JP2003018984A - Method for producing cell having multiple differentiation potency - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide cells derived from a muscle tissue which is taken out from an adult body of a mammalian animal and having a multiple differentiation potency to mesdermic cells capable of differentiating to the muscle cells, bone cells, fat cells, etc. SOLUTION: This method for producing the cells having the multiple differentiation potency to mesodermic cells is provided by culturing a group of cells containing mesodermic stem cells derived from the muscle tissue which is taken out from the adult body of the mammalian animal, in the presence of a cytekine belonging to the fibroblast growth factor and/or IL-6 family.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for obtaining cells having pluripotency into mesodermal cells from a cell group containing mesodermal stem cells derived from mammalian muscle tissue, and a method for obtaining the pluripotency. The present invention relates to a method for inducing differentiation of a desired cell into a desired mesodermal cell such as a cell having bone-forming ability, and a tissue such as a substitute bone obtained thereby. Further, the present invention relates to a screening method for factors affecting bone formation by using the differentiation induction method.

[0002]

2. Description of the Related Art Among various cells, cells having proliferation ability and ability to form differentiated cells (sometimes referred to as "stem cells") have been found. Stem cells are cells that are present in tissues and the like in an immature and undifferentiated state, and exist to maintain a steady state by proliferating / differentiating / maturating when tissue repair is necessary.For example, neural stem cells are Differentiate into neurons and glial cells (R. McKay, Science, 276, 66
(1997)). However, it has been reported that these neural stem cells have so-called pluripotency, which is ectopically differentiated into skeletal muscle and blood cells (CRRBjornson, et al., Science 283,534).
(1999), R. Galli, et al., Nature Neurosci. 3, 986 (200
0)). On the other hand, it has been reported that even bone marrow mesenchymal stem cells have pluripotency to differentiate into osteogenic cells, skeletal muscle cells, nerve cells, etc. (DJProckop, Science 276, 7
1 (1997)). Although stem cells do not necessarily have pluripotency, if stem cells are separated from adult-derived cells to obtain cells with pluripotency and a method for differentiating these into desired cells is developed, transplantation or It is expected that new methods can be provided in the field of regenerative medicine.

From histopathological studies to date, skeletal muscle-derived mesodermal stem cells have been shown to undergo mitotic stimulation during the regeneration period of muscles to fuse with each other and differentiate into myotubes or muscle fibers. (Y. Saito, i. Nonaka, A
cta Neuropathol. 88, 252 (1994)). However,
There is no example showing that muscle stem cells present in such normal tissues are taken out as a uniform population and have a pluripotency capable of differentiating into cells other than muscle cells. C2C12 cells, an immortalized mouse muscle cell line thought to be derived from muscular tissue-derived mesodermal stem cells, differentiate into not only myotubes but also osteoblasts and adipocytes by selecting the culture conditions There is a report that it has the ability (T.Katagiri, et.
al., J. Cell Biol. 127, 1775 (1994), L. Teboul et a
L., J. biol. Chem. 270, 28183 (1995)). However, the immortalized mouse cell line is a kind of tumor cell, and it is highly likely that it is different from normal muscle tissue-derived cells in the adult in controlling gene expression, etc. The response to the stimuli does not always reflect normal cells, and it is considered that the findings obtained using such cell lines cannot be directly applied to normal cells. In addition, in the experiment using this immortalized cell line,
For example, although it shows the properties of osteoblasts, there is no report that the formation of bone matrix (extracellular calcium accumulation) has been reached. Even if the immortalized mouse muscle cell line can be differentiated into osteoblasts, it is not possible to use the differentiated cells as they are for transplantation therapy for bone regeneration, because of the problem of organ rejection due to xenogeneic animals or tumors. There are problems such as the pros and cons of administration of cells, and it cannot be implemented.

[0004] In particular, since osteoporosis due to aging, bone fracture, difficult recovery after bone damage, and decrease in bone density after menopause have become social problems in recent years, the safety of excision surgery as a source of cells If a method of culturing cells derived from muscular tissue that is highly effective and easy to collect and induces differentiation into bone cells is established, it will be a great gospel in the fields of bone transplantation and bone regenerative medicine. Be expected.

However, there has been no report of culturing muscle tissue-derived cells taken out from adult mammals, inducing them to differentiate into cells having osteogenic ability, and forming bone matrix, and the development of such a method has not been reported. There are great expectations for its application.

[0006]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for culturing a cell group derived from muscle tissue to obtain cells having pluripotency into mesodermal cells. Further, it is an object of the present invention to provide desired mesodermal cells, such as cells having osteogenic ability, which can be used as cells for transplantation treatment of bone diseases such as osteoporosis, from the cells having pluripotency. Another object of the present invention is to provide a method for screening a substance that affects the osteogenic ability such as a new osteogenesis promoting factor, and a method for producing cells having the osteogenic ability using the substance obtained by the screening. Furthermore, a method for producing an alternative bone tissue using the above-mentioned cells having osteogenic ability, and an alternative bone tissue obtained by the method, and the above-mentioned cells having pluripotency, or stem cells, and belonging to the TGF-β superfamily It is an object to also provide an osteogenesis promoting agent containing a combination of proteins.

[0007]

Means for Solving the Problems The present inventor has conducted extensive studies as to a method for culturing mouse muscle tissue-derived cells to solve the above problems, and as a result, a culture in which muscle tissue-derived mesodermal stem cells specifically proliferate. By culturing under the conditions of basic fibroblast growth factor which is a fibroblast growth factor and / or leukocyte inhibitory factor which is a cytokine belonging to the IL-6 family, myocytes and bone cells are obtained. We succeeded in obtaining cells with pluripotency into mesodermal cells such as fat cells and adipocytes.
(bone morphogenetic protein) and / or β-glycerophosphate, the cells were successfully obtained that were positive for alkaline phosphatase and / or osteocalcin, which is a marker for bone cells. By further continuing the culture, it was confirmed that a bump-shaped cell mass was observed, calcium was accumulated in the extracellular matrix, and the cell had osteogenic ability.

Further, the present inventors applied the above-mentioned method to humans and obtained bone-cell marker-positive cells from cells derived from human muscle tissue by the same method and continued culturing. It was confirmed that the cell-like cell mass was found, calcium was accumulated in the extracellular matrix, and the cells had osteogenic ability. The present invention has been completed as described above, and its gist is as follows.

(1) A group of cells containing mesodermal stem cells derived from mammalian muscle tissue is treated with fibroblast growth factor and / or IL-6.
A method for producing cells having pluripotency into mesodermal cells, which comprises culturing in the presence of a cytokine belonging to the family. (2) The method according to (1), wherein a cell group containing muscle tissue-derived mesodermal stem cells is cultured in the presence of a fibroblast growth factor and a cytokine belonging to the IL-6 family. (3) Culturing a cell group containing muscular tissue-derived mesodermal stem cells in the absence of fibroblast growth factor and IL-6 family cytokine under culture conditions in which mesodermal stem cells selectively proliferate Then, a colony composed of undifferentiated cells is recovered from the appeared colonies, and the cells contained in the recovered colony are treated with fibroblast growth factor and / or
The method according to (1) or (2), which comprises maintaining the culture in the presence of a cytokine belonging to the IL-6 family. (4) A cell group containing mesoderm stem cells derived from muscle tissue is added in the presence of a fibroblast growth factor and / or a cytokine belonging to the IL-6 family under culture conditions in which mesodermal stem cells selectively grow. After culturing, colonies composed of undifferentiated cells among the appeared colonies are collected, and the cells contained in the collected colonies are treated with fibroblast growth factor and / or
Alternatively, the method according to (1) or (2), which comprises maintaining the culture in the presence of a cytokine belonging to the IL-6 family. (5) The method according to any one of (1) to (4), wherein the fibroblast growth factor is a basic fibroblast growth factor. (6) The cytokine belonging to the IL-6 family is a leukocyte inhibitory factor (LIF), (1) to
The method according to any one of (5). (7) The method according to any one of (1) to (6), wherein the cell having pluripotency into mesodermal cells is positive for a transcription factor specific to mesodermal cells. (8) Transcription factors specific for mesodermal cells are MyoD and Runx
2 or the method according to (7), which is PPARγ. (9) The method according to any one of (1) to (8), wherein the mesodermal cells are any of muscle cells, bone cells, and adipocytes. (10) A cell having a pluripotency into mesodermal cells, which is produced by the method according to any one of (1) to (9). (11) A method for producing a desired mesodermal cell or a tissue derived from the cell group, which comprises culturing the cell or the stem cell according to (10) under conditions for differentiating into a desired mesodermal cell. . (12) The method according to (11), wherein the desired mesodermal cell is a cell having osteogenic ability. (13) The condition for differentiating into a cell having osteogenic ability is
The cell or the stem cell according to (10), 1 × 10 ³ to
It is cultured in the presence of proteins belonging to the TGF-β superfamily at a density of 1 × 10 ⁶ / cm ² (12).
The method described in. (14) The condition for differentiating into a cell having osteogenic ability is
The cell or the stem cell described in (10) is 1 × 10 ³ to 1
The method according to (12), which comprises culturing in the presence of BMP-2 and / or β-glycerophosphate at a density of × 10 ⁶ / cm ² . (15) The method according to any one of (11) to (14), wherein cells having bone-forming ability are obtained by using as an index positive for alkaline phosphatase and / or positive for osteocalcin. (16) The method according to any one of (11) to (14), wherein cells having bone formation ability are obtained using the bone matrix formation ability as an index. (17) A cell obtained by the method according to any one of (11) to (16) or a tissue derived from the cell group. (18) Administering a test substance to the cells according to (10),
A method for screening a substance affecting osteogenic ability, which comprises evaluating the osteogenic ability of induced cells. (19) A substance selected by the screening method described in (18). (20) The cell or the stem cell according to (10),
A method for producing cells having osteogenic ability, which comprises culturing in the presence of the substance according to 9). (21) A method for producing an alternative bone tissue, which comprises forming the bone tissue by attaching the cells according to (17) to a support and culturing the cells. (22) An alternative bone tissue obtained by the method according to (21). (23) An osteogenesis promoter comprising a combination of the cell according to (10) or any of the stem cells and a protein belonging to the TGF-β superfamily.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below.
The method of the present invention is a method for producing cells having pluripotency into mesodermal cells from a cell group containing muscular tissue-derived mesodermal stem cells. Here, stem cells are present in tissues in a juvenile / undifferentiated state, and when tissue repair is required,
It refers to cells present to proliferate, differentiate, mature and maintain a steady state. For example, mesodermal stem cells present in muscle tissue are differentiated into muscle cells and repaired when the muscle is damaged due to injury. The method of the present invention is a cell group containing such muscular tissue-derived mesodermal stem cells, cells having the ability to differentiate into mesodermal cells other than muscle cells, that is,
It is characterized by producing cells having pluripotency into mesodermal cells.

As the cell group containing muscular tissue-derived mesodermal cells used in the method of the present invention, muscle tissue-derived cells taken out from an adult mammal are preferably used as a material. Examples of mammals include laboratory animals such as mice, rats, dogs, pigs, monkeys, and humans. As an adult,
It is an individual who has grown enough to collect muscle tissue. When performing transplantation medicine, it is generally preferable to use one's own tissue rather than that of another person in order to avoid rejection. In order to repair damage or dysfunction of one's own tissue with one's own tissue, it is preferable to use adult-derived muscle tissue to produce cells having pluripotency into mesodermal cells of the present invention.

As a collection source of the cell group containing muscular tissue-derived mesodermal stem cells used in the present invention, any muscle tissue in which the cells are present may be used, and skeletal muscle, smooth muscle, myocardium, etc. may be used. Among them, skeletal muscle tissues such as gastrocnemius muscle tissue and subcutaneous muscle tissue are preferably used.

The muscle tissue-derived cell group as described above includes mesodermal stem cells, fibroblasts, muscle fibers, nerve cells, blood cells / hematopoietic stem cells, and the like. From such a cell group,
In order to obtain the mesodermal stem cells, it is preferable to culture by a culture method in which the mesodermal stem cells selectively grow. Specifically, at the start of culture, the cell density in the culture medium is 2
00 pieces / cm ² or less, preferably 15 to 100 pieces / cm ²
Examples of the method include culturing cells so that the above, and a method of adding a supplement to a commonly used cell culture medium. Supplements to be added include Ultraser G (Bios
epra), bovine pituitary extract, etc.
It is preferable to use traser G. Further, in this culture, selective culture of mesodermal stem cells can be effectively performed by performing the culture in the presence of a substance that promotes cell growth and suppresses differentiation. As a substance that promotes cell growth and suppresses differentiation, fibroblast growth factor or IL-6
Examples of cytokines belonging to the family include bFGF
And leukocyte inhibitory factor (LIF) are preferably used. Of the colonies appearing by such culture, colonies composed of undifferentiated cells are collected, and the cells contained in the obtained colonies are used as fibroblast growth factor and / or IL-6.
By culturing in the presence of a cytokine belonging to the family, cells having pluripotency into mesodermal cells can be obtained. Further, the cells thus obtained can be cultured and maintained in the presence of fibroblast growth factor and / or cytokine belonging to the IL-6 family. Below, the specific example of the method mentioned above is shown.

The gastrocnemius muscle of an adult mouse, preferably an 8-12 week old mouse, or an adult human subcutaneous subcutaneous muscle tissue piece is taken out and placed in a petri dish to remove excess adipose tissue and the like and cut into small pieces.

The cut muscle tissue is dispersed with a cell dispersion containing collagenase V (manufactured by Sigma) and trypsin (manufactured by Lifetech) and centrifuged to collect the supernatant. This operation is repeated several times, preferably five times, to separate the cells.

[0016] pmGM medium [Primary Myocyte
Growth Medium; 20% fetal bovine serum, 2% Ultraser G (Bios
hDMEM containing epra) (containing 4.5 mg / ml glucose)
Dulbecco's modified Eagle medium)]
In a dish (made by Sumilon) coated with Lagen,
Belongs to the fibroblast growth factor and / or IL-6 family
Culture in the presence of the cytokine. At this time, cell tightness
200 / cm ²Below, preferably 15 to 100
/ Cm²Adjust as follows. After 3-4 days of culture,
Ronnie is formed, and thereafter, the proliferation accelerates. Culture 7
Undifferentiated cells among colonies that appeared after 10 days
The colony composed of is collected.

A preferred embodiment of the present invention is characterized in that colonies composed of undifferentiated cells are recovered from the thus-appearing colonies. That is, although various cells as described above are mixed in the cell group derived from muscle tissue, it is possible to obtain mesodermal cells of the present invention by collecting a colony composed of such undifferentiated cells. It is possible to remove cells other than cells having the ability to be activated. A colony composed of undifferentiated cells can be obtained by selecting a scattered colony composed of spherical cells.

Here, as a method for collecting colonies, the ring method (tissue culture technique (Asakura Shoten), P34-35)
Etc. are used. Specifically, for example, a colony to be separated is selected and marked under an inverted microscope, the medium is discarded, and the bottom surface is covered with a stainless steel cylinder (10 mm x 10 mm) coated with silicon grease that has been sterilized in advance with high pressure.
A small amount of trypsin EDTA solution can be put therein with a syringe or a Pasteur pipette, and the cells can be peeled and collected.

Next, the cells contained in the colony obtained by the above method are seeded on a 35-mm diameter petri dish (manufactured by Sumilon Co., Ltd.) which has been previously coated with type I collagen to give fibroblast growth factor and / or Alternatively, cells having pluripotency into mesodermal cells can be obtained by maintaining the cells in culture in the presence of cytokines belonging to the IL-6 family. Fibroblast growth factor and cytokines belonging to the IL-6 family function as factors that promote the proliferation and maintenance of undifferentiated state of cells. As the fibroblast growth factor, basic fibroblast growth factor (bFGF) is preferable. As the cytokine belonging to the IL-6 family, leukocyte inhibitory factor (LIF) is preferable. The fibroblast growth factor and the cytokine belonging to the IL-6 family can be used alone, but when these are used together, an additive effect is observed.

The bFGF is usually used at a concentration of about 5 ng / ml to 50 ng / ml, preferably about 10 ng / ml to 20 ng / ml. The leukocyte inhibitory factor (LIF) is usually 10 ² u / ml to 10 ⁴ u / ml, preferably 10 ³ u / ml.
Used at a concentration of about u / ml. In addition, examples of the medium used for culture include pmGM [Primary Myocyte Growth Medium].

[0021] As described above, spontaneous differentiation is suppressed while maintaining the proliferative property of muscular tissue-derived mesodermal stem cells,
It is possible to obtain cells that have maintained an undifferentiated state, that is, cells that have a pluripotency into mesodermal cells. The cells having pluripotency into mesodermal cells of the present invention obtained as described above can be confirmed by using the positive transcription factor specific to mesodermal cells as an index. Examples of such factors include MyoD (transcription factor that controls muscle cell differentiation), Runx2 (transcription factor involved in bone formation), and PPARγ (transcription factor involved in adipocyte differentiation). The mesodermal cells capable of differentiating the cells include at least muscle cells,
Examples thereof include bone cells and adipocytes. The thus obtained cells having pluripotency into mesodermal cells can be cultured and maintained in the presence of fibroblast growth factor and / or cytokines belonging to the IL-6 family. For mouse-derived cells, subculturing is relatively easy, but for human-derived cells, the subculture should be 15 or less, preferably 10 or less.

Next, the cells having pluripotency into mesodermal cells or the stem cells produced by the method of the present invention are cultured under the conditions for differentiating into desired mesodermal cells, and desired mesodermal cells are obtained. Alternatively, a tissue derived from the cell group can be obtained. Stem cells that can be used in addition to cells having pluripotency into mesodermal cells produced by the method of the present invention include embryonic stem cells (ES cells), neural stem cells,
Hair follicle stem cells, small intestinal epithelial stem cells and the like can be mentioned. The desired mesodermal cells include, for example, cells having osteogenic ability,
Examples thereof include muscle cells and adipocytes, and examples of the tissue derived from the cell group include bone matrix, bone tissue, muscle tissue, adipose tissue and the like. That is, the tissue derived from the cell group may be not only a tissue composed of the cell group but also a tissue containing a substance derived from the cell group such as bone matrix. Here, the bone matrix means a substance formed by cells having bone-forming ability and accumulating substances secreted to the outside of the cell, and its main component is calcium phosphate and the like, and osteocalcin, osteopontin, and osteonectin. , Proteins such as collagen.

As a method of differentiating into cells having osteogenic ability, for example, cells having pluripotency into mesodermal cells or stem cells produced by the method of the present invention
A method of culturing in the presence of a protein belonging to the F-β superfamily is preferably used. Proteins belonging to the TGF-β superfamily include BMP-2 and BMP
-4, BMP-5, BMP-7 and the like, preferably BMP-2, BMP
-4, particularly preferably BMP-2 is used. The cell density at the time of culturing is preferably a density at which a protein belonging to the TGF-β superfamily acts effectively, and it is preferable to seed at a high density such that inhibition does not occur in the growth of cells. The cell density is preferably adjusted to 1 × 10 ³ to 1 × 10 ⁶ / cm ² , preferably 1 to 3 × 10 ⁴ / cm ² . Further, in order to suppress excessive growth, the concentration of fetal calf serum (FCS) in the culture medium is 0 to 5%, preferably 2 to 3
% Is preferably adjusted. When culturing, a protein expression activating substance contained in the bone matrix, a phosphoric acid source, or a collagen synthesis promoting substance may be added. Dexamethasone and Vitamin D are substances that activate the expression of proteins contained in bone matrix.
3 and the like, β-glycerophosphoric acid and the like as a phosphoric acid source, and ascorbic acid and the like as a collagen synthesis promoting substance. These can be appropriately combined and selected according to the type and state of cells to be used, and the concentration to be added may be determined according to the cells to be used.

More specifically, for example, in the presence of BMP-2 and / or β-glycerophosphate, the cell density in the culture medium at the start of culture is 1 × 10 ³ to 1 × 10 ⁶ / cm ² ,
Preferably, a method of culturing so as to have a density of 1 to 3 × 10 ⁴ / cm ² or a concentration of fetal calf serum (FCS) added to a commonly used cell culture medium is 0 to 5%, preferably 2 to
A method of setting it to 3% can be mentioned. In this method, for example, BMP-2 alone can be used for human-derived cells, but β-glycerophosphate is preferably used in combination for mouse-derived cells. Here, when obtaining cells having osteogenic ability, it is possible to use alkaline phosphatase-positive and / or osteocalcin-positive as an index. In addition, cells having bone forming ability can be obtained using the bone matrix forming ability as an index. The bone matrix forming ability can be confirmed by using extracellular calcium accumulation detected by an alizarin staining method or the like as an index.

By culturing the cells of the present invention, which have a pluripotency into mesodermal cells, in the presence of gamma-linolenic acid, they can be differentiated into adipocytes having many lipid droplets in their cytoplasm. As the medium, for example, DM supplemented with 10% fetal bovine serum in addition to gamma-linolenic acid
EM and the like are preferably used. Further, the cells having a pluripotency into the mesodermal cells were treated with a medium for muscle differentiation containing transferrin, insulin and selenium (pmDM medium; 2% fetal bovine serum, 5 μg / ml transferrin, 10 μg).
/ Ml insulin, hDME with addition of 10 nM selenium
Induction into myotube cells is observed by culturing in M) or the like. This can be confirmed by using muscle-specific creatine kinase positive, muscle-specific myosin heavy chain positive and the like as indexes.

A method for obtaining cells having osteogenic ability will be exemplified below. After obtaining cells having pluripotency into mesodermal cells of the present invention by the method described above, the cell density is 1 × 10 ³ to 1 × 10 ⁶ / cm ² , preferably 1 to 3
Seed at × 10 ⁴ / cm ² . Human Recombinant BMP-2 (S
trathmanBiotech Co., Ltd.) 200ng / ml~1000ng / ml, preferably medium was replaced with 300ng / ml~500ng / ml containing medium for muscle differentiation (pmDM), 4~ with 10% CO _2, 37 ℃, 100% humidity Incubate for 6 days. There is no need to change the medium during this culture period. In the case of mouse-derived cells, β-glycerophosphate (manufactured by Sigma) is further added to the above medium at 5 mM to 20 mM, preferably 10 mM.
Add to M. As a result, cells positive for alkaline phosphatase and osteocalcin, that is, cells having osteogenic ability can be obtained. By further culturing, a bump-shaped cell mass (nodule)
Are formed in the central part of the dish, and the bone matrix accumulates around it. This can be easily confirmed by the fact that the central part does not transmit light and thus appears dark.

As described above, cells having osteogenic ability can be obtained by using, for example, the fact that they are positive for alkaline phosphatase and / or positive for osteocalcin. It is possible to screen for substances that give That is, a test substance is administered to cells having pluripotency of the mesodermal cells of the present invention, and the osteogenic potential of the induced cells is evaluated using the above-mentioned index to obtain the test substance. The effect of the substance on the bone formation ability can be detected. The substance thus selected is a substance that affects the bone formation ability, and is effective against bone diseases such as osteoporosis, bone fracture and bone damage, and post-menopausal decrease in bone density.

The bone-forming cells produced by the method of the present invention can be adhered to a support and cultured to form a bone tissue to produce a substitute bone tissue. For example, a method of Ueda et al. (Journal of the Japanese Society of Oral Surgery, 47, 1, 1-7 (2001)) and the like are attached to a support serving as a scaffold for the cells and cultured to produce an alternative bone tissue You can As the support, an artificial extracellular matrix or a porous ceramic, or a resorbable biopolymer such as gelatin, collagen or cellulose can be used, and these can be used in the form of sponge, powder, gel, web or the like. By implanting the manufactured alternative bone tissue, osteogenic healing can occur at the implantation site. Such substitute bone tissue may further contain drugs such as antibiotics, anti-inflammatory agents, immunosuppressants, preservatives, excipients and the like. In addition, the combination of cells or stem cells having pluripotency into mesodermal cells of the present invention and a protein belonging to the TGF-β superfamily can be used as an osteogenesis promoter. The osteogenesis promoter further comprises an expression activator of a protein contained in the bone matrix,
A phosphoric acid supply source, a collagen synthesis promoting substance, or the like can also be included. Examples of the protein expression activating substance contained in the bone matrix include dexamethasone, vitamin D3 and the like, phosphate source such as β-glycerophosphate, and collagen synthesis promoting substance such as ascorbic acid.

The osteogenesis promoting agent of the present invention may be in any physiologically acceptable form, but is preferably an injection or the like, and when administered as an injection. Is prepared pyrogen-free. The form of the injection includes, for example, encapsulating the cells of the present invention, or stem cells and proteins belonging to the TGF-β superfamily for delivery to damaged parts such as bone, cartilage, and tissues, or having a viscosity. It can be prepared as a liquid. Further, the cells or stem cells and a matrix for controlling the release of the protein may be contained, and the matrix may be absorbed into the body. The administration rules such as the dose of the osteogenesis promoter should be considered in consideration of the weight of bone desired to be formed, the site of bone damage, the state of injury, the age and sex of the patient, the administration period, the dosage form and other clinical factors. Then, it may be appropriately determined. In addition, when such an osteogenesis promoter is administered, it is preferable to regularly monitor the degree of bone growth and / or repair in order to grasp the effect. For example, the effect can be grasped by monitoring using X-ray or histomorphological measurement.

[0030]

EXAMPLES The present invention will be described in more detail with reference to the following examples, but the following examples should be regarded as one way of helping to gain a concrete understanding of the present invention, and the scope of the present invention is as follows. It should not be limited in any way by example.

[0031]

[Example 1] Preparation of cells having pluripotency into mesodermal cells (1) Aseptic gastrocnemius muscle tissue having a length of about 3 mm and a diameter of about 3 mm was sterilized from female ICR mice aged 8 to 12 weeks. I took it out.
Three 100 mm diameter petri dishes containing 10 ml of sterilized PBS (phosphate buffered saline) were prepared, and the muscle tissues were soaked and washed in order. Excessive adipose tissue was removed as carefully as possible with tweezers.

In the case of human-derived muscle tissue, the excised human dorsal subcutaneous muscle tissue piece was wrapped in gauze soaked with sterile PBS, placed in a sterile petri dish, and then cooled with ice. Within 10 hours after extraction, cut out a part of it (about 10 mm x 10 mm in area),
The skin was removed with a scalpel. The following operations were exactly the same for mice and humans.

(2) The obtained muscle tissue was transferred to a 50 ml tube and cut into as small pieces as possible with ophthalmic scissors.

(3) The cell dispersion (0.
05% trypsin (manufactured by DIFCO), 0.025% collagenase V
5 ml of hDMEM (manufactured by Sigma) (Dulbecco's modified Eagle medium containing 4.5 mg / ml glucose, manufactured by Asahi Techno-Glass)) was added and gently mixed by hand at room temperature for 5 minutes, and then 2 at 100 rpm. Centrifuge for minutes. 50 ml of fresh supernatant
Transfer to a tube and add 1 ml of fetal bovine serum. This operation was repeated 4 more times.

(4) Of about 30 ml of the collected supernatant, 3 ml of basic fibroblast growth factor (bFGF) (Peprotech) and leukocyte inhibitory factor (LIF) (Chemicon) were each added to a final concentration of 10 ng. / ml, 1000u / ml
mGM medium [Primary Myocyte Growth Medium; hDMEM (4.5% containing 20% fetal bovine serum, 2% Ultraser G (Biosepra))
12 ml of Dulbecco's modified Eagle medium (Asahi Techno Glass Co., Ltd.) containing mg / ml glucose was added thereto and uniformly suspended. 3 ml of this suspension was placed in advance with 7 ml of pmGM medium to which bFGF and LIF were added so that the final concentrations were 10 ng / ml and 1000 u / ml, respectively, and the I of 100 mm in diameter was added.
Petri dish coated with type collagen (collagen typ
e I-coated dish (Sumiron)). Hereinafter, unless otherwise stated, in the culture process for obtaining cells having pluripotency into mesodermal cells, the cells were cultured in pmGM medium containing bFGF and LIF.

(5) Small colony formation was observed after 3 to 4 days of culture, and thereafter the growth accelerated. After 7 to 10 days, ring method (tissue culture technology (Asakura Shoten), P34-35)
The colony cells composed of undifferentiated cells were treated with PBS containing 0.05% trypsin (DIFCO) and 1 mM EDTA.
Peel it off from the petri dish and collect it.
It was evenly seeded on a ype I-coated dish (Sumiron).

(6) After 2 to 4 days, when the cells became 20 to 30% confluent, half of the cells were collided with 100 mm in diameter.
It was sown on an agen type I-coated dish (manufactured by Sumiron). Two
Do not reduce the cell density too much every 3 days (diameter 10
(1 to 2 × 10 ⁴ cells per 0 mm dish), and the culture was continued while reseeding in a new dish.

(7) The cells obtained in (6) were seeded at a cell number of 1 × 10 ⁵ / dish on a 35-mm-diameter Petri dish (manufactured by Sumilon Co.) previously coated with type I collagen to prepare bFGF and The cells were cultured in pmGM medium containing LIF for 20-24 hours, and were used for the following experiment for differentiation into mesodermal cells.

[0039]

Example 2 Examination of Addition Effect of FGF and / or LIF 1/100 amount of the cells obtained in (3) of Example 1 was seeded on a 35-mm type I collagen coated dish (manufactured by Sumilon). The medium used was pmGM supplemented with bFGF at a final concentration of 20 ng / ml and LIF at a final concentration of 1000 u / ml. Culture 7
After a day, the number of colonies formed and the morphology of the obtained colonies were observed with a microscope. As a result, the number of colonies obtained in the case of pmGM medium alone was 10, the number of colonies obtained in the medium containing bFGF was 9, and the number of colonies obtained in the case of adding LIF alone was 6, b.
The number of colonies obtained when FGF and LIF were added was 12.

When the colonies obtained under the respective conditions were observed under a microscope, the presence of myotube cells was observed in all the obtained colonies when the pmGM medium alone was used, and the number of cells in each individual colony was small. , Most of the cells were differentiated into myoblasts (Fig. 7; 7-1). Here, myoblasts are spindle-shaped cells that fuse with further differentiation to form myotubes (cells). Next, bFGF
In all cases, myotubes were found to be present in all the obtained colonies, but the number of cells in each colony tended to increase, and the ratio of myotubes in the colonies also tended to decrease. (Fig. 7; 7-2). In the case of LIF alone, the percentage of myotube cells found in the obtained colonies was low, and the percentage of myotube cells present in the colonies also tended to decrease (Fig. 7; 7-3). However, in the case where these bFGF or LIF were added alone, spindle-shaped myoblasts which were being differentiated into myotubes were found in colonies in which myotubes were not found. On the other hand, when cultured in the presence of both bFGF and LIF, the proportion of colonies in which the presence of myotubes was found was extremely low among the obtained colonies, and the number of cells in each colony was large. Despite the increase, almost no myoblasts and myotubes appeared in the colony, which consisted of spherical cells (Fig. 7; 7-4).

[0041]

Example 3 Detection of MyoD and Runx2 The cells (1 × 10 ⁵ cells / 35 mm dish) obtained in (7) of Example 1 were washed with PBS after 2 days and then fixed with 4% paraformaldehyde. did. Triton X-100
After washing with phosphate buffered saline (T-PBS) containing 0.5%, blocking was performed with 1% normal donkey serum at room temperature for 10 minutes. Next, anti-MyoD antibody (Novocastra, 20-fold diluted) and 4
After reacting overnight at ℃, wash and Cy3 labeled anti-mouse IgG antibody (manufactured by Jackson Immuno Laboratory, 500-fold dilution)
Was reacted at room temperature for 1 hour.

The cells were fixed in the same manner as above, blocked with normal donkey serum, reacted with anti-Runx2 antibody (hybridoma culture supernatant) overnight at 4 ° C., washed, and washed with biotin-labeled anti-mouse IgG antibody ( The reaction was carried out for 1 hour at room temperature with a 500-fold dilution (manufactured by Jackson Immuno Laboratory).
Furthermore, it was reacted with horseradish peroxidase-labeled streptavidin (NEN, 100-fold diluted) at room temperature for 20 minutes. Then, it was reacted with FITC-labeled tyramide at room temperature for 10 minutes. The anti-Runx2 antibody was kindly provided by Professor Yoshiaki Ito of Kyoto University School of Medicine. (For antibody preparation, see Yu-Wen
Zhang, et al., Proc. Natl. Acad. Sci. USA 97, 1054
9 (2000). ) In both cases, fluorescence observation was performed using a falling fluorescence microscope.
It was confirmed that each transcription factor was positive.

[0043]

[Example 4] Preparation of cells having osteogenic ability (1) The cells obtained in Example 1 were used as a muscle differentiation medium containing human recombinant BMP-2 (300 to 500 ng / ml) (Strathman Biotech). Change the medium to (pmDM) and add 10% CO ₂ , 37
Cultivation was carried out at ℃ and 100% humidity for 4 to 6 days. The medium was not changed during the culture period. In the case of cells obtained from mouse muscle tissue, β-glycerophosphate (manufactured by Sigma) was further added to the above medium so that the concentration was 10 mM.

(2) After 3 to 4 days of culture, a nodular cell mass (nod
ule) was formed in the center of the dish. After 4 to 6 days of culture, the bone matrix further accumulated around the cell mass, and the central part became dark because it did not transmit light (FIG. 1 for mouse, FIG. 4 for human).

[0045]

[Example 5] Detection of alkaline phosphatase activity and detection of calcium accumulation (1) The cells obtained in (1) of Example 4 were fixed with 4% paraformaldehyde (manufactured by Merck) at 4 ° C for 10 minutes. After that, 100 mM Tris-HCl (pH 9.6), 100 mM NaCl, 20 mM
MgCl ₂ , 0.01% naphtol AS-MX (manufactured by Sigma), 0.5 mg / m
The reaction was performed for 20 minutes in Fast Blue RR (manufactured by Sigma), and alkaline phosphatase activity was detected histochemically according to a conventional method.

(2) The cells fixed with paraformaldehyde in (1) were stained with Alizarin Red S (Sigma) for 30 minutes and then washed with PBS for 30 to 60 minutes. 2 and 4
As shown in, the bone matrix was stained with alizarin red S, and it was found that calcium was accumulated extracellularly.

[0047]

Example 6 Detection of Osteocalcin The anti-mouse osteocalcin sheep antibody (400-fold dilution, was added to the cells fixed with paraformaldehyde in (1) of Example 5).
Biomedical Tech) diluted with PBS containing 0.1% saponin (Sigma) was added, and the mixture was reacted at 4 ° C for 12 to 20 hours. Then, after reacting with a FITC-labeled anti-sheep IgG antibody (200-fold diluted, manufactured by Jackson Immuno Laboratory) at room temperature for 1 hour, it was observed with a fluorescence microscope. As a result, osteocalcin accumulated in the same extracellular matrix part as calcium (FIG. 3).

[0048]

Example 7 Induction of Differentiation into Adipocytes The mouse-derived cells obtained in Example 1 were 1 × 10 ⁵ cells / 100 mm.
It was seeded on a type I collagen coated dish (manufactured by Sumilon). Add this to pmGM medium [Primary Myocyte Growth Mediu
m; 20% fetal bovine serum, 2% Ultraser G (Biosepra)
HDMEM (Dulbecco's modified Eagle medium containing 4.5 mg / ml glucose)], and 20-24 hours later, the medium was added with 100 μmole of gamma-linolenic acid (Sigma) and 10% fetal bovine serum. 10% CO ₂
When the culture was continued at 37 ° C in the presence, adipocytes having many lipid droplets appeared in the cytoplasm after 6 to 9 days (Fig. 5).

[0049]

[Example 8] Induction of differentiation into myotube cells The mouse-derived cells obtained in Example 1 were transformed into 1 x 10 ⁵ cells / 100.
It was seeded on a mmI type collagen coated dish (manufactured by Sumilon). HDMEM medium containing 2% fetal bovine serum, 5 μg / ml transferrin (manufactured by Sigma), 10 μg / ml insulin (manufactured by Sigma), and 10 nM selenium (manufactured by Sigma) (p
When cultured in mDM medium), induction into myotube cells was observed (Fig. 6).

[0050]

INDUSTRIAL APPLICABILITY The present invention provides a method for producing cells having pluripotency into mesodermal cells from a cell group containing muscular tissue-derived mesodermal stem cells. Also provided is a method for producing a desired mesodermal cell or a tissue derived from the cell group by differentiating the pluripotent cell or stem cell. The method of the present invention provides cells for use in osteogenic cell transplant therapy in bone disease. Since the acquisition of muscle tissue is simpler and safer than the acquisition of bone marrow cells or the like, it is possible to obtain cells having osteogenic ability using cells derived from the muscle tissue of the patient himself. As a result, autologous cell transplantation without immune rejection can be realized. In addition, it is possible to detect and screen new osteogenesis promoting factors and the like using these cells.

[Brief description of drawings]

FIG. 1 is a photograph showing bone matrix formation by cells having pluripotency into mesodermal cells obtained from cells derived from mouse muscle tissue by the method of the present invention.

FIG. 2 is a photograph showing calcium accumulation in cells having pluripotency into mesodermal cells obtained from cells derived from mouse muscle tissue by the method of the present invention. Stained with Alizarin Red S.

FIG. 3 shows the results of immunostaining with an anti-mouse osteocalcin antibody on a bone matrix formed in cells having pluripotency into mesodermal cells obtained from cells derived from mouse muscle tissue by the method of the present invention. Photo.

FIG. 4 is a photograph showing calcium accumulation in cells having pluripotency into mesodermal cells obtained from cells derived from human muscle tissue by the method of the present invention. Stained with Alizarin Red S.

FIG. 5 is a photograph of adipocytes having a large number of lipid droplets in the cytoplasm, which are differentiated from cells having pluripotency into mesodermal cells obtained from cells derived from mouse muscle tissue by the method of the present invention.

FIG. 6 is a photograph of myotube cells derived from cells having pluripotency into mesodermal cells obtained from cells derived from mouse muscle tissue by the method of the present invention.

FIG. 7 is a photograph showing the effect of adding bFGF and / or LIF. 7-1: When bFGF and LIF were not added. 7-2; when only bFGF was added. 7-3: When only LIF was added. 7-4; when bFGF and LIF were added.

Front page continuation (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 33/15 C12N 5/00 E 33/50 A61K 37/24 F term (reference) 2G045 AA40 CB01 4B063 QA01 QA18 QQ08 QQ20 QQ33 QR57 QR77 QS03 QX01 4B065 AA91X AA93X BB19 BB34 CA44 CA60 4C084 AA02 AA03 BA44 DB61 MA02 ZA961 ZA962 ZC032 4C087 AA01 AA02 BB47 BB64 CA04 MA02 ZA96

Claims (23)

[Claims] 1. Mesoderm, which comprises culturing a cell group containing mesodermal stem cells derived from mammalian muscle tissue in the presence of a fibroblast growth factor and / or a cytokine belonging to the IL-6 family. A method for producing cells having pluripotency into cells. 2. The method according to claim 1, wherein a cell group containing muscular tissue-derived mesodermal stem cells is cultured in the presence of fibroblast growth factor and a cytokine belonging to the IL-6 family. 3. A group of cells containing mesoderm stem cells derived from muscle tissue, in the absence of fibroblast growth factor and cytokines belonging to the IL-6 family under culture conditions in which mesodermal stem cells selectively proliferate. The colonies composed of undifferentiated cells among the emerged colonies are recovered, and the cells contained in the recovered colonies are collected in the presence of fibroblast growth factor and / or cytokines belonging to the IL-6 family. The method according to claim 1 or 2, wherein the method is maintained in culture. 4. The presence of a fibroblast growth factor and / or a cytokine belonging to the IL-6 family under a culture condition in which a cell group containing mesoderm stem cells derived from muscle tissue is selectively proliferated by mesodermal stem cells. Cultivated under, collect colonies composed of undifferentiated cells among the appeared colonies,
The method according to claim 1 or 2, wherein the cells contained in the recovered colony are cultured and maintained in the presence of fibroblast growth factor and / or a cytokine belonging to the IL-6 family. 5. The method according to any one of claims 1 to 4, wherein the fibroblast growth factor is a basic fibroblast growth factor. 6. The method according to any one of claims 1 to 5, wherein the cytokine belonging to the IL-6 family is leukocyte inhibitory factor (LIF). 7. The cell having pluripotency into mesodermal cells is positive for a transcription factor specific to mesodermal cells, according to any one of claims 1 to 6. Method. 8. A transcription factor specific to mesodermal cells is My
The method according to claim 7, which is oD, Runx2 or PPARγ. 9. The method according to claim 1, wherein the mesodermal cells are any of muscle cells, bone cells, and adipocytes. 10. A cell having a pluripotency into a mesodermal cell, which is produced by the method according to any one of claims 1 to 9. 11. A desired mesodermal cell or a tissue derived from the cell group, which is characterized in that the cell or the stem cell according to claim 10 is cultured under a condition for differentiating into a desired mesodermal cell. how to. 12. The method according to claim 11, wherein the desired mesodermal cell is a cell having osteogenic ability. 13. The condition for differentiating into a cell having an osteogenic ability is the cell according to claim 10 or 1 × the stem cell.
The method according to claim 12, which comprises culturing in the presence of a protein belonging to the TGF-β superfamily at a density of 10 ³ to 1 × 10 ⁶ / cm ² . 14. The condition for differentiating into a cell having osteogenic ability is 1 × 1 of the cell of claim 10 or the stem cell.
13. The method according to claim 12, which comprises culturing in the presence of BMP-2 and / or β-glycerophosphate at a density of 0 ³ to 1 × 10 ⁶ / cm ² . 15. An alkaline phosphatase-positive and / or osteocalcin-positive index is used as an index.
The method according to any one of claims 11 to 14, wherein cells having osteogenic ability are obtained. 16. The method according to any one of claims 11 to 14, wherein cells having bone-forming ability are obtained using the bone matrix-forming ability as an index. 17. A cell obtained by the method according to claim 11, or a tissue derived from the cell group. 18. A method for screening a substance that affects the osteogenic ability, which comprises administering a test substance to the cell according to claim 10 and evaluating the osteogenic ability of the induced cell. 19. A substance selected by the screening method according to claim 18. 20. A method for producing a cell having osteogenic ability, which comprises culturing the cell according to claim 10 or the stem cell in the presence of the substance according to claim 19. 21. A method for producing an alternative bone tissue, wherein the cell according to claim 17 is adhered to a support and cultured to form a bone tissue. 22. Alternative bone tissue obtained by the method of claim 21. 23. An osteogenesis promoter comprising a combination of the cell according to claim 10 or any one of the stem cells and a protein belonging to the TGF-β superfamily.