WO2011083752A1

WO2011083752A1 - Gpr40-positive bone marrow stem cell

Info

Publication number: WO2011083752A1
Application number: PCT/JP2010/073911
Authority: WO
Inventors: 山嶋哲盛
Original assignee: 国立大学法人金沢大学
Priority date: 2010-01-05
Filing date: 2010-12-28
Publication date: 2011-07-14
Also published as: JPWO2011083752A1

Abstract

Disclosed is a G-protein coupled receptor 40 (GPR40)-positive bone marrow-derived mesenchymal stem cell which can be differentiated into a neural cell when induced with a polyunsaturated fatty acid (PUFA). Specifically disclosed are: a GPR40-positive bone marrow-derived mesenchymal stem cell which is derived from a mammal; and a CD29-positive, CD90-positive, CD73-positive, CD105-positive, CD3-negative, CD4-negative, CD8-negative, CD14-negative, CD34-negative, CD45-negative and CD31-negative bone marrow-derived mesenchymal stem cell.

Description

GPR40 positive bone marrow stem cells

The present invention relates to a stem cell derived from bone marrow, an isolation method thereof, and use thereof.

G-protein coupled receptor 40 (GPR40) is a member of a subfamily of G-protein coupled receptor (GPCR), which is a 7-transmembrane receptor, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), polyunsaturated fatty acids (PUFA) such as arachidonic acid are endogenous ligands of the GPR40 receptor (also called fatty acid receptor, FFAR1).
Among PUFAs, DHA has been reported to be involved in the survival and neurite formation of cerebral cortical neurons and hippocampal neurons (see Non-Patent Documents 1 and 2).
Since the GPR40 gene was identified in the human pancreas and brain in 2003, the role of GPR40 has been mainly studied for its involvement in insulin secretion. However, the role of GPR40 in the brain has not been elucidated at all in the past. For example, there has been a report that GPR40 is not expressed in the rodent brain (see Non-Patent Document 3). According to the analysis at the gene level, GPR40 is expressed in various sites in the primate brain (see Non-Patent Document 4), and is most frequently expressed in the medulla, and is reported to be moderately expressed in the hippocampus. It had been. On the other hand, the expression at the protein level has not been confirmed regardless of the species, but recently, it has been reported that GPR40 protein is expressed in primate neural stem cells (see Non-Patent Document 5). . However, there are still many unclear points regarding the role of GPR40 in the brain and neurogenesis niche. Although GPR40 receptor has been reported to be detected in both whole bone marrow samples and bone marrow-derived stromal cell lines (see Non-Patent Document 6), it has been shown that specific cells express GPR40. Absent.
Furthermore, there is a report that DHA binds to GPR40 on the cell surface, promotes Ca ²⁺ influx, and stimulates cell protein kinase C (see Non-Patent Documents 3 and 4).
Bone marrow is known to contain bone marrow mesenchymal stem cells, which are tissue stem cells, and these cells differentiate into mesenchymal tissues such as bone and blood vessels, and are expected to be applied to regenerative medicine. . However, bone marrow mesenchymal cells are tissue stem cells and have a limited ability to differentiate.

An object of the present invention is to provide GPR40-positive bone marrow-derived mesenchymal stem cells that can be differentiated into nerve cells by induction of PUFA, and further to provide a method for using the stem cells.
The present inventor paid attention to the fact that GPR40 protein is expressed in the brain and that polyunsaturated fatty acids (PUFA) such as docosahexaenoic acid (DHA) function as a ligand for GPR40. We have made an extensive study on the relationship. As a result, the present inventor has found that bone marrow-derived mesenchymal stem cells expressing GPR40 exist, and further, the bone marrow-derived mesenchymal stem cells are transferred to nerve cells and others via the PUFA-GPR40 signaling system. It was found that the cells can be differentiated into cells. Thus, the present inventor isolated a new “GPR40 positive bone marrow-derived mesenchymal stem cell” and completed the present invention.
That is, the present invention is as follows.
[1] GPR40-positive bone marrow-derived mesenchymal pluripotent stem cells derived from mammals.
[2] The bone marrow-derived mesenchymal pluripotent stem cell according to [1], which is further CD29 positive, CD90 positive, CD73 positive and CD105 positive.
[3] The bone marrow-derived mesenchymal pluripotent stem cell according to [1] or [2], which is further CD3 negative, CD4 negative, CD8 negative, CD14 negative, CD34 negative, CD45 negative and CD31 negative.
[4] The bone marrow-derived mesenchymal pluripotent stem cell according to any one of [1] to [3] having the following characteristics:
(I) does not become cancerous;
(Ii) has the ability to differentiate into three germ layers; and (iii) has the ability to self-renew (self-replicating).
[5] The bone marrow-derived mesenchymal pluripotent stem cell according to any one of [1] to [4], which can be differentiated into a nerve cell by stimulation with a polyunsaturated fatty acid.
[6] The bone marrow-derived mesenchymal pluripotent stem cell of [5], wherein the polyunsaturated fatty acid is docosahexaenoic acid (DHA).
[7] (i) Adherent culture of bone marrow mononuclear cells, recovering the adhered cells,
(Ii) obtaining a single cell-derived colony from the collected cells;
(Iii) The cells of the obtained colony are cultured at a low density under low-concentration serum conditions in a medium prepared for expansion, expanded,
(Iv) The expanded cell of (iii) is further subcloned to obtain the cell as a single clone,
(V) confirming the expression of GPR40, CD29, CD90, CD73 and CD105 on the cell surface of the obtained cells, and obtaining cells in which expression of GPR40, CD29, CD90, CD73 and CD105 is observed as stem cells, [1] A method for isolating bone marrow-derived mesenchymal stem cells according to any one of [1] to [6].
[8] A method for isolating bone marrow-derived mesenchymal pluripotent stem cells according to [7], further comprising confirming the expression of CD3, CD4, CD8, CD14, CD34, CD45 and CD31 on the cell surface, and CD3 Obtaining a negative, CD4 negative, CD8 negative, CD14 negative, CD34 negative, CD45 negative and CD31 negative.
[9] The method for isolating bone marrow-derived mesenchymal pluripotent stem cells according to any one of [1] to [6], comprising isolating GPR40 positive cells from bone marrow mononuclear cells.
[10] The method for isolating bone marrow-derived mesenchymal pluripotent stem cells according to [9], further comprising isolating cells that are CD29 positive, CD90 positive, CD73 positive and CD105 positive.
[11] The bone marrow-derived mesenchymal pluripotent stem cell of [10] further comprising isolating CD3 negative, CD4 negative, CD8 negative, CD14 negative, CD34 negative, CD45 negative and CD31 negative cells. Separation method.
[12] A method for inducing differentiation into a nerve cell by contacting the bone marrow-derived mesenchymal pluripotent stem cell of any one of [1] to [6] with a polyunsaturated fatty acid in vitro.
[13] The method of [12], wherein the polyunsaturated fatty acid is docosahexaenoic acid (DHA).
[14] A composition for tissue regeneration comprising bone marrow-derived mesenchymal pluripotent stem cells according to any one of [1] to [6].
[15] The tissue regeneration composition according to [12], wherein the tissue is nerve tissue, adipose tissue, bone tissue, or cartilage tissue.
The GPR40-positive bone marrow-derived mesenchymal stem cells of the present invention have pluripotency to differentiate into not only neurons but also adipocytes, osteoblasts, chondrocytes and the like. In particular, the stem cells are differentiated into nerve cells via a GPR40-polyunsaturated fatty acid signal transduction system by stimulation of polyunsaturated fatty acids such as DHA. The stem cells of the present invention can be used for nerve regeneration in combination with polyunsaturated fatty acids, and can also be used for regeneration of other tissues.
This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2010-000736 which is the basis of the priority of the present application.

FIG. 1 is a view showing the properties of GPR40-positive bone marrow-derived mesenchymal stem cells. FIG. 1A shows the formation of calcified pieces that are stained purple by von Kossa staining on the 21st day, and FIG. Due to induction of fat formation, formation of lipid droplets dyed red by oil red 0 staining on day 35, and further, FIG. 1C shows formation of cartilage mass dyed blue by Arshan blue staining on day 35 by induction of ossification. Indicates.
FIG. 2 is a diagram showing expression of various expression markers in GPR40-positive bone marrow-derived mesenchymal stem cells. Among various markers, CD105, CD29, and CD90 are positive.
FIG. 3 is a diagram showing colony forming ability of GPR40 positive bone marrow-derived mesenchymal stem cells. The clonal expand group has a stronger colony-forming ability than the non-clonal group.
FIG. 4 is a diagram showing neuronal differentiation and GPR40 expression (cultured day 7) of bone marrow-derived mesenchymal stem cells in the clonal expand group. In GPR40 immunostaining, the target culture group (C) is slightly positive (green) as shown in FIG. 4A. FIG. 4B shows the results of a culture group (bFGF) to which bFGF was added. The culture group showed a strong positive (green), and neurite formation was also observed. FIG. 4C shows the results of the DHA-added culture group (DHA), which shows a decrease in the expression of GPR40 (green). This is a phenomenon called “receptor internalization” specific to GPCRs in which, when a receptor binds to a ligand and fulfills its role, its localization is changed and expression is reduced. FIG. 4D shows the result of the culture group (bFGF / DHA) to which both bFGF and DHA are added, and cells showing an increase in expression of GPR40 (green) and cells showing a decrease are mixed.
FIG. 5 is a diagram showing the results of investigating GPR40 gene expression of GPR40-positive bone marrow-derived mesenchymal stem cells by RT-PCR, and GPR40 gene expression shows the same tendency as protein expression by immunostaining.
FIG. 6 is a view showing the results of examining the expression of GPR40 in GPR40-positive bone marrow-derived mesenchymal stem cells by Western blot, and the protein expression of GPR40 shows the same tendency as in immunostaining and RT-PCR.
FIG. 7 shows the results of quantitative evaluation of GPR40 gene expression in GPR40-positive bone marrow-derived mesenchymal stem cells.
FIG. 8 is a diagram showing the expression (day 7 of culture) of the stem cell marker nestin and the young nerve cell marker βIII-tubulin in GPR40-positive bone marrow-derived mesenchymal stem cells differentiated into young nerve cells. FIG. 8A shows the result of double immunostaining of nestin and βIII-tubulin (Nestin / βIII-tubulin), and the target culture group (C) shows strong nestin positive (green). FIG. 8B shows the results of the culture group (bFGF) to which bFGF was added, in which a decrease in the expression of nestin (green) and an increase in the expression of βIII-tubulin (red) were observed. FIG. 8C shows the results of the DHA-added culture group (DHA), and both nestin (green) and βIII-tubulin (red) are weakly expressed. FIG. 8D shows the results of the culture group (bFGF / DHA) to which both DbFGF and DHA were added, while the expression of nestin (green) is hardly observed, whereas the expression of βIII-tubulin (red) is remarkable. .
FIG. 9 is a diagram showing the results of examining the gene expression of nestin and βIII-tubulin in GPR40-positive bone marrow-derived mesenchymal stem cells by RT-PCR, and the gene expression of nestin and βIII-tubulin is related to protein expression by immunostaining. The same tendency is shown.
FIG. 10 is a diagram showing the results of examining the gene expression of nestin and βIII-tubulin in GPR40-positive bone marrow-derived mesenchymal stem cells by Western blotting. It shows the same tendency as RT-PCR.
FIG. 11 is a diagram showing quantitative evaluation of nestin and βIII-tubulin gene expression of GPR40 positive bone marrow-derived mesenchymal stem cells.
FIG. 12 is a diagram showing expression of mature neuronal markers NF-M and Map2 (cultured on the 14th day) by GPR40-positive bone marrow-derived mesenchymal stem cells differentiated into mature neurons, and NF-M and Map2 2 Co-positive cells (yellow) seen by heavy immunostaining (Map2 / NF-M) have extended long neurites (arrows).
FIG. 13 is a diagram showing the results of RT-PCR examining the expression (day 14 of culture) of mature neuronal markers NF-M and Map2 by GPR40-positive bone marrow-derived mesenchymal stem cells differentiated into mature neurons. NF-M and Map2 gene expression is not observed in the subject group (C), whereas mild expression is observed in the DHA group, moderate expression in the bFGF group, and strong expression in the bFGF / DHA group.
FIG. 14 is a diagram showing the results of quantitative evaluation of gene expression of NF-M and Map2 in GPR40-positive bone marrow-derived mesenchymal stem cells differentiated into mature neurons.
FIG. 15 is a diagram showing the results of examining the expression of mature neuronal markers NF-M and Map2 (cultured on day 14) by GPR40-positive bone marrow-derived mesenchymal stem cells differentiated into mature neurons by Western blotting; The gene expression of NF-M and Map2 shows the same tendency as RT-PCR.
FIG. 16 shows the results of cell cycle analysis of GPR40-positive bone marrow-derived mesenchymal stem cells. FIG. 16A shows the results of the DHA-added culture group (DHA), with the majority of BrdU− / Ki67 + (green) cells and a slight mixture of BrdU + / Ki67 + cells (yellow). FIG. 16B shows the results of the bFGF-added culture group (bFGF), BrdU− / Ki67 + (green) cells decreased, BrdU + / Ki67 + cells (yellow) tended to increase, and BrdU + / Ki67− (red) cells. Is starting to appear. FIG. 16C shows the results of the culture group (bFGF / DHA) to which both bFGF and DHA were added. BrdU + / Ki67− (red) cells accounted for the majority, and a small number of BrdU + / Ki67 + cells (yellow) were also observed. A1 and C1 are enlarged photographs of A and C, respectively.
FIG. 17 shows the results of quantitative evaluation of BrdU + / Ki67− cells in the cell cycle analysis of GPR40-positive bone marrow-derived mesenchymal stem cells.
FIG. 18 is a diagram showing an outline of neuronal differentiation of GPR40 positive bone marrow-derived mesenchymal stem cells.
FIG. 19 is a schematic diagram comparing a process in which GPR40-positive bone marrow-derived mesenchymal stem cells differentiate into neurons (upper half) and a process in which neural stem cells differentiate (lower half).

Hereinafter, the present invention will be described in detail.
The stem cell of the present invention is a stem cell having GPR40 (G-protein coupled receptor 40) -positive pluripotency isolated from bone marrow.
GPR40 is a member of a subfamily of G-protein coupled receptor (GPCR), a seven-transmembrane receptor (Yamashima T., Progress in Neurobiology 84 (2008) 105-115; Briscoe C P. et al., The Journal of Biological Chemistry, 278 (2003) 11130-13113).
In the present invention, the “stem cell” refers to a cell having self-renewal ability and multipotency. Stem cells are usually able to regenerate the tissue when the tissue is damaged.
In the present invention, the term “stem cell” refers to a cell population containing at least a certain amount of stem cells, and includes, for example, a cell population containing 90% or more, preferably 95% or more of stem cells.
The stem cells of the present invention are positive for GPR40, express CD29 (integrin) and CD90 relatively strongly (CD29 positive, CD90 positive), and weakly express CD73 and CD105 (CD73 weakly positive, CD105 weakly positive). . Furthermore, lymphocyte markers such as CD3, CD4 and CD8, hematopoietic stem cell markers such as CD14, CD34 and CD45, and CD31 which is an endothelial cell marker are negative (CD3 negative, CD4 negative, CD8 negative, CD14 negative, CD34 negative, CD45 negative, CD31 negative). Here, a certain antigen being positive means having an antigen that is a cell surface antigen, that is, expressing the surface antigen on the cell surface. Whether these surface antigens are positive or negative or weakly expressed is whether the cells were stained with a secondary antibody labeled with a chromogenic enzyme or a fluorescent compound after reacting with the primary antibody against these antigens. It can be determined by examining whether or not by microscopic observation or the like. For example, cells can be immunostained using these antibodies to determine the presence or absence of surface antigens, or can be determined using magnetic beads to which the antibodies are bound. It can also be determined whether surface antigens are present using a FACS or flow cytometer. As the FACS and flow cytometer, for example, FACS vantage (manufactured by Becton Dickinson), FACS Calibur (manufactured by Becton Dickinson) and the like can be used.
The bone marrow-derived stem cells of the present invention have pluripotency and can differentiate into three germ layers (endoderm, mesodermal, ectoderm). The bone marrow-derived stem cells of the present invention proliferate from one cell to form a clone. Furthermore, the bone marrow-derived stem cells of the present invention do not have infinite proliferation and do not become cancerous.
The stem cells of the present invention can be isolated from bone marrow. As the bone marrow, bone marrows such as humans, non-human primates, mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, pigs, cows, horses, goats and the like can be used.
The stem cells of the present invention can be isolated from bone marrow by the following method. First, BM-MSC (Bone marrow-derived mesenchymal stromal cell) is isolated from the bone marrow. BM-MSC can be isolated by a known method. For example, bone marrow cells (BMC) are collected from the above animals by bone marrow puncture or the like, and density gradient centrifugation is performed to obtain a mononuclear cell fraction. The obtained mononuclear cell fraction was adherently cultured on a plastic dish for culturing, and the cells adhering to the dish were removed by trypsin treatment to obtain BM-MSC (Bone marrow-derived mesenchymal stromal cell). to recover. The recovered BM-MSC is cultured by the limiting dilution method, and the proliferated cells are further cultured by the limiting dilution method to obtain a clone as a single cell-derived colony. The colony cells thus obtained are cultured in a medium prepared for expansion at low density under low-concentration serum conditions and expanded. Expanded cells are further subcloned to obtain cells as a single clone. About the obtained cells, the expression of GPR40 on the cell surface can be confirmed by flow cytometry or the like, and only those in which GPR40 expression is recognized can be selected and used as the isolated stem cells of the present invention. At this time, the medium to be used is not limited, and a known medium such as MEM (Minimum Essential Medium) or DMEM (Dulbecco's modified Eagle's medium) medium containing animal serum such as fetal bovine serum (FBS) may be used. it can. A known stem cell culture medium can also be used, such as a mesenchymal stem cell basal medium (MSCBM) (Cambrex Bio Science) or a proliferation medium mesenchymal cell cell growth medium (MSCBM). ) (Cambrex Bio Science) or the like can also be used. To the medium, antibiotics such as penicillin and streptomycin, various physiologically active substances, vitamins and the like may be appropriately added. Note that the medium (growth medium) used when BM-SMC is first cultured to obtain clones as single cell-derived colonies contains 10 to 15% serum, but the obtained clones are expanded. Further, although the serum concentration of the medium (expanded medium) used for subcloning is lowered, it is specifically 1 to 5%, and if possible, about 3%. Thus, the stem cells of the present invention differ from embryonic stem cells (ES cells) in that they are isolated from bone marrow cells of a subject or a mature individual.
Furthermore, the stem cells of the present invention can also be isolated using direct flow cytometry without culturing from bone marrow cells or BM-MSC populations using GPR40 expression as an indicator. At this time, a magnetic field obtained by binding cells positive for lymphocyte markers such as CD3, CD4 and CD8, hematopoietic stem cell markers such as CD14, CD34 and CD45 and CD31 which is an endothelial cell marker from bone marrow cells in advance with antibodies against these antigens. GPR40 positive cells may be isolated from the remaining cell population by removal using beads.
As described above, the stem cell of the present invention can be obtained without introducing a foreign gene or protein into the bone marrow.
The isolated stem cells of the present invention can be induced to differentiate into specific tissue cells in vitro. That is, since the stem cell of the present invention has pluripotency, it can be induced to differentiate into any tissue cell of 3 germ layer origin. Media for inducing differentiation are commercially available, and differentiation can be induced into various tissues using these commercially available media. For example, Neurobasal Media (NM) (Invitrogen) or the like can be used to induce differentiation into nerve cells. In addition, in the case of inducing differentiation into bone cells, a bullet kit osteoblast differentiation medium (induction medium) (Cambrex Bio Science) can be used. Further, when differentiation induction into adipocytes is performed, Brett kit adipocyte differentiation medium (induction medium) (Cambrex Bio Science) may be used. When differentiation induction into chondrocytes is induced, Brett kit chondrocyte differentiation medium (induction medium). ) (Cambrex Bio Science). When differentiation is induced into nerve cells, the efficiency of differentiation is increased by adding polyunsaturated fatty acids (PUFA) to the medium. The bone marrow-derived stem cells of the present invention express GPR40 having a fatty acid as a ligand on the surface, and when polyunsaturated fatty acid binds to GPR40, signal transduction that induces differentiation occurs. As the polyunsaturated fatty acid, those of n-3 (ω3) line and n-6 (ω6) line can be used. Examples of polyunsaturated fatty acids such as n-3 series include DHA (docosahexaenoic acid; docosahexaenoic acid), EPA (eicosapentaenoic acid; eicosapentaenoic acid), α-linolenic acid, and the like. Arachidonic acid, linoleic acid, γ-linolenic acid and the like. Among these, DHA has been confirmed to be able to induce differentiation of the stem cells of the present invention into neurons at a low concentration, and is an ideal ligand. Also, bFGF and a polyunsaturated fatty acid may be used in combination because differentiation into nerve cells is induced by using bFGF (basic fibroblast growth factor).
Cells differentiated into these tissue cells can be further cultured to regenerate the tissue. The GPR40-positive bone marrow stem cells of the present invention are up-regulated and highly expressed when proliferating, but when differentiated into neurons, the expression of GPR40 is down-regulated and the expression is reduced or stopped. This decrease can be explained by a phenomenon called “receptor internalization”, which is characteristic of G-protein coupled receptor (GPCR).
Whether or not the stem cells of the present invention have been induced to differentiate into tissue cells can be determined by examining the expression of markers specific to each tissue cell. For example, the differentiation into nerve cells is based on the expression of NF-M, NF-H (neurofilament medium or heavy chain; medium, high molecular weight neurofilament), NSE (neuronic specific γ-enolase; nerve specific enolase), etc. You can investigate. Furthermore, production of mature neural microtubules and filaments can be used as an index. Differentiation into osteoblasts can be examined with von Kossa staining, differentiation into chondrocytes with Alshan Blue staining, and differentiation into adipocytes with positive findings for oil red 0 staining as indicators.
The bone marrow-derived stem cells of the present invention can be applied to regenerative medicine for various cerebrovascular disorders, brain tumors, neurodegenerative diseases, brain trauma and the like. For example, the bone marrow-derived stem cells of the present invention can be administered to the brain damaged by cerebral infarction, degeneration or trauma. Specific examples include cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage, brain tumor, Alzheimer's disease, Parkinson's disease, Huntington's disease, neurodegenerative diseases such as amyotrophic lateral effect, and cerebral contusion. For example, if cells are administered into a blood vessel such as the carotid artery, the stem cells reach the brain via the artery. It may also be administered directly into the brain. In any case, the polyunsaturated fatty acid may be systemically administered by intravenous injection or oral administration after or simultaneously with the administration of stem cells to the brain. Alternatively, the stem cells of the present invention may be preliminarily contacted with a polyunsaturated fatty acid in vitro to induce initial differentiation and then administered to the brain. Furthermore, after regenerating nerve tissue in vitro, the regenerated tissue can be transplanted to a damaged site of the brain. By administering to the brain, stem cells are differentiated into nerve cells, and damaged nerves and brains can be regenerated.
In these regenerative medicine, in order to avoid rejection by the recipient of the transplanted cells or tissues, bone marrow is collected from the patient who has a neurological disease to receive regenerative medicine, and the bone marrow of the present invention is derived from the bone marrow. GPR40 positive stem cells can also be isolated and autotransplanted. Furthermore, after the isolated and proliferated stem cells are contacted with a polyunsaturated fatty acid such as DHA, stimulation is performed to differentiate into nerve cells, or further nerve tissue is constructed, and then these nerve cells and tissues are transferred to the patient. Brain regeneration treatment can also be performed by transplanting into the cerebral organ. In addition, by eliminating the problem of rejection by the immune system with an immunosuppressant or the like, a third party bone marrow-derived stem cell of the present invention can be administered or transplanted to a patient and used for treatment.
Further, adipose tissue, bone tissue or cartilage tissue can be similarly regenerated from the stem cells of the present invention.
The present invention also includes a composition containing the stem cells of the present invention and used for the purpose of regeneration of nerve tissue such as brain, adipose tissue, bone tissue or cartilage tissue.
Furthermore, the bone marrow-derived stem cells of the present invention can be differentiated into specific tissues such as nerve tissue, adipose tissue, bone tissue, and cartilage tissue in vitro and used for drug effect determination and screening for specific drugs.
In addition, when the stem cells of the present invention are collected from a large number of subjects and then cryopreserved and the tissue needs to be regenerated, subjects who need tissue regeneration from the stored stem cells A cell having genetic characteristics suitable for the cell can be selected, and regenerative treatment can be performed using the cell without causing rejection. In order to use the stem cells of the present invention in this way, a cell bank may be constructed for stem cells collected from a large number of subjects.
Furthermore, the present invention encompasses a regenerative treatment method comprising administering the stem cells of the present invention to a patient in need of treatment for the treatment of a disease.
The present invention will be specifically described by the following examples, but the present invention is not limited to these examples.

1. Examination of GPR40 positive bone marrow-derived mesenchymal pluripotent stem cells Method Bone Marrow Cell Collection Kusida T. et al. From 3 Japanese macaques (Macaca fuscata), 2 to 3 years old and 3 to 4 kg in weight, provided by Japan BioResource (National BioResource Project, Okazaki Institute of Physiology). et al, I. et al. Stem Cells. (2002) 20: 155-162.
Isolation of Bone Marrow-Derived Stem Cells from Monkey BM-MSC (Bone Marlow-Derived Messychimalal Cell) Table 1 shows how to use the medium in this example.

BM-MSC-derived clones were cultured in serum-free neurobasal medium supplemented with N-2 supplements (R & D Systems). The experimental group was a group induced by bFGF or DHA alone (bFGF alone group or DHA alone group) and a group induced by DHA and bFGF (DHA + bFGF group).
Fresh pipetted bone marrow cells (BMC) were gently overlaid on Density Gradient Media (Axis-Shield) to obtain a single cell suspension, which was centrifuged for 30 minutes at 150 × 10 rpm, 20 ° C. Next, the mononuclear bone marrow cells collected from the interface of the whole bone marrow cell sample obtained by centrifuging the mononuclear cell fraction were adhered to plastic to obtain a BM-MSC (Bone marrow-derived mechanicalmal cell). That is, bone marrow mononuclear cells collected by density gradient centrifugation were treated with 5 ml of growth medium (GM) (Minimum Essential Medium (MEM), Alpha Medium GlutaMAX (Invitrogen, GIBCO), 10-15% FBS (fetal bovine serum) and Resuspended at 1 × 10 ⁴ cells / cm ² in growth medium (GM) containing 1% Antibiotic-Antilytic (Invitrogen, GIBCO) and cultured for 24-48 hours, half of the medium every 24 hours. Thereafter, cells other than single-adherent cells showing the form of fibroblasts were discarded, and half of the medium was replaced every 72 hours using GM and cultured for 9 ± 2 days. Reached ~ 60% confluence.
The cells were then cloned and clones were obtained. Adhered bone marrow mononuclear cells were detached using 0.25% trypsin containing EDTA and serially diluted into 48 well plates containing GM. After 8 hours, wells containing 10 or fewer adherent cells were further expanded. Colonies formed at 7 ± 1 days, then trypsinized and transferred to 24-well plates and 3 cm dishes. Three clones were established from each of the three Japanese macaques (hereinafter referred to as clonal expands).
Clonal expansion was performed under low-concentration serum conditions by seeding cells at low density in an expansion medium (EM). Non-cloned strains of BM-MSC were cultured at a high density in GM containing 10% FBS, and used as a control (non-clonal expand) group.
Details of the clonal expansion method are described below.
0.5-1 × 10 ³ / cm ² of BM-MSC is cultured in a culture flask containing 5 ml of EM (5% CO ₂ , 37 ° C.) every 72 hours until the cells reach 50-60% confluence. The medium was changed. The cells were suspended in 0.25% trypsin containing EDTA, washed twice with DPBS, and resuspended in EM at a density of 0.5-1 × 10 ³ / cm ² . The obtained cells were designated as passage 1.
Details of the non-clonal expand are described below.
1 × 10 ⁴ / cm ² of BM-MSC is cultured in a culture flask containing 5 ml of GM (5% CO ₂ , 37 ° C.), and the medium is changed every 72 hours until cells reach 50-60% confluence. did.
Cell viability was calculated using trypan blue.
It was confirmed to have at least 90% viability prior to each subcloning during expansion.
The cells obtained by clonal expansion were confirmed to be positive for GPR40 expression, and then used as bone marrow-derived stem cells of the present invention.
Differentiation of bone marrow-derived mesenchymal stem cells into neurons 0.5 to 1 × 10 ⁵ bone marrow-derived mesenchymal stem cells are cultured in a cell culture flask (25 cm ² ) containing 5 ml of FBS-free neuron medium (NM). (5% CO ₂ , 37 ° C.). NM is Neurobasal Media (Invitrogen, GIBCO, L-alanyl-L-glutamine a dipeptide substate for L-glutamine (Invitrogen, GIBCO, GLUTAMAX, -2505S). Anticotic (Invitrogen, GIBCO) was included, and culture was performed under conditions including bFGF (basic fibroblast growth factor) (bFGF alone group) and conditions including bFGF and DHA (DHA + bFGF group).
bFGF (Sigma-Aldrich) was used at a concentration of 10 ng / ml. DHA was used at a final concentration of 10 μM in the form of a complex with BSA (bovine serum albumin) (BSA: DHA complex). That is, 1 ml of 99% ethanol was added to a container containing 25 mg of dry DHA (molecular weight 328.5), dispensed into four 1 ml sterile Eppendorf tubes, and evaporated in a vacuum evaporator (6. Including 25 mg DHA). 0.5 g of free fatty acid free BSA powder was dissolved in 3.13 ml of sterile double distilled water (2.4 mM). 6.25 mg of DHA is dissolved in 25 μl of 0.1 M NaOH, 1.9 ml of 2.4 mM free fatty acid free BSA is added, incubated at 50 ° C. for 5 minutes, 10 mM (× 1000) BSA: A DHA complex stock solution was obtained. The stock solution was stored frozen at −20 ° C. and used within 2 weeks.
Differentiation of bone marrow-derived mesenchymal stem cells into adipocytes, osteoblasts and chondrocytes differentiated into adipocytes Bone marrow-derived mesenchymal stem cells from adipocyte induction / maintenance medium (Cambrex Bio Science) ) Was used for 3 cycles of incubation. That is, the cells were cultured for 4 days using an adipocyte induction medium, and then cultured for 3 days using an adipocyte maintenance medium. This induction / maintenance was repeated for 3 cycles, and further cultured for 7 days using an adipocyte maintenance medium.
Differentiation into osteoblasts Bone marrow-derived mesenchymal stem cells that did not reach confluence were cultured for 5 weeks using an osteoblast induction medium (Cambrex Bio Science) until morphological changes were observed. The osteoblast induction medium was changed every 3-4 days.
Differentiation into chondrocytes Bone marrow-derived mesenchymal stem cells were added to a cartilage medium (Cambrex Bio Science), and the medium was changed every 2-3 days and cultured for 5 weeks.
FACS analysis of bone marrow-derived mesenchymal stem cells Bone marrow-derived mesenchymal stem cells were washed with DPBS and detached using 0.25% trypsin containing EDTA. The collected cells were treated with FITC (fluorescein isothiocyanate) or PE (phycoerythrin) -conjugated anti-human primate CD3, CD4, CD8, CD14, CD29, CD31, CD34, CD45 (BD Biosciences) antibody, anti-human CD90 (Biotrend), CD73 (BD). Pharmingen) and CD105 (Serotec) antibodies and stained using FACScan (Becton Dickinson).
Immunofluorescence analysis Proliferated bone marrow-derived mesenchymal stem cells were fixed with 4% paraformaldehyde (PFA) in methanol at room temperature or 20 ° C. for 15 minutes, and immunofluorescent staining was performed.
Primary antibodies used were rabbit polyclonal anti-GPR40 antibody (1: 100), mouse monoclonal anti-GPR40 antibody (1: 250, TransGenic Inc), mouse monoclonal anti-nestin antibody (1: 200, Chemicon, Millipore), mouse monoclonal anti-neuron. βIII-tubulin antibody (1: 500, Covance, Clone Tuj1, MMS-435P), mouse monoclonal anti-neurofilament medium chain (NF-M) antibody (1: 1000, Chemicon, Millipore), and rabbit anti-Map2 antibody. Secondary antibodies were green fluorescent anti-mouse Alexa Fluor 488 (Invitrogen) and anti-rabbit Alexa Fluor 488 (Invitrogen) and red fluorescent anti-rabbit Alexa Fluor 546 (Invitrogen) and anti-mouse Alexa Fluor 546 (Invitrogen).
Digital images were obtained using a confocal laser microscope (LSM510; Carl Zeiss) and LSM510 software (version 3.2SP2, Carl Zeiss). All fields of positive cells were obtained as a percentage of the total number of DAPI-labeled cell nuclei.
Western blot analysis Cell pellets were obtained by centrifugation from bone marrow-derived mesenchymal stem cells peeled using 0.25% trypsin containing EDTA, and then used with RIPA buffer (Sigma-Aldrich) and Protease Inhibitor Cocktail (Sigma-Aldrich) (In the case of culture containing GPR40, 0.1% Triton-X was further added). It was then centrifuged to extract the protein. Protein concentration was measured by Bradford assay.
Western blot analysis was performed with the amount of protein in each sample combined. The antibodies used were mouse monoclonal anti-GPR40 antibody (1: 1000, TransGenic Inc., Clone No. G16), rabbit polyclonal anti-GPR40 antibody (1: 1000), mouse monoclonal anti-nestin antibody (1: 1000, Chemicon, Millipore). Mouse monoclonal anti-βIII tubulin antibody (1: 500, Sigma-Aldrich, Clone SDL.3D10, T8660), rabbit anti-Map2 antibody (1: 2000, Chemicon, Millipore), and mouse monoclonal anti-NF-M antibody (1: 1000). Chemicon, Millipore).
Cell cycle analysis Bone marrow-derived mesenchymal stem cells were incubated with 10 μM BrdU (Sigma-Aldrich) for 2 hours, washed twice with DPBS for 10 minutes, and immunostained with rat anti-BrdU and mouse anti-Ki67 primary antibodies did. Evaluation of phenotypic distribution was performed using a laser confocal microscope 24 and 72 hours after induction of differentiation.
Semi-quantitative RT-PCR
Total RNA was collected from 1 × 10 ⁶ bone marrow-derived stem cells using TRI Reagent (SIgma-Aldrich), and reverse transcription was performed from 1 μg of total RNA using First-Strand Synthesis System (Invitrogen, SuperScript III). . Gene-specific primers can be obtained from the NCBI Primer-BLAST software (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) and the NCBI reference sequence ((Macaca multitta Genome5 The whole genome shotgun sequence (http://www.hgsc.bcm.tmc.edu/projects/rmaqueque/) was used to create RT-PCR, which is Platinum Taq DNA Polymerase High Fidelity (Invitrogen) and Thr. This was done using a Cycler (Applied Biosystems). The primer sequences are shown in Table 2.

2. Results Characteristics of Bone Marrow-Derived Mesenchymal Stem Cells Obtained by Clonal Expand Bone marrow-derived mesenchymal stem cells obtained by clonal expand are pluripotent cell standards (Dominici M et al., Cytotherapy) determined by Donimici et al. 9, 315-7, 2006). That is, it had characteristic surface molecules, differentiated into 3 germ layers, and had self-renewal (self-replication) ability over 50 passages.
The results of evaluation of bone marrow-derived mesenchymal stem cells using a flow cytometer are shown in FIG. It was negative for lymphocyte markers such as CD3, CD4, CD8, CD31 which is an endothelial cell marker, and CD14, CD34 and CD45 which are hematopoietic cell markers. On the other hand, CD29 and CD90 showed strong positive, and CD73 and CD105 showed weak positive. That CD14 and CD31 are negative indicates that macrophages and endothelial cells are not contained in the cells obtained by clonal expansion.
Colonies obtained from single cells of Japanese monkey bone marrow-derived mesenchymal stem cells expanded at low density under low serum conditions versus non-clonal expands obtained at high density at 10% serum conditions In addition to showing remarkable colony formation, proliferation ability was recognized after subculture after 3 passages (FIG. 3). Bone marrow-derived mesenchymal stem cells obtained by clonal expansion showed the characteristics of pluripotent stromal cells and maintained the differentiation ability into three germ layers for about 50 passages (FIGS. 1A, B, and C). That is, differentiation into chondrocytes was confirmed by Alcian blue staining using paraffin-embedded sections (FIG. 1A). After 4 weeks of adipocyte induction, differentiation into adipocytes was confirmed by oil red 0 staining (FIG. 1B). Furthermore, differentiation into osteoblasts was confirmed by von Kossa staining (FIG. 1C).
Expression of GPR40 receptor in proliferating or differentiated bone marrow-derived mesenchymal stem cells Expression of GPR40 mRNA and protein in proliferating cells and differentiated cells into neurons was determined by immunohistochemistry (FIGS. 4A, B, C, D), RT- Analysis was by PCR (Figures 5 and 7) and Western blot (Figure 6). Stable GPR40 expression was observed in cells growing in EM. On the other hand, when neuron medium was used, GPR40 production was attenuated (FIG. 4A). There was a difference in GPR40 expression between nerve induction by bFGF alone group (FIG. 4B) and bFGF + DHA group (FIG. 4D). That is, in the case of bFGF alone, the immunostaining property of GPR40 was the highest, and the immunostaining property was lowered when bFGF + DHA was used. It was understood that this was because DHA was bound to the GPR40 receptor and the GPR40 receptor was activated, so that it was transferred into the cell (receptor internalization). Regarding GPR40 expression during the process of neuronal differentiation induced by cytokines, stem cells were cultured in EM or NM before nerve induction and the results were compared in order to examine the extent of DHA-dependent neuronal phenotype acquisition.
On the other hand, in the case of DHA alone, the expression of GPR40 was significantly low (FIGS. 5 to 7). There was little distribution of cytoplasm, and no immunofluorescent signal was observed on the cell membrane (FIG. 2C). Even in the case of DHA + bFGF, no significant change was observed compared to the control of GPR40 expression. When comparing the DHA + bFGF group and the DHA single group, GPR40 expression was significantly lower in the DHA + bFGF group (FIGS. 5 to 7).
Expression of immature neuronal markers in proliferating or differentiated bone marrow-derived mesenchymal stem cells Expression of BM-MSC nestin and neural βIII-tubulin (Tuj1) during proliferation in EM was weak. Expression of both nestin and βIII-tubulin was observed in bone marrow-derived mesenchymal stem cells grown in serum-free NM for 72 hours (FIGS. 8A, B, C, D). From the results of immunofluorescence staining, it was found that there was a difference in the results of nerve induction under two conditions. No changes in marker expression were observed in the DHA alone group (FIG. 8C, FIGS. 9 to 11). On the other hand, bone marrow-derived mesenchymal stem cells grown with bFGF in EM showed up-regulation of nestin and slight expression of βIII-tubulin, whereas bone marrow-derived mesenchymal stem cells induced with bFGF + DHA are precursor markers. The expression of nestin was weak and up-regulation of immature neuronal marker βIII-tubulin was observed (FIG. 10). RT-PCR (FIGS. 9 and 11) and Western blot (FIG. 10) results were consistent with the immunofluorescence staining results, indicating significantly different levels of expression of nestin and βIII-tubulin. Bone marrow-derived mesenchymal stem cells grown in NM before nerve induction were not significantly different between bFGF alone and bFGF + DHA induction conditions.
Expression of mature nerve markers in differentiated bone marrow-derived mesenchymal stem cells Expression of mature nerve markers NF-M and Map2 was examined one week after nerve induction under bFGF alone and bFGF + DHA induction conditions. In both bone marrow-derived mesenchymal stem cells cultured in a normal medium and bone marrow-derived mesenchymal stem cells cultured in DHA alone, expression of the mature nerve marker was not observed particularly at the protein level (FIGS. 13 to 15). However, expression of mature nerve markers was observed in the bFGF alone group and the bFGF + DHA group (FIGS. 13 to 15). The bFGF alone group and the bFGF + DHA group induced NF-M and Map2 mRNA, and the bFGF alone group did not induce significant Map2 protein expression. Furthermore, the NF-M level was significantly higher than the Map2 level in the DHA + bFGF group (FIG. 14). Bone marrow-derived mesenchymal stem cells grown in EM prior to induction show a unique morphology with longer neurites extending than small cell bodies (Figure 12), whereas bone marrow-derived mesenchymal stem cells grown in NM Leaf stem cells had a fibroblast-like morphology and short processes.
Cell cycle analysis of differentiated bone marrow-derived mesenchymal stem cells BrdU is taken up by cells in S phase, and Ki67 is taken up by all cells in proliferation. Ki67− / BrdU + cells out of the cell cycle were counted by double immunostaining (FIGS. 16A, B, C, E). After 24 hours of BrdU incorporation, no significant difference was observed between the control and the 4 groups of bFGF, DHA and bFGF / DHA. However, 72 hours after induction of neural differentiation, the proliferation rate of bone marrow-derived mesenchymal stem cells decreased in the bFGF alone group (FIG. 16B) compared to the control group, and this tendency was particularly remarkable in the bFGF + DHA group ( FIG. 16C). That is, cells out of the cell cycle increased dramatically in the bFGF + DHA group compared to the control group (FIG. 17).
Reference example Role of GRP40 in neurogenesis in vivo GPR40 and phosphorylated cAMP response element-binding protein using a hippocampal neurogenesis monkey model promoted by transient ischemic stress We investigated the transcriptional factors involved in the positional relationship with pCREB), adult neurogenesis, learning and memory. Furthermore, brain-derived neurotrophic factor (BDNF) and its receptor tropomyosin receptor kinase B (TrkB) (both of which are present downstream of the gene transcript of pCREB) were also examined.
When the expression of pCREB was searched by Western blot, it was remarkably up-regulated after transient global cerebral ischemia and correlated temporally with increased neurogenesis in the hippocampus. It was found by immunofluorescence microscopy that the localization patterns of GPR40 and pCREB correspond completely. Both were co-expressed in mature and newborn neurons, as well as in astrocytes present in the lower dentate gyrus (SGZ). GPR40 / pCREB co-positive cells were markedly increased in SGZ 15 days after ischemia. ProBDNF was maximal on day 9 after ischemia, but mature BDNF (mBDNF) and TrkB receptors were not significantly altered when confirmed by Western blot. Immunofluorescence microscopy revealed that BDNF was expressed in newborn neurons but not TrkB. These results indicate that PUFA, GPR40, pCREB and BDNF are closely involved in the same signal transduction system and are involved in neuronal differentiation in the adult primate neurogenesis niche.
With regard to signals that promote adult neurogenesis in SGZ that occurs after transient ischemic stress, the inventors have suggested an association with GPR40, a free fatty acid receptor. In the hippocampal neurogenesis niche, GPR40 was found as a progenitor (neural or neuronal) in mature neurons as well as in astrocytes present in SGZ of normal (non-ischemic) adult Japanese monkeys. Furthermore, using an ischemia-promoting adult neuronal neonatal monkey model, it was found that GPR40 is upregulated after ischemia, particularly in SGZ neonatal neurons. These findings suggest that GPR40 is involved in neurogenesis in adults and is a major factor leading to PUFA signals. In addition, in recent studies, ARA or DHA has been used to transform PC12 cells or rat embryonic neural stem cells transformed with the GPR40 gene through activation of the GPR4-PLC / IP3 (phospholipase C / inositol triphosphate) signaling system. , It was found to increase intracellular Ca ²⁺ .
The expression of GPR40 in the neurogenesis niche was recently found using an ischemic monkey model of adult neurogenesis. Similar experiments profiled the expression pattern of pCREB and found that the intracellular distribution of pCREB was similar to that in the hippocampal neurogenesis niche of GPR40. GPR40 and pCREB proteins were co-distributed in almost all mature neurons of the dentate gyrus (DG) granule cell layer (GCL) and some of the newborn neurons, and also in SGZ astrocytes. Furthermore, both pCREB and GPR40 were similarly up-regulated 2 weeks after ischemia, peaking at day 15. These findings indicate that GPR40 and pCREB are functionally related and can act in the same signal transduction system leading to the effects of PUFA.
In the above study, the GPR40 receptor receives a PUFA signal typified by DHA, and phosphorylates CREB (cAMP response element-binding protein), which is one of important transcription factors. The production of brain-derived neurotrophic factor (BDNF) indicates that it is highly likely to be involved in the differentiation of new neurons and synaptic plasticity.
That is, GPR40 also transmits PUFA signals and differentiates neural stem cells in vivo, and GPR40-positive bone marrow-derived mesenchymal pluripotent stem cells are involved in neurogenesis in vivo. It was.

The stem cells of the present invention can be used for medical treatment such as regenerative medicine, and can also be used as a tool for developing new medicines such as determination of drug effects.
All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

A mammalian-derived GPR40-positive bone marrow-derived mesenchymal pluripotent stem cell.
Furthermore, the bone marrow-derived mesenchymal pluripotent stem cell according to claim 1, which is CD29 positive, CD90 positive, CD73 positive and CD105 positive.
Furthermore, the bone marrow-derived mesenchymal pluripotent stem cells according to claim 1 or 2, which are CD3 negative, CD4 negative, CD8 negative, CD14 negative, CD34 negative, CD45 negative and CD31 negative.
The bone marrow-derived mesenchymal pluripotent stem cell according to any one of claims 1 to 3, which has the following characteristics:
(I) does not become cancerous;
(Ii) has the ability to differentiate into three germ layers; and (iii) has the ability to self-renew (self-replicating).
The bone marrow-derived mesenchymal pluripotent stem cell according to any one of claims 1 to 4, which can be differentiated into a nerve cell by stimulation with a polyunsaturated fatty acid.
The bone marrow-derived mesenchymal pluripotent stem cell according to claim 5, wherein the polyunsaturated fatty acid is docosahexaenoic acid (DHA).
(I) Adherent culture of bone marrow mononuclear cells, collecting the adhered cells,
(Ii) obtaining a single cell-derived colony from the collected cells;
(Iii) The cells of the obtained colony are cultured at a low density under low-concentration serum conditions in a medium prepared for expansion, expanded,
(Iv) The expanded cell of (iii) is further subcloned to obtain the cell as a single clone,
(V) confirming the expression of GPR40, CD29, CD90, CD73, and CD105 on the cell surface of the obtained cells, and obtaining stem cells that express GPR40, CD29, CD90, CD73, and CD105. A method for isolating bone marrow-derived mesenchymal pluripotent stem cells according to any one of claims 1 to 6.
A method for isolating bone marrow-derived mesenchymal pluripotent stem cells according to claim 7, further comprising confirming the expression of CD3, CD4, CD8, CD14, CD34, CD45 and CD31 on the cell surface, and negative for CD3, Obtaining a CD4 negative, a CD8 negative, a CD14 negative, a CD34 negative, a CD45 negative and a CD31 negative.
The method for isolating bone marrow-derived mesenchymal pluripotent stem cells according to any one of claims 1 to 6, comprising isolating GPR40 positive cells from bone marrow mononuclear cells.
The method for isolating bone marrow-derived mesenchymal pluripotent stem cells according to claim 9, further comprising isolating cells that are CD29 positive, CD90 positive, CD73 positive, and CD105 positive.
The isolation of bone marrow-derived mesenchymal pluripotent stem cells according to claim 10, further comprising isolating cells that are CD3 negative, CD4 negative, CD8 negative, CD14 negative, CD34 negative, CD45 negative and CD31 negative. Method.
A method for inducing differentiation into a nerve cell by contacting the bone marrow-derived mesenchymal pluripotent stem cell according to any one of claims 1 to 6 with a polyunsaturated fatty acid in vitro.
The method according to claim 12, wherein the polyunsaturated fatty acid is docosahexaenoic acid (DHA).
A composition for tissue regeneration comprising bone marrow-derived mesenchymal pluripotent stem cells according to any one of claims 1 to 6.
The tissue regeneration composition according to claim 12, wherein the tissue is nerve tissue, adipose tissue, bone tissue or cartilage tissue.