WO2005116194A1

WO2005116194A1 - Method of culturing myoblst or myoblast-like cell for forming myofiber in vitro

Info

Publication number: WO2005116194A1
Application number: PCT/JP2005/010000
Authority: WO
Inventors: Nobuhiro Morishima; Keiko Nakanishi
Original assignee: Riken
Priority date: 2004-05-25
Filing date: 2005-05-25
Publication date: 2005-12-08
Also published as: JPWO2005116194A1

Abstract

It is intended to efficiently prepare shrinkable myofiber by differentiating myoblasts. Before inducing differentiation, myoblasts are exposed to endoplasmic reticulum (ER) stress specific to endoplasmic reticulum followed by the induction of differentiation to thereby efficiently differentiate the myoblasts into myofiber.

Description

Myoblast or myoblast-like cell culture method for in vitro muscle fiber formation

The present invention belongs to the technical field of animal cell differentiation and culture, particularly muscle cell differentiation and culture. Background art

It has been reported that culturing myoblasts (adherent cells) in a nutrient-rich growth medium to increase cell density, and then replacing the cells with a nutrient-free differentiation medium halts cell growth and initiates muscle differentiation. ing. It is known that cell death (apoptosis) occurs in some cells immediately after the cells are transferred to the differentiation medium, but the majority of surviving cells form myotubes and also myofibers (Non-patent literature). 1, 2).

It is said that the proliferation and differentiation of skeletal muscle myoblasts are exclusive, do not differentiate during proliferation, and once differentiated cells do not proliferate again. Skeletal myogenesis is induced by removing the serum from the culture and culturing myoblasts to form multinucleated myotubes that have irreversibly stopped cell division. The coordinated induction of muscle-specific gene products coincides with these morphological changes. MyoD family members of muscle-specific transcription factors (MyoD, myogenin, Myf-5 MRF4) control these processes.

On the other hand, ectopic expression of MyoD family members in various non-muscle cells can induce myogenesis. Forced expression of MyoD in vitro can inhibit cell cycle progression independent of differentiation.

The basis of the antagonism between cell proliferation and differentiation remains unclear, but recently it has been reported that the cell cycle inhibitor P21 is induced during myogenesis, and how cell cycle arrest is linked to muscle differentiation One part of was clarified. With respect to muscle differentiation and control of the cell cycle, the temporal context of the step in which proliferating myoblasts transition to myocytes has become clear. When myoblasts proceed through the differentiation pathway, they first combine myogenin. After induction of myodidinin, induction of p21 and consequent arrest of the cell cycle, and then expression of proteins involved in muscle contraction, followed by fusion of myocytes to multinucleated myotube cells (myotube) ) Is formed in a very orderly sequence (Non-Patent Document 3).

Non-Patent Document 1 Exp.Cell Res.Vol. 24, 508-512. (1961).

Non-Patent Document 2 Cell Vol. 15, 855-864. (1978).

Non-Patent Document 3 J. Cell Biol. Vol. 132, 657-666. (1996) Disclosure of the Invention

In order to form muscle fibers capable of muscle contraction by the conventional method, the muscle tissue of a living body is often used as a material, the tissue is separated, and myoblasts (primary cultured cells) are often taken out and used. However, it is not easy to stably collect high quality primary cultured cells, and the differentiation efficiency may not always be sufficient. In addition, it was necessary to remove muscle tissue from the individual each time the material was obtained. '

On the other hand, the established myoblasts are available in large quantities, but when the established myoblasts are differentiated by the conventional method, there is a disadvantage that the ability to form muscle fibers is inferior to that of primary cultured cells. It is relatively easy to form myotubes from established myoblasts, but the efficiency of differentiation into muscle fibers is low, and cells showing muscle contraction are rare. Also, the fibers formed are much smaller than muscle fibers in vivo, or muscle fibers produced in vitro by primary cultured cells.

Therefore, an object of the present invention is to differentiate myoblasts and efficiently prepare contractile muscle fibers.

The inventor of the present invention succeeded in promoting myoblast apoptosis at the early stage of differentiation by exposing to endoplasmic reticulum (ER) stress before inducing differentiation, and promoting the subsequent differentiation process. Myoblasts have been found to undergo cell fusion to form multinucleated myotubes, and to efficiently differentiate into muscle fibers to cause muscle contraction.

This description includes part or all of the contents as disclosed in the description and / or drawings of Japanese Patent Application No. 2004-154813, which is a priority document of the present application. Brief Description of Drawings

Figure 1 'shows that ER-specific ER stress inducer enhances apoptosis of C2C12 cells (day 1 of differentiation induction). Proliferating means proliferating cells; UT means differentiation after no pretreatment; TUN means differentiation after tunicamycin pretreatment; TG means differentiation after thapsigargin pretreatment.

FIG. 2 shows activation of caspase 12 during differentiation culture of C2C12 cells. GM is a growth medium, D1 is a sample on day 1 of differentiation, D2 is a sample on day 2 of differentiation, L is live cell, D is dead cell, UT is untreated, TUN / DM is pre-treated with tanyumycin, TG / DM means pretreatment with thapsigargin, and TG means thapsigargin pretreatment. The arrow indicates procaspase 12 and the arrowhead indicates the active form of caspase 12.

Figure 3 shows a comparison of muscle fiber size with and without pretreatment with an ER-specific ER stress inducer. UT means untreated, TUN means tunicamycin pretreatment, and TG means thapsigargin pretreatment. The scale bar is 200 μηι. Many muscle fibers contracted spontaneously in the differentiation culture of pretreated cells.

Figure 4 shows the control of apoptosis and survival by photocrine factor after pretreatment with an ER-specific ER stress inducer. IGF-II in the conditioned medium was detected by immunoblotting using an anti-IGF-II antibody. UT means untreated, TUN means tunica mycin pretreatment, TG means thapsigargin pretreatment, DM means an unused differentiation-inducing medium, and 18 means days 1 to 8 in place of the differentiation-inducing medium.

Fig. 5 shows suppression of apoptosis at the early stage of differentiation by adding 1 ^ g / m1 of IGF-II to the differentiation induction medium (day 1 of differentiation). UT means untreated, TUN means okkamycin pretreatment. The scale bar is 200 μπι.

Fig. 6 shows the detection of procatebcin Β secreted into the differentiation-inducing medium by immunoblotting. The daricosyl and non-daricosyl forms of cathepsin are indicated by arrows and arrowheads, respectively. UT is untreated, TUN is pretreatment with tsuyuikimycin, TG is thapsigargin pretreatment, 1-3 is day 1 to day 3 instead of the differentiation induction medium, and DM is an unused differentiation induction medium.

Figure 7 shows the promotion of apoptosis by conditioned medium prepared from C2C12 cells pretreated with an ER-specific ER stress inducer. 効率 Efficient formation of large muscle fibers. Proliferating C2C12 cells were transferred to a conditioned medium and cultured for 24 hours, and then cultured for 12 days in a differentiation-inducing medium. TUNCM is a conditioned medium prepared using cells pretreated with tanyumycin, TGCM is a conditioned medium prepared using cells pretreated with thapsigargin, and CCM is a culture of differentiation of control cells (no pretreatment). Means the conditioned medium prepared. The scale par is 200 μιη.

Figure 8 shows the resistance of a differentiating C2C12 cell to apoptosis. Proliferating C2C12 cells (GM), differentiated C2C12 cells (TUN;) pretreated with Tuyumycin, differentiated C2C12 cells (TG) pretreated with thapsigargin, and untreated C2C12 cells Cells (DM) were treated with any of the apoptosis-inducing agents of 2 g Zm 1 tsuyuikimycin, ΙμΜ thapsigargin, 100 g / m 1 etoposide, and 0.2 μΜ staurosporine. All the differentiating cells are 3 days after the start of differentiation. The scale bar is 200 ^ m. 'Figure 9 shows Bcl-xl levels in C2C12 cells undergoing differentiation. L means live cells, D means dead cells, GM means proliferating cells, UT means untreated, TUN means tsuyuikimycin pretreatment, and TG means thapsigargin pretreatment. Cells from day 1 to day 7 of differentiation initiation were analyzed.

FIG. 10 shows the induction of myodidinin in the differentiation medium. Myogenin in C2C12 cells from day 1 to day 10 after the start of differentiation was detected by immunoplotting. UT, no pretreatment; TUN, tukamycin pretreatment; TG, thapsigargin pretreatment. BEST MODE FOR CARRYING OUT THE INVENTION

The present invention provides a method for differentiating myoblasts or myoblast-like cells into muscle fibers.

The inventor of the present application discloses that myoblasts or myoblast-like cells are exposed to endoplasmic reticulum (ER) stress before or immediately after culturing in a differentiation-inducing medium. The inventors have found that cells undergo cell fusion to form multinucleated muscle fibers and cause muscle contraction, thereby completing the present invention. The present inventors have detected the occurrence of ER stress during the myogenic differentiation process in the myoblast culture system in vitro and in the muscle tissue of the mouse embryo, and have shown that the stress induces apoptosis in the early stage of differentiation. I came out. Conventionally, in muscle differentiation of culture systems, differentiation was induced by lowering the serum concentration of the culture medium.In addition to this procedure, the possibility of increasing ER stress by applying ER stress to cells may be increased. And completed the invention of the present application.

The invention of the present application includes a pretreatment for exposing the myoblasts to endoplasmic reticulum stress before the differentiation treatment of the myoblasts in the differentiation induction medium or immediately after the start of the culture, and then culturing the cells in the differentiation induction medium. And the resulting muscle fibers.

In the endoplasmic reticulum (ER), the protein synthesized by the ribosome takes a correctly folded conformation through, for example, sugar chain modification and disulfide bond formation, and is then transferred to the Golgi apparatus. However, proteins that do not fold correctly due to protein mutations or abnormal modifications accumulate in the endoplasmic reticulum.If the accumulation is excessive, the endoplasmic reticulum is stressed, and force spase 12 is involved. It is said that apoptosis (cell death) is caused.

In the differentiation from established myoblasts according to the conventional method, even if the myosite is differentiated, the formation of myotube cells by cell fusion from myocytes and the subsequent formation of muscle fibers are not sufficient. Muscle contraction failed to occur.

The present inventors transiently apply endoplasmic reticulum stress that causes cell death to myoblasts before differentiation induction or immediately after the start of differentiation induction culture, thereby enhancing apoptosis in the early stage of the differentiation process and increasing stress. This is the first finding that myoblasts can be differentiated very efficiently by artificially removing weak cells and selecting stronger and better cells. Transient application of endoplasmic reticulum stress to myoblasts before or at the start of differentiation induces myoblasts that differentiate from myoblasts to cause cell fusion to form myotube cells and muscle fibers This seems to have had a very significant effect on the stage.

1. [Myoblasts or myoblast-like cells]

As the myoblasts or myoblast-like cells used in the present invention, primary cultured myoblasts and established myoblasts derived from various animals such as birds or mammals can be used. For example, primary cultured myoblasts and established myoblasts or myoblast-like cells derived from chickens, humans, monkeys, rats, mice, pigs, or mice can be used. As established myoblasts or established myoblast-like cells In some cases, those established from normal muscle tissue and those established from tumor cells such as rhabdomyosarcoma can be used, specifically, those established from mice.

C2C12, mouse F-6, mouse C2BP5, rat F-12, rat L6, human RD (examples of established myoblast-like cells, such as the cell CCL-136 described in the ATCC catalog). These can be obtained from RIKEN BioResource Center, American Type Culture Collection (ATCC), etc.

Optionally, cells obtained by culturing myoblasts from an experimental animal or a patient can also be used. For example, a muscle isolated from a biopsy is treated with trypsin-EDTA to prepare a primary culture, and DMEM (Dalbecco's modified Eagle's Eagle) supplemented with 10% FBS, 2n ML-glutamine, and antibiotics Medium) Myoblasts can be isolated by flow cytometry using both anti-myosin heavy chain monoclonal antibody and anti-5.1.H11 antibody cultured in growth medium (Ann. Transplant (1999) 4: 103-108) ₀ Also, for example, the method described in The Journal of Cell Biology (1994); 125: 1275-1287, or Circulation (2000); 102 (19 Suppl. 3): III210-215. The method described in. Can also be used.

2 [ER stress]

Means for applying ER stress can be achieved by various known means, for example, heat shock, or by exposing myoblasts or myoblast-like cells to, for example, an ER stress inducer. Preferably, means for imparting stress specifically to the endoplasmic reticulum, specifically, an endoplasmic reticulum-specific endoplasmic reticulum stress inducer can be used. Further, after culturing myoblasts or myoblast-like cells for a short time in a growth medium or a differentiation medium to which an endoplasmic reticulum-specific endoplasmic reticulum stress inducer has been added, an endoplasmic reticulum-specific endoplasmic reticulum stress inducer is included. Apoptosis promoting effect equivalent to that of pretreatment with an endoplasmic reticulum-specific endoplasmic reticulum stress inducer using the supernatant (conditioned medium) separated from the differentiation induction medium, A formation promoting effect can be obtained.

3. [ER-specific ER stress inducer]

Examples of the endoplasmic reticulum stress inducer used in the present invention include various agents that give stress to the endoplasmic reticulum. Preferably, an endoplasmic reticulum-specific endoplasmic reticulum stress inducer that specifically gives stress to the endoplasmic reticulum can be used. For example, specific to the ER Includes inhibitors of the enzymes present, as well as inhibitors of ER-specific mechanisms. For example, an agent that inhibits N-glycosylation in the endoplasmic reticulum, specifically, an inhibitor of tunicamycin or corynetoxin, a glycosidase, specifically, an inhibitor of castanospermine or mannosidase, specifically, a funensine ) Or deoxymannojirimycin, an endoplasmic reticulum-specific inhibitor of Ca ⁺⁺ ATPase, specifically thapsigargin, or an inhibitor of ryanodine receptor, specifically Can be. Hereinafter, an endoplasmic reticulum-specific endoplasmic reticulum stress inducer will be described. Other endoplasmic reticulum stress inducers can be used in the same manner as long as the differentiation of myoblasts or myoblast-like cells can be promoted.

As a means for exposing myoblasts or myoblast-like cells to ER-specific ER stress using an ER-specific ER stress inducer, the above ER-specific ER stress inducer is used in a medium. Myoblasts after addition, or pretreatment with a medium containing an endoplasmic reticulum-specific ER stress inducer, or a culture supernatant obtained by culturing myoblast-like cells in a differentiation medium Can be used as The concentration when the vesicle-specific ER stress inducer is added to the medium is, for example, based on the concentration at which about half of myoblasts or myoblast-like cells undergo apoptosis and die after 24 hours. Concentration can be set. For example, the final concentration is 0.01 to 1 and 0.000 g / m1, preferably 0.5 to 10 g / ml in the case of tsuyumycin, and the final concentration is 0.1 and 10 in the case of thapsigarnoresin. It can be added so as to be 0.1 to 1, 000 / zM, preferably 0.5 to 10 μΜ.

Exposure of myoblasts or myoblast-like cells to endoplasmic reticulum-specific endoplasmic reticulum stress includes various cell defense reactions due to the occurrence of stress in the endoplasmic reticulum in myoblasts or myoblast-like cells. Tress response), but not enough to initiate apoptosis. For example, when the above agent is added to the growth medium, the myoblasts or myoblast-like cells are reduced to the ER-specific cells at the above concentration for about 0.5 to 48 hours, preferably for about 1 to 6 hours. It can be achieved by culturing in a medium containing an endoplasmic reticulum stress inducer.

When an ER-specific ER stress inducer is added to the growth medium and used, the medium is removed after exposure to ER stress, and the ER-specific ER stress inducer is included. Add no differentiation induction medium. The endoplasmic reticulum-specific endoplasmic reticulum stress pretreatment does not require the use of a growth medium. When the myoblasts or myoblast-like cells are transferred from the normal growth medium to the differentiation medium, the endoplasmic reticulum-specific endoplasmic reticulum It is also possible to add a tress inducer and culture for 0.1 to 24 hours, preferably for about 0.5 to 3 hours. As a differentiation induction medium, a conventionally known standard differentiation induction medium for myoblasts or myoblast-like cells can be used. For the differentiation of mammalian myoblasts or myoblast-like cells, for example, a medium obtained by adding 2% poma serum (Invitrogen Gene) to DMEM can be used. At this time, for example, 1 lg / ΧΆ1 insulin (Sigma-Aldrich) may be further added.

4. [Induction of differentiation]

After exposure to endoplasmic reticulum-specific endoplasmic reticulum stress, myoblasts or myoblast-like cells are cultured in a differentiation-inducing medium for at least 4 or 5 days, preferably for at least 1 week, to reduce myotubes to myofibers. Can be differentiated. Optionally, culturing in a differentiation-inducing medium can be continued for an additional 3 weeks or more. Examples of the differentiation-inducing medium include, for example, DMEM (supplemented with 50 units / ml of penicillin (Invitrogen) and 50 g / ml of streptomycin (Invitrogen)), and serum of serum (Invitrogen) in the range of 0 to 50%. A medium having a growth factor concentration lower than that of a standard growth medium, such as a medium supplemented with 0% to 2% of 5% or bovine serum (Invitrogen) (or fetal bovine serum (Invitrogen)) can be used. At this time, for example _0. 1~10μ ε / πι1 insulin (in vitro Zhen) or 0. 055~5. 5 g / ml of transferrin (Invitrogen) may be e pressurized.

5. [Conditioned medium (conditioned medium: conditioned medium)]

A medium in which myoblasts or myoblast-like cells, which have been pretreated for a short time with a medium to which the ER-specific ER stress inducer has been added, are cultured in a differentiation medium containing no ER-specific ER stress inducer Alternatively, endoplasmic reticulum stress can be specifically applied to myoblasts or myoblast-like cells using the supernatant separated from the culture as a conditioned medium.

Alternatively, after transferring myoblasts or myoblast-like cells to a differentiation medium, an endoplasmic reticulum-specific endoplasmic reticulum stress is applied in the same medium for a short time, and an endoplasmic reticulum-specific endoplasmic reticulum stress inducer is contained. A conditioned medium generated by a differentiation medium and a differentiation medium that does not contain an endoplasmic reticulum-specific endoplasmic reticulum stress inducer used for subsequent culture may be used.

More specifically, for example, pretreatment of myoblasts or myoblast-like cells in a growth medium obtained by adding an endoplasmic reticulum-specific ER stress inducer to cells growing in a growth medium for 1 to 6 hours After that, the cells were differentiated by continuing the culture for 6 to 48 hours, preferably 12 to 36 hours, more preferably 24 hours, instead of the differentiation inducing medium (containing no inducer). The medium of the differentiation-inducing medium or a supernatant thereof can be used as a conditioned medium. Preferably, the used medium can be recovered and centrifuged at low speed (2,000 rpm, 20 minutes) to remove large impurities and the like.

The pretreatment of the myoblasts or myoblast-like cells using the conditioned medium can be performed, for example, for 0.5 to 48 hours, preferably for 12 to 36 hours. After the pretreatment with the conditioned medium, the differentiation into muscle fibers can be induced by continuing the culture in place of the normal differentiation medium.

After pretreatment of myoblasts or myoblast-like cells with an endoplasmic reticulum-specific stress inducer, first replace the medium with a differentiation-inducing medium and culture for 24 hours. It is preferable to apply a mechanical stress, but the conditioned medium obtained by culturing the differentiation-inducing medium after the pretreatment after the second exchange into the differentiation-inducing medium, and the conditioned medium obtained by subsequent It can also be used in the same way as.

6. [Culture kit]

The present invention includes the following muscle fiber culture kit.

(1) Myoblast or myoblast-like cell differentiation kit

(B) a primary culture of established myoblasts or myoblast-like cells or myoblasts, (b) a myoblast comprising an endoplasmic reticulum-specific endoplasmic reticulum stress inducer, and (c) a differentiation-inducing medium. A kit capable of differentiating muscle fibers from vesicles or myoblast-like cells.

As the myoblast or myoblast-like cell, the endoplasmic reticulum-specific endoplasmic reticulum stress inducer and the differentiation induction medium, the above-described myoblast or myoblast-like cell, the inducer and the culture medium are used.

Specifically, as the established myoblasts or myoblast-like cells, mouse C2C12, mouse F-6, mouse C2BP5, rat F-12, rat L6, rat H9C2, Or a human rhabdomyosarcoma-derived cell RD. Examples of the ER-specific ER stress inducer include tunicamycin or thapsigargin.

(2) Muscle fiber culture kit

(B) a kit comprising myofibroblasts or myofibroblast-like cells, myofiber cells generated by exposure to endoplasmic reticulum stress, and (mouth) a differentiation-inducing medium.

7. [Screening method]

The kit of the present invention can be used for screening of a drug acting on muscle fibers and for gene expression using muscle fibers.

The kit and the screening method of the present invention can be used 1) as a muscle regeneration model for prevention and treatment development of muscle death and muscle destruction due to exercise load and aging. 2) Since muscle tissue is the main sugar-consuming tissue in the body, the formed muscle fiber can be used for pathological studies of diabetes, screening and testing of preventive and therapeutic drugs.

(1) Prevention of muscle death and muscle rupture due to exercise load and aging, and screening for potential therapeutic agents.

(1-1) a compound or reagent to be tested is administered to myoblasts or myoblast-like cells to form muscle fibers by the method of the present invention, and (a) a compound or reagent that increases the speed of muscle fiber formation, or (Mouth) 'A compound or reagent that further increases the formation efficiency, or (C) A compound or reagent to be tested on muscle fibers formed by the method of the present invention is administered to myoblasts or myoblast-like cells, Compounds or reagents that make muscle fibers longer (live longer) can be screened as drug candidates for prevention or treatment against muscle necrosis or muscle dystrophy. Whether the searched prophylactic or therapeutic drug candidate actually has a preventive or therapeutic effect on exercise load or muscle canopy can be confirmed by clinical trials on experimental animals and humans. According to the screening method of the present invention, it is possible to narrow down the search to drug candidates that directly affect muscle fibers.

(1-2) One aspect of exercise load can be reproduced by injecting, for example, lactic acid into muscle fibers formed by the method of the present invention. Therefore, after injecting lactic acid into the muscle fiber of the present invention, a reagent or a compound which is a candidate for screening for a candidate for the prevention or treatment of muscle necrosis or muscle rupture is administered to the muscle fiber, and the muscle fiber is administered to the muscle fiber. Drugs to prevent death, It can be selected as a prophylactic or therapeutic candidate for muscle death or muscle destruction.

(2) Muscle tissue takes up the largest amount of sugar in the body, is important as a place for utilizing and storing sugar, and affects the concentration of bran in the blood. Therefore, the formed muscle fiber should be used as a material for studying the mechanism of controlling sugar uptake and metabolism by muscle cells, and also as a test system for screening of drugs that can artificially control sugar uptake and metabolism. Can be.

Specifically, a reagent or a compound to be screened is administered to the muscle fiber formed by the method of the present invention, and sugar uptake or sugar metabolism in the muscle fiber is measured, and sugar uptake into muscle cells is measured. In addition, drug candidates that inhibit or activate metabolism can be selected.

8. [Transformed muscle fiber]

Differentiation can be induced in vitro from myoblasts or myoblast-like cells into which a foreign gene has been introduced to form myofibers. Transformed muscle fiber can be used as a vector for stable gene expression and gene therapy by transplanting it into a living body and assimilating it into muscle tissue.

More specifically, a cDNA encoding a target gene or a recombinant expression vector recombined with the cDNA is introduced into myoblast culture cells, muscle fibers are formed by the method of the present invention, and this is transplanted into a living body. As with transformed myoblasts, continuous gene expression can be achieved in muscle tissue by assimilation into muscle tissue (Vandenburgh, H. et al., 1996, Tissue-engineered s). eletal muscle organoids for reversible gene therapy. Hum. Gen. Ther. 7, 2195-2200). Transplantation using conventionally transformed myoblasts increases the risk of forming a tumor. However, as in the present invention, the use of already differentiated transformed myofibers leads to such a risk. Can be avoided.

The type of gene to be introduced into myoblasts or myoblast-like cells may be a gene encoding a protein that functions in muscle tissue, or a gene encoding a protein that functions in other tissues. For example, since the vascular system is developed in muscle tissue, expressing a secretory protein can have a systemic effect by blood flow. Reagents used in the following examples. For antibodies: tunicamycin, thapsigargin, staurosporine and etoposide were purchased from Calbiochem. Recombinant IGF-II was obtained from GroPep. Anti-caspase-12 is from (Nakagawa, et al. (2000) Nature 403, 98-103). Anti-IGF-II was obtained from R & D Systems, Anti-Cathebucin B was obtained from Upstate Biotechnology, etc., Anti-Bcl ~ xLf and Transduction Laboratories, etc.

[Example 1] Effect of pretreatment of C2C12 with ER stress inducer

C2C12 cells (RIKEN Cell Bank) were seeded on gelatin-coated plates. 10% (v / v) © Shi calf serum (Sigma-Aldrich), using DMEM supplemented with 50 units / ml penicillin及Pi 50μ§ / ηι1 streptomycin (Invitrogen) (Inbitorogen), 5% C0 ₂ present The culture was performed at 37 ° C below. When the C2C12 cells had grown to near confluence, the medium was replaced with fresh DMEM. Then, add the specific endoplasmic reticulum stress inducer, tsuyuikimycin (purchased from Calbiochem) or thapsigargin (purchased from Calbiochem), to the final concentration of tsuyuikimycin (2 μg / m1) or Added to make thapsigargin (1 μΜ). Note that no ER-specific ER stress inducer was added to the control medium. After culturing for 1 hour in a medium containing an endoplasmic reticulum-specific endoplasmic reticulum stress inducer, the medium was replaced with a differentiation induction medium. The differentiation-inducing medium was DMEM (50 units / ml of penicillin and 5 (^ _g / ml of streptogen) supplemented with 2% sera (Invitrogen) and lg / ml insulin (Sigma Aldrich). (Invitrogen) The differentiation-inducing medium was replaced with a fresh medium at intervals of 24 hours.

Note that the same pre-processing as described above is also performed in Examples 2-3 below.

(1) After pretreatment with tunicamycin or thapsigargin, the apoptosis rates after 1 day, 2 days and 3 days after replacing the medium with the differentiation inducing medium are as follows. table 1

Pretreatment of C2C12 cells with endoplasmic reticulum-specific stress inducing apoptosis

As seen in Figure 1, apoptotic cells have a significantly reduced cell volume, become round and small cells, and have poor adhesion to culture plates. A small number of round, poorly adherent cells are found in the growth medium, but these are cells in the mitotic phase, not apoptotic cells. As shown above, after pretreatment with tsuyumycin or thapsigargin, about 40% and 20% of the C2C12 cells in the culture plate were replaced with the differentiation induction medium on days 1 and 2, respectively. Became apoptotic. This rate of apoptosis induction is 2-3 times higher than that of control cells in the differentiation medium (day 1, 15%, day 2, less than 10%).

(2) When cultured in a differentiation-inducing medium for about 2 weeks, the length and width of muscle fibers and the number of contracting muscle fibers (per 3. 5 cm dish) are as follows. Table 2 Comparison with and without pretreatment with ER-specific stress inducer

Fig. 3 shows the photograph.

The size of the pretreated myotubes increased for about 2 weeks. In the meantime, cell fusion is taking place, eventually reaching a length of about 1-3 mm and a width of about 0.2-0.5 mm. It is about the same size as in vivo muscle fibers, but more than 10 times larger than control cells. These pretreated myotubes began to contract spontaneously on day 7 (approximately 1-2 cycles / sec) in place of the differentiation induction medium. This indicates that these cells contained myofibrils and were functional myofibers. 3. In a 5 cm dish, over 50 muscle fibers were reproducibly formed, and most of them continued to contract in differentiation medium for 25 days. On the other hand, control cells formed a few muscle fibers per dish and started to contract around day 9 in place of the differentiation-inducing medium. However, it stopped contraction on day 15 instead of the differentiation induction medium. (3) Expression of insulin-like growth factor II (IGF-II)

Induction of insulin-like growth factor II (IGF-II) was detected within one hour after transfer of C2C12 myoblasts to the differentiation induction medium. Insulin-like growth factor II (IGF-II) acts as an autocrine survival factor that binds to the IGF-1 receptor and indirectly activates Akt-dependent anti-apoptotic pathways during the transition from growth to differentiation (Stewart and Rotwein J. Biol. Chem. Vol. 271, pp. 11330-11338; Lawlor and Rotwein J. Cell Biol. Vol. 151, pp. 1131-1141).

To examine the quantitative variation of IGF-II secreted from the cells into the medium, cells were transferred to a differentiation induction medium and conditioned medium (hereinafter referred to as CCM) prepared daily from control culture. After pretreatment with O. nikkimycin, the cells were conditioned medium (hereinafter abbreviated as TUNCM), which is the supernatant from the culture transferred to the differentiation induction medium, or thapsigargin pretreatment, followed by differentiation induction. A conditioned medium (hereinafter, abbreviated as TGCM), which was a supernatant from the culture transferred to the medium, was collected, aliquoted in a predetermined amount, and treated with SDS-PAGE: sample buffer. The treated medium was analyzed by SDS-PAGE and Western plot method, and IGF-II contained in the medium was detected. The Eastern blot prepared by a conventional method was incubated with an anti-IGF-II antibody (R & D Systems) and then with a horseradish peroxidase-labeled anti-IgG antibody (Jackson ImmunoResearch Laboratories), and the IGF-II signal was measured. Detection was performed using ECL Plus reagent (Amersham-Pharmacia).

IGF-II in conditioned medium (CCM) prepared from control differentiation cultures reached levels detectable by Western blotting at 20th place in place of differentiation induction medium, but was pretreated with tunicamycin IGF-II in conditioned medium (TUNCM) from conditioned cultures or conditioned medium (TGCM) from cultures pre-treated with thapsigargin was detected from day 4 onward after pretreatment, replacing the differentiation-inducing medium. (Figure 4). This result suggests that the autocrine survival factor in the early stage of differentiation was reduced and the apoptosis was promoted by the effect of the pretreatment with nikkimycin or thapsigargin. This is supported by the following observations. As shown in Figure 5, by adding recombinant IGF-II to the culture medium and increasing the concentration of survival factors, C2C12 undergoing differentiation almost completely, regardless of the presence or absence of ER stress-inducing pretreatment. Can suppress cell apoptosis It was.

Since IGF-II acts not only as a survival factor, but also as an autocrine differentiation factor (Florini et al., J. Biol. Chem. Vol. 266, pp. 15917-15923), as shown later, IGF-II Decreased expression leads to a delay in the induction of muscle-specific proteins. (See Example 2.)

(4) Mechanism of fluctuation of extracellular IGF-II level by endoplasmic reticulum stress pretreatment: amount of pro-tepsin B

The level of extracellular IGF-II is regulated by being taken up and degraded by the IGF-II / Man-6P (mannose-6-phosphate) receptor. The original ligand of IGF-II / Man-6P is Man-6-P modified, for example, procatebcin B (Hanewinkel et al., J. Biol. Chem. Vol. 262, pp. 12351-12355). It was expected that the quantitative variation of Man-6-P modified protein would affect the efficiency of degradation of IGF-II. '

Prepared daily from control cultures after transferring cells to differentiation induction medium. Supernatant from cultures transferred to differentiation induction medium after conditioned medium (CCM) and tweakmycin pretreatment After conditioned medium (TUNCM) or thapsigargin pretreatment, the conditioned medium (TGCM), which is the supernatant from the culture transferred to the differentiation induction medium, was collected and examined for quantitative changes in procatebcin B. . A fixed amount of each conditioned medium was analyzed by SDS-PAGE and Western blotting. Western plots were incubated with an anti-Catebsin B antibody (Upstate Biotechnology) followed by a horseradish peroxidase-labeled anti-IgG antibody (Jackson ImmunoResearch Laboratories). Procathepsin signal was detected using ECL Plus reagent (Amersham-Pharmacia).

As a result, Man-6P-modified procatebcin B was detected at a constant level in control conditioned medium (CCM) from day 1 to day 3, but Man-6P-modified in the pretreated culture. It was found that the type was greatly reduced on day 1 in place of the differentiation induction medium (FIG. 6). In the case of TGCM, almost no procatebcin B was detected, and in TUNCM, only non-dalicosylated propothepsin B which was not modified with Man-6-P was detected.

This result indicates that extracellular Man-6P-modified proteins such as procatebcin B This suggests that it is reduced by the influence of ER stress pretreatment, and instead of Man-6-P protein, IGF-II binds to the IGF-Π / Man-6P receptor and becomes easier to degrade. Things are possible.

(5) Use of conditioned medium

The effects of promoting apoptosis and muscle fiber formation can be obtained without using an endoplasmic reticulum-specific ER stress inducer directly on cells. After pretreatment of C2C12 myoblasts with an endoplasmic reticulum-specific endoplasmic reticulum stress inducer for 1 hour, the cells were transferred to a differentiation-inducing medium and cultured. The differentiation induction medium was collected every 24 hours and replaced with a new differentiation induction medium. The recovered medium was centrifuged at 200 rpm for 20 minutes to remove cell dead cells and precipitates, and the supernatant was obtained and used as a conditioned medium (TUNCM and TGCM described above). ). Separately prepared C2C12 cells were grown to subconfluent and pretreated with TUNCM or TGCM for 24 hours. Thereafter, the cells were transferred to a fresh differentiation induction medium and cultivation was continued. By adding pretreatment with a conditioned medium prepared using cells pretreated with an endoplasmic reticulum-specific ER stress inducer in this way, compared to the normal differentiation induction method in which cells are directly transferred from the growth medium to the differentiation medium Thus, apoptosis and promotion of muscle fiber formation were observed. The level reached a level similar to that of cells pretreated with an ER-specific ER stress inducer (Fig. 7). Such a promoting effect was not observed when pretreatment was performed with CCM instead of TUNCM or TGCM.

[Example 2] Confirmation of caspase-12 activation by ER-specific ER stress inducer and expression of myodidinin

(1) Western blot analysis

After pretreatment with an endoplasmic reticulum-specific endoplasmic reticulum stress inducer in the same manner as in Example 1, living or apoptotic C2C12 cells were replaced on day 1 or day 2 in place of the differentiation induction medium, respectively. The cells were collected and dissolved in SDS-PAGE sample buffer to make a cell extract. Proteins in the extract were separated by SDS-PAGE (14% acrylamide) and transferred to Immobilon-P membrane (Millipore, Bedford, MA).

After blocking, the above membrane was coated with an anti-caspase-12 antibody (Nakagawa, et al. (2000)

(Nature 403, 98-103), followed by horseradish peroxidase-labeled anti-IgG antibody (Jackson ImmunoResearch Laboratories). Kasuno ヽ¹ ~--1 The signal of 2 was detected using ECL Plus reagent (Amersham-Pharmacia).

As shown in FIG. 2, caspase-12, a protease that induces endoplasmic reticulum stress-specific apoptosis, is activated in apoptotic cells, indicating that endoplasmic reticulum stress has occurred.

(2) Expression of myodidinin

Myogenin is a muscle-specific protein.

After pretreatment with an endoplasmic reticulum-specific endoplasmic reticulum stress inducer in the same manner as in Example 1, C2C12 cells cultured from day 1 to day 10 every day instead of the differentiation induction medium were prepared, A Western plot was prepared as described in (1) above (FIG. 10).

After blocking, the membranes were incubated with an anti-myogenin antibody (Santa Cruz) and then with a Western: Wasabi peroxidase-labeled anti-IgG antibody (Jackson ImmunoResearch Laboratories). Myogenin signal was detected using ECL Plus reagent (Amersham-Pharmacia). '

In control cells, expression of myogendin was confirmed from day 2, whereas in cells pretreated with an endoplasmic reticulum-specific ER stress inducer, expression of myogenin was confirmed only on day 4. Pretreatment of endoplasmic reticulum stress reduces the amount of differentiation-inducing factor, IGF-II, which is consistent with the slowing of the differentiation process.

[Example 3] Resistance of differentiated C2C12 cells

After pretreatment of C2C12 cells with tsuyumycin or thapsigargin, differentiation was initiated, and on day 3 their resistance was examined and compared to proliferating C2C12 cells (Figure 8).

(1) 2 g / m1 tube for proliferating C2C12 cells, differentiated C2C12 cells pretreated with tunicamycin, differentiated C2C12 cells pretreated with thapsigargin, and differentiated C2C12 cells not pretreated One of the apoptosis-inducing agents, forcemycin, 1 M thapsigargin, 100 g Zml etoposide, and 0.2 μΜ staurosporine was allowed to act. Differentiating cells are all three days after the start of differentiation. Cells were observed microscopically 3 hours after drug addition for staurosporine and 24 hours after other apoptosis inducers.

These apoptosis inducers are highly effective on growing C2C12 cells, Induces apoptosis in half of the cells. On the other hand, cells in the pre-treated differentiation induction medium were almost completely resistant to ER stress inducers (twinikimycin, thapsigargin), etoposide and staurosporine. This result indicates that cells that have undergone endoplasmic reticulum stress and have survived apoptosis have become more resistant to apoptotic stimuli.

Control cells (without pretreatment) in differentiation-inducing medium showed slightly higher resistance than proliferating cells. These results indicate that there is a positive correlation between the degree of stress and apoptosis received by cells and the drug resistance of the cells (pretreated differentiated cells)> differentiated cells without pretreatment. Cells in the order of growing cells).

(2) Bcl-xL expression level

The amounts of Bcl-xL in proliferating C2C12 cells, differentiated C2C12 cells pre-treated with Tuyumycin, differentiated C2C12 cells pre-treated with thapsigargin, and untreated differentiated C2C12 cells were determined. This was examined by Western blotting (Figure 9). Cell extract

(Example 2, (1)) was electrophoresed and transferred to Immobilon-P membrane (Millipore, Bedford, MA).

(transduction Laboratories) and then with horseradish peroxidase-labeled anti-IgG (Jackson ImmunoResearch Laboratories). Cygnale was detected using ECL Plus reagent (Amersham-Bualmacia).

(Result)

Bcl-xL is an anti-apoptotic protein with potent anti-apoptotic activity. The amount of Bel-xL in living cells (in growth, in differentiation medium) does not differ significantly between the three. However, Bel-xL levels in apoptotic cells were significantly lower than in viable cells. Thus, the low resistance exhibited by apoptotic cells is at least partially explained by low levels of Bcl-xL.

Multiple single clones were isolated from cultures of C2C12 cells, expanded in growth medium and transferred to differentiation medium for apoptosis. As a result, the cell population was divided into apoptotic cells and cells that survived and differentiated, even from a single clone. This was initially uniform in vulnerability and Bel-xL expression C2C12 This shows that the culture was divided into dead cells and surviving cells. The exact mechanism of such sorting is currently unknown, but apoptosis here contributes to the removal of cells that are weakly stressed, plays a role in selecting cells that can form large muscle fibers and withstand the stress of muscle contraction. There may be.

The above selection is likely to be a phenomenon specific to endoplasmic reticulum stress. For example, when another apoptosis-inducing stimulus (eg, etoposide) is given to myoblasts, apoptosis during the differentiation process is enhanced but efficient. Muscle fiber formation was not seen. Industrial applicability

According to the method of the present invention, (1) the efficiency of muscle fiber formation can be dramatically increased as compared with the conventional method, and the formation rate can be increased by about 10 times or more when the same number of cells is used as a starting material. (2) Further, the size of the formed muscle fiber can be formed, on average, as long as 10 times the length and width as compared with the conventional method. (3) In addition, the life of the muscle fiber is about 2 weeks in the conventional method, but the muscle fiber prepared according to the present invention can survive for 3 weeks or more.

The muscle fiber of the present invention is effective for the development of muscle regeneration technology, screening of reagents affecting muscle, and gene therapy using muscle fiber.

All publications, patents, and patent applications cited in this specification are incorporated herein by reference in their entirety.

Claims

The scope of the claims

1. A method for differentiating myoblasts or myoblast-like cells comprising the following steps.

(1) exposing myoblasts or myoblast-like cells to endoplasmic reticulum stress; and

(2) a step of further culturing myoblasts or myoblast-like cells exposed to endoplasmic reticulum stress in a differentiation-inducing medium.

2. A method for differentiating myoblasts or myoblast-like cells comprising the following steps.

(1) A step of culturing myoblasts or myoblast-like cells in a growth medium to which an endoplasmic reticulum-specific ER stress inducer is added, or inducing myoblasts or myoblast-like cells from a growth medium Immediately after transfer to the culture medium, the step of adding an endoplasmic reticulum-specific ER stress inducer to the differentiation induction medium and culturing

(2. The step of further culturing the myoblast or myoblast-like cells cultured in the step (1) in a differentiation-inducing medium.

3. The ER-specific ER stress inducer is an ER-specific N-glycosylated transferase inhibitor, ER-specific mannosidase inhibitor or ER-specific

Ca ^H ATP ase er specific ER stress inducer der Ru claim 2 method of differentiated myoblasts or myoblasts like cells according selected from inhibitors of.

4. The final concentration of tunicamycin as an endoplasmic reticulum-specific endoplasmic reticulum stress inducer is 0.01 to: L0000 g / m1, or thapsigargin is 0.01 to 1 Ο 1 Ο / iM. 4. The method according to claim 2 or 3, wherein the compound is added so that

5. The myoblast or myoblast-like cell is a myoblast cell line, a cell line derived from a human myosoma cell line, or a primary cultured myoblast cell prepared from a mammalian or avian biological tissue. 5. The method for differentiating myoblasts or myoblast-like cells according to any one of claims 4 to 4.

6. The cell line according to claim 5, wherein the established myoblasts or myoblast-like cells are selected from mouse C2C12, mouse F-6, mouse C2BP5, rat F-12, rat L6, H9C2, and RD. Myoblast or myoblast-like cell differentiation method.

7. A method for differentiating myoblasts or myoblast-like cells comprising the following steps.

(1) (a) Obtained by culturing myoblasts or myoblast-like cells in a growth medium to which an endoplasmic reticulum-specific ER stress inducer has been added, and then changing to a differentiation medium and continuing the culture. Separating the supernatant from the culture medium, or (Mouth) supernatant from a medium obtained by culturing myoblasts or myoblast-like cells using a differentiation medium containing an endoplasmic reticulum-specific ER stress inducer. Separating the

(2) culturing myoblasts or myoblast-like cells using the supernatant separated in (1) as a conditioned medium, and

(3) A step of culturing in a differentiation-inducing medium.

8. The ER-specific ER stress inducer is an ER-specific N-glycosylation transfer: r-rase inhibitor, ER-specific mannosidase inhibitor or ER-specific

Ca ^H ATP ase er specific ER stress inducer der Ru 7. The method of differentiated myoblasts or myoblasts like cells according selected from inhibitors of.

9. As a ER-specific ER stress inducer, add tunicamycin to a final concentration of 0.01 to: 1000 g Zm 1 or thapsigargin to a final concentration of 0.01 to 100. Item 7. The method according to Item 7 or 8.

10. The method according to any one of claims 7 to 9, wherein the culture is performed for 0.5 to 48 hours in a conditioned medium prepared using myoblasts or myoblast-like cells pretreated with endoplasmic reticulum-specific stress. Method.

11. The claim that the myoblasts or myoblast-like cells are established myoblasts, established human myosoma-derived cells, or primary cultured myoblasts prepared from biological tissues such as mammals or birds. 7. The method for differentiating myoblast or myoblast-like cell according to any one of 7 to 10.

12. The established myoblast or myoblast-like cell is selected from mouse C2C12, mouse F-6, mouse C2BP5, rat F-12, rat L6, H9C2 or RD. The method for differentiating myoblast or myoblast-like cell according to the above.

13. Muscle fibers obtained by differentiating myoblasts or myoblast-like cells by the method according to any one of claims 1 to 12.

14. (a) Primary culture of established myoblasts or myoblast-like cells or myoblasts, (mouth) endoplasmic reticulum-specific endoplasmic reticulum stress inducer, and (c) differentiation induction medium, Kit consisting of.

15. A kit comprising myofibroblasts or myoblast-like cells differentiated from myoblasts or myoblast-like cells exposed to endoplasmic reticulum stress, and (mouth) a differentiation-inducing medium.

16. A preventive agent against muscle necrosis or muscle destruction is administered to the muscle fiber according to claim 13 and cultured under conditions that cause muscle necrosis or muscle destruction in the muscle fiber. A method for screening a prophylactic agent candidate.

1. A method for administering a therapeutic agent for improving glucose metabolism to a muscle fiber according to claim 13, and screening the therapeutic agent for a glucose metabolism disorder such as diabetes.

18. A myoblast or myoblast-like cell differentiation inducer containing, as an active ingredient, an endoplasmic reticulum-specific endoplasmic reticulum stress inducer or an apoptosis-inducing component secreted into the medium by cells after pretreatment with endoplasmic reticulum-specific stress.

1 9. After culturing myoblasts or myoblast-like cells in a growth medium containing an endoplasmic reticulum-specific endoplasmic reticulum stress inducer, replacing the medium with a differentiation medium and continuing the culture, Centrifugal supernatant obtained from the conditioned medium.

20. A transformed muscle fiber obtained by introducing a target gene into the muscle fiber according to claim 13. 21. A gene therapy agent comprising the transformed muscle fiber according to claim 20.