter. Thereafter, centrifugation was performed as necessary.

5) Put 15 ml of Ficoll (registered trademark) into a 50 ml tube, and slowly add 15-20 ml of cell solution as much as possible to form a layer on it.

6) The tube was centrifuged at 400 xg for 30 minutes. (18-20 ° C)

7) After centrifugation, 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.

8) Add serum-containing PBS (10% FBS or PBS containing 10% human serum) to the collected cell suspension to make 50 ml, mix by pipetting, and centrifuge (400 X g, 5 minutes) ) did.

9) The supernatant was aspirated, again added with serum-added PBS to 50 ml, mixed by pipetting, and then centrifuged (400 X g, 5 minutes).

10) The supernatant was aspirated and a cell group containing the precipitated stem cells was collected.

[0138] (Π) Preparation method 2

1) In the clean bench, use the suction tube to suck the liquid part of the sucked material by liposuction. The filtrate was sealed in a closed separation bag through a reservoir with a filter (pore size; 120 / zm).

2) Using a cell separator (blood component separator ASTEC204, Amco Corporation, Tokyo, Japan), centrifuge three times to obtain platelet components with low specific gravity, red blood cells with high specific gravity, and granulocyte components. Removed as much as possible.

3) A fraction (about 30 to 40 ml) containing a high concentration of stem cells was collected. The specific gravity of the isolated cells ranged from 1.050 to 1.075.

[0139] Rough cell specific gravity is determined by mixing density gradient centrifuge separation media such as Percoll ^™ , Readygrad ^™, 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.

[0140] (Example 3: Characterization of recovered stem cells)

The stem cells collected in Example 2 were characterized using FACS according to the following procedure:

[0141] Approximately 5 ml of the cell suspension was washed twice with staining medium (SM; PBS containing 0.5% ushi serum albumin and 0.05% NaN3). Cells were counted as needed.

[0142] About 1 to: LO X 10 ⁶ cells A final concentration of 0.001-0.

A gZml labeled antibody (phycoerythrin (PE), allophycocyanin (APC) and Z or fluorescein isothiocyanate (FITC) was used for labeling) was added.

[0143] After incubating the mixture for about 30 minutes on ice, the cells were washed, and the concentration of the cell suspension was adjusted to about 5 X 10 ⁵ cells Zml using SM.

[0144] 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.

[0145] 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.

(Table 4: Expression of various CDs in stem cells after 2 passages) CD expression level

Three

Four

11c

13 + +

14

15

16

19

29 + +

31 +

33

34 +

36 + +

38

44 +

45 +

49d ++

54 +

56

58 +

61

62E

62P

69

71 + + 73 + +

90 + +

104

105 + +

106

117 +

135

144

146 +

151 + +

235a-SH3 +

STRO- 1 +

“One” = expression not detected

"+" = Detected in 20% or less of cells

"++" = detected in more than 20% of cells

From the above results, 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.

[0147] (Example 4: Characterization of stem cells collected from the liquid portion of the aspirate obtained by liposuction of multiple subjects)

Furthermore, stem cells were collected from the liquid portion of the aspirate obtained by multiple subject force liposuction and characterized. The results are shown below.

[0148] (Table 5: Multiple subject forces Stem fines collected from the liquid portion of aspirate obtained by liposuction (Results of cell characterization)

[Table 5]

Numbers indicate the percentage of stem cells expressing each protein in the cell population.

“One” = expression not detected, “+” = expression detected, N. T. = not tested.

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.

[0151] 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.

[0152] When 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. Conversely, the expression of CD105 expressed by this population of stem cells was not normally seen in fibroblasts. In addition, the expression of CD49d expressed by this stem cell population was not normally seen in bone marrow-derived mesenchymal stem cells.

[0153] In addition, 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]

(Isolation of human adipose-derived stem cells)

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. In summary, 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. Alternatively, 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.

[0155] (Cell culture of neurosphere)

For neurosphere culture, 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

2

Cultured in a culture medium. 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 μgZml 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.

[0156] (Result)

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. began (data not shown.) 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.

[0157] (Example 6: Quantitative real-time RT-PCR (reverse transcriptase polymerase chain reaction)) 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.

[0158] Expression of the undifferentiated neural stem cell marker genes Nestin and Musashi-1 and the expression of the adipogenic differentiation marker Leptin were performed using conventional real-time data using ABI PRISM 7700 (Applied Biosyst ems, Foster City, CA). Analyzed by quantitative RT-PCR. The gene expression of the target sequence was normalized for the expression of the housekeeping gene j8-actin. The transcription level in the control (undifferentiated adipose-derived stem cells) group was expressed as 1. TaqMan theory and assays with designed primers and probe sets were used for human Nestin, Musashi-1, Leptin and j8-actin. All primer and probe sets were purchased from Applied Bio systems. The primer sets used are as follows. TaqMan Gene Expression Assays system (Applied Biosystems), β-actin: 1 ml of Hs99999903; Nestin: Hs00707120-si; Musashi: Hs00159291-ml; Leptin: Hs001748 77-ml.

[0159] (Result)

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.

[0160] (Summary)

In this example, 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. In this example, 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.

[0161] (Example 7: Whole mouse embryo culture and transplantation of Eurosphere-like cells)

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). 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. In this example, 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.

[0162] Mouse whole embryo culture was performed as described below: Nagase T et al., J Anat 2 003; 203: 77-88, Nagase T et al., J Craniofac Surg 2005; 16: 80-88, Na gase T et al., Genes Cells 2005; 10: 595-604 and Yamada Y et al., J Cranio fac Surg 2005; 16: 1055-1061.

[0163] Nine mouse embryos (8-day embryos (E8)) were removed without destroying the yolk sac. A micropipette was then used to transfer GFP-transferred eurosphere cells into the head region of the embryo. These embryos were cultured for about 40 hours and examined for the presence of GFP-positive transplanted cells under a fluorescence microscope.

[0164] (Result) GFP positive cells transplanted into mouse embryos were clearly observed in 2 out of 9 embryos and seemed to survive. The GFP-positive cells were incorporated into the craniofacial region and the heart and trunk of these two embryos (see Figure 3). _{0 In} particular, the transplanted cells lined up along the second arch. (See arrows in Figures 3B and C.) ₀ This was very similar to the pattern of neural crest cells migrating in the second arch. This result suggests an interesting possibility that neurosphere cells derived from adipose-derived stem cells have neural crest cell-like properties.

(Summary)

Attempts to transfer neurospheres into cultured mouse embryos have shown that some of these cells migrate along the second arch and contribute to craniofacial morphogenesis. This cell migration pattern was very similar to that of the cranial crest cells, as previously reported by the inventors (Nagase T et al., J Anat 2003; 203: 77-88). This neural crest cell is an embryonic cell population characterized by extensive migration and a unique differentiation repertoire. Neural crest cells are often considered as stem or progenitor cells for peripheral and Schwann cells. The craniofacial skeleton mesenchyme also guides the neural dyke. Therefore, according to this example, it was confirmed that the transplanted-eurosphere had the same migration ability as the neural crest cell which is a neural progenitor cell in vivo.

[0165] 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.

[0166] Normally, it takes a period of about one month to form neural stem cell force-eurosphere, but 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.

[0167] From the above results, 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.

Therefore, 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]

(Isolation of mouse adipose-derived stem cells)

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.

[0170] (Cell culture of neurosphere)

Using the same method as in Example 5, 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.

[0171] Adipose-derived stem cells were treated with neurosphere culture medium (serum-free, EGF and basic

When cultured in (including FGF), it is confirmed that floating adipose-derived stem cells begin to form small clumps. Aggregation of neurosphere-like cells is observed. The number and size of this sphere will gradually increase. After passage, it is confirmed that a new spherical cell mass is formed. This suggests the neurosphere's ability to self-replicate.

(Example 9: Quantitative real-time RT—PCR (reverse transcriptase polymerase chain reaction)) Using the same method as in Example 6, except that the mouse-eurosphere cultured in Example 8 was used. Then, the expression of undifferentiated neural stem cell marker genes Nestin and Musashi-1 and the expression of the adipogenic marker Leptin are analyzed.

[0173] It is confirmed that the expression of the Nestin and Musashi-l genes is significantly up-regulated in the neurosphere compared with the control fat-derived stem cells cultured in the neurosphere culture medium. This result indicates that the neurosphere has the characteristics of neural progenitor cells. In contrast, we confirm that Leptin expression decreases dramatically in neurospheres. This result indicates that the neurosphere's ability to produce fat has decreased.

(Example 10: Mouse whole embryo culture and mouse-eurosphere-like cell transplantation) A method similar to that in Example 7 is used except that the mouse-spheres cultured in Example 8 are used.

[0175] 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. In particular, 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.

[0176] Normally, it takes a period of about one month to form neural stem cell force-loss spheres, but the mouse adipose-derived stem cells obtained in this example form the spheres in about one week. To be observed. Furthermore, it is confirmed that 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.

[0177] From the above results, it can be confirmed that 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.

Accordingly, it is understood that 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.

(Example 11: Effect of medium in cell culture of Eurosphere)

Instead of using DMEMZHAMF12 (1: 1) as the medium for neurosphere culture, use 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.

[0180] Adipose-derived stem cells were treated with neurosphere culture medium (serum-free, EGF and basic

When cultured in (including FGF), it is confirmed that floating adipose-derived stem cells begin to form small clumps. Aggregation of neurosphere-like cells is observed. The number and size of this sphere will gradually increase. After passage, a new spherical cell mass is formed. Is confirmed. This suggests the neurosphere's ability to self-replicate.

[0181] (Quantitative real-time RT—PCR (reverse transcriptase polymerase chain reaction))

Except for using mouse-eurosphere cultured in this example, expression of undifferentiated neural stem cell marker genes Nestin and Musashi-1 and expression of adipogenic 分 marker Leptin were performed using the same method as in Example 6. Analyze expression.

[0182] It is confirmed that Nestin and Musashi-l gene expression is significantly up-regulated in neurospheres compared to control adipose-derived stem cells cultured in neurosphere culture medium. This result indicates that the neurosphere has the characteristics of neural progenitor cells. In contrast, we confirm that Leptin expression decreases dramatically in neurospheres. This result indicates that the neurosphere's ability to produce fat has decreased.

(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.

[0184] 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.

[0185] (Summary)

Based on the above results, cultivated in any medium of BME, MEM, DMEM or HAMF12-Even when using Eurospheres, the results are the same as when using VMEM with DMEM / HAMF12 (1: 1) medium I can confirm that.

(Example 12: Effect of maintenance-cultured adipose-derived stem cells)

In Example 5, the human adipose-derived stem cells isolated in the above are passaged 5 times in DMEM medium to prepare and culture eurosphere.

[0187] (Quantitative real-time RT—PCR) The expression of the neural stem cell marker Nestin and Musashi-1 gene and the adipogenic marker Leptin is analyzed using the same method as in Example 6 except that the spheres of this example are used.

[0188] It is confirmed that the expression of the Nestin and Musashi-l genes is significantly up-regulated in the neurosphere compared with the control fat-derived stem cells cultured in the neurosphere culture medium. This result indicates that the neurosphere has the characteristics of neural progenitor cells. In contrast, we confirm that Leptin expression decreases dramatically in neurospheres. This result indicates that the neurosphere's ability to produce fat has decreased.

[0189] (Whole embryo culture and transplantation of mouse-rothsphere-like cells)

The same method as in Example 7 is used except that the spheres of this example are used.

[0190] 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. In particular, 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.

[0191] (Summary)

From the above results, it was proved that 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.

[Example 13: -Expression of neural crest cell marker in Eurosphere]

Prepared in Examples 5 8 and 11-Using the spheres, the quantitative real-time RT-PCR method similar to that in Example 6 was used to determine the neural crest cell marker gene CRA BP1 (NM004378 (human)), AP2 ( NM002097 (human)), Slug (NM003068 (human)), SoxlO (NM006941 (chicken)), Snail (NM005985 (chicken)), Twist (NM000474 (chicken) )), Pax3 (NM000438 (human)), Pax7 (NM002584 (human)), HNKl (NM004854 (human)), p75NTR (NM002507 (human)), TRP2 (NM006267 (human)), Wntl (NM0 05430 (human)) ), PO (NM002723 (human)), tPA (NM000930 (human)) are analyzed.

[0193] 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.

[0194] It is also possible to confirm that neurospheres express neural crest cell markers by specific staining methods, immunohistochemical methods, in situ hybridization methods, and Western blotting methods for each marker.

[0195] As described above, it is understood that the scope of the present invention should be construed only by the scope of the claims by exemplifying the present invention using the preferred embodiments of the present invention. The patents, patent applications, and literature cited herein are to be incorporated by reference in their entirety, as if the contents themselves were specifically described herein.

Industrial applicability

[0196] 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.

Claims

The scope of the claims

[I] Neural crest cells derived from fat.

[2] The neural crest cell according to claim 1, induced by adipose-derived stem cell force.

[3] The neural crest cell according to claim 1, wherein the neural crest cell has a migration ability.

[4] The method according to claim 3, wherein the migration ability is confirmed by transplanting to the head region of the embryo and then observing the migration ability to the mesenchyme of the frontal nasal bump, maxillary bump, or arch. The described neural dyke cell.

[5] The neural crest cell expresses at least one neural stem cell marker

The neural crest cell according to claim 1.

6. The neural crest cell according to claim 5, wherein the neural stem cell marker is selected from the group consisting of Nestin, Musashi-1, CD133, notch1, Hesl, Mashl, Neurogenin, Pax6, CD15 and PDGFR.

[7] The neural crest cell according to claim 6, wherein the neural stem cell marker includes Nestin and Musashi-1.

[8] The neural crest cell expresses at least one neural crest cell marker

The neural crest cell according to claim 1.

[9] The neural crest cell marker is CRABP1, AP2, Slug, SoxlO, Snail, Twist, Pax

The neural crest cell according to claim 8, which is selected from the group consisting of 3, Pax7, HNK1, p75NTR, TRP2, Wntl, PO and tPA force.

[10] In the neural crest cells, Leptin expression is lower than that of adipose-derived stem cells.

The neural crest cell according to claim 1, characterized in that it is V ヽ.

[II] The neural crest cell according to claim 1, wherein the neural crest cell expresses Nestin and Musashi-1, and the expression level of Leptin is lower than that of an adipose-derived progenitor cell. .

12. The neural crest cell according to claim 1, wherein the fat is derived from a mammal.

13. The neural crest cell according to claim 12, wherein the fat is derived from a human.

[14] A method for preparing neural crest cells comprising the following steps:

A) the step of obtaining fat stem cells as well B) Step of subjecting the stem cells to conditions for inducing neural crest cells

Including the method.

15. The method according to claim 14, wherein the anterior stem cell is an adipose-derived stem or stromal cell.

16. The method according to claim 14, wherein the step of obtaining stem cells includes collagenase treatment and centrifugation treatment.

17. The method according to claim 14, wherein the stem cell is selected from the group consisting of PLA cell and LAF cell force.

[18] The method according to claim 14, wherein in step B), the adipose-derived progenitor cells are seeded at a density of about 1 × 10 ⁴ cells / ml to about 1 × 10 ⁶ cells Zml.

[19] The method according to claim 18, wherein in the step B), the adipose-derived stem cells are seeded at a density of about 1 × 10 ⁵ cells Zml.

[20] The method according to claim 14, wherein in step B), the adipose-derived stem cells are cultured in a medium containing at least epidermal growth factor (EGF).

[21] The step (B), wherein 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 method described in 1.

[22] The nerve-inducing factor is piotin, L-carcin, corticosterone, ethanolamine, D (+) galactose, dartathione (reduced form), linoleic acid, linolenic acid, progesterone, retin-noreacetate , Selenium, triodothyronine (T3), DL-a tocopherol, DL-a-tocopherol acetate, albumin (usi), catalase, insulin, superoxide dismutase, transferrin The method described.

[23] The method according to claim 14, wherein the step B) comprises medium strength glutamic acid and aspartic acid used for culturing the stem cells.

[24] Medium power used for culturing the stem cells after the step B) Eagle basic medium (BME), minimum essential medium (MEM), Dulbecco's modified Eagle medium (DMEM) or HAMF12 medium The method according to claim 14, or a mixed medium thereof.

[25] Medium power Dulbecco used for culturing the stem cells in the step B) 15. The method according to claim 14, wherein the modified eagle medium (DMEM) / F12 (l: 1).

[26] The method according to claim 14, wherein the step B) includes replacing the medium with a new medium on days 2 to 6.

[27] The method according to claim 14, wherein the step B) comprises replacing the medium with a new one on the 4th to 5th days.

[28] The method according to claim 14, wherein the step B) includes passaging on day 6 to day 10.

[29] The method of claim 14, wherein the fat is derived from a mammal.

[30] The method of claim 29, wherein the fat is derived from a human.

[31] In the step B), the fat-derived stem cells are human-derived,

The adipose-derived stem cells are cultured in a medium containing epidermal growth factor, basic fibroblast growth factor, nerve inducer, penicillin and streptomycin as auxiliary components, wherein the nerve inducer comprises piotin, L— Carcin, corticosterone, ethanolamine, D (+) —galactose, dartathione (reduced form), linoleic acid, linolenic acid, progesterone, lettyl acetate, selenium, tolydothyronine (T3), DL— a-tocopherol, DL-a-tocopherol acetate, albumin (usi), catalase, insulin, superoxide dismutase, transferrin, and

15. The method according to claim 14, wherein the medium is Dulbecco's Modified Eagle Medium (DMEM) ZF12 (1: 1).

[32] The method according to claim 14, further comprising the step of confirming whether the stem cells express a neural stem cell marker.

[33] The method according to claim 32, wherein the neural stem cell marker is selected from the group consisting of Nestin, Musashi-1, CD133, notch1, Hesl, Mashl, Neurogenin, Pax6, CD15, and PDGFR.

[34] The method according to claim 33, wherein the neural stem cell marker comprises Nestin and Musashi-1.

[35] The step of confirming whether the stem cell expresses a neural crest cell marker is further included. 15. The method of claim 14, comprising.

[36] The method according to claim 14, wherein the cell expresses at least one neural crest cell marker.

[37] 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 force. The method described in 1.

[38] The method according to claim 14, further comprising the step of confirming the level of expression of the adipocyte marker in the stem cells.

[39] The method according to claim 38, wherein the confirmation of the level of the adipocyte marker is confirmation of a decrease in Leptin.

[40] The method according to claim 14, wherein the neural crest cells become a fat-derived neural crest cell precursor containing neural crest cells and adipose-derived stem cells by the step B).

[41] The method further includes the step of confirming whether the stem cell expresses a neural stem cell marker and a neural crest cell marker, and the step of confirming the level of expression of the adipocyte marker by the stem cell. The method described in 1.

[42] The method according to claim 14, further comprising the step of confirming whether or not the stem cells have migration ability.

[43] The method according to claim 42, wherein the migration ability is confirmed by transplanting to a head region of an embryo and observing the subsequent migration ability.

[44] The method according to claim 41, wherein the neural stem cell marker is Nestin and Musashi-1, and the fat cell marker is Leptin.

[45] A composition for cell transplantation for the treatment of a nervous system disease, disorder or condition, comprising the cell of claim 1.

[46] The composition according to claim 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.

[47] The composition according to claim 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.

[48] The fat is in a heterogeneous relationship with the individual subject to the nervous system disease, disorder or condition 46. The composition of claim 45, wherein the composition is derived from an individual.

[49] The composition according to claim 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.

[50] A method for cell transplantation for the treatment of a nervous system disease, disorder or condition comprising the step of administering the cell of claim 1.

[51] The method according to claim 50, 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.

[52] Use of the cell of claim 1 for the preparation of a medicament for treating or preventing a nervous system disease, disorder or condition.

[53] The use according to claim 52, 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.

[54] A fat-derived neural crest cell mixture comprising neural crest cells and adipose-derived stem cells.

[55] The fat-derived neural crest cell mixture according to claim 54, further comprising neural stem cells.