KR101636531B1 - The method for preparing myogenic stem cell using transcription factors including Six 1 - Google Patents

Abstract

The present invention relates to a composition for preparing muscle stem cells comprising four transcription factors of Six1, Eya1, Esrrb and Pax3, a method for producing muscle stem cells using the same, and muscle stem cells prepared by the method. The muscle stem cells prepared according to an embodiment of the present invention have stable muscle differentiation ability.

Description

Technical Field [0001] The present invention relates to a method for producing muscle stem cells having muscle differentiation ability using transcription factors including Six1,

The present invention relates to compositions and kits for preparing muscle stem cells comprising four transcription factors of Six1, Eya1, Esrrb and Pax3, a method for producing muscle stem cells using the same, and muscle stem cells prepared by the method.

Although various treatments for congenital muscular diseases such as dysmenorrhea are being tried, there is no effective treatment. In addition, as the patient enters the aging society, the number of patients with sarcopenia is increasing, and muscle damage due to exercise is also increasing. Stem cell therapy has been attempted for the treatment of these congenital and acquired muscle diseases.

Currently, there are three types of stem cells that are being tried for the treatment of muscle diseases: myoblasts, muscle satellite cells, induced pluripotent stem cells, adipose tissue derived stem cells ) And the like.

However, in the case of myoblasts and muscle satellite cells, it is necessary to take samples from the muscles, which is very invasive to remove the muscle tissue. On the other hand, when the STEMs are applied to muscle diseases, differentiation through appropriate muscle differentiation medium and selection through cell surface markers are suggested. In the case of adipose-derived stem cells, the regulation of niche A method of promoting differentiation into muscle has been proposed. However, stem cells and adipose-derived stem cells are rapidly dividing and proliferating, which is good for securing the cell count for stem cell treatment, but it is limited to the possibility of differentiation into cells other than the target cells, or incomplete differentiation into muscles have.

Accordingly, the present inventors have made intensive efforts to acquire suitable stem cells that can be utilized in the treatment of muscle diseases in consideration of the above-mentioned points. As a result, the inventors of the present invention have found that somatic cells are involved in specific muscle differentiation during embryo stage, Of the present invention can directly establish a muscle stem cell line that differentiates into muscle, thus completing the present invention.

It is an object of the present invention to provide compositions and kits for the preparation of muscle stem cells.

Another object of the present invention is to provide a method for producing muscle stem cells capable of stably differentiating into muscle.

It is still another object of the present invention to provide a muscle stem cell line produced by the above production method.

One aspect of the present invention relates to a Sine oculis homeobox homolog 1 protein or a polynucleotide encoding the same; Eya1 (Eyes absent homolog 1) protein or a polynucleotide encoding the same; Esrrb (Estrogen-related receptor beta) protein or a polynucleotide encoding the same; And a composition for producing muscle stem cells from somatic cells comprising Pax3 (Paired box 3) protein or a polynucleotide encoding the same.

As used herein, the term "myogenic stem cell" refers to a cell that has the characteristics of muscle stem cells, including all of the ability to proliferate without any transformation, infinite proliferation, self-renewal ability, and ability to differentiate into muscle , Self-renewal ability or infinite proliferative capacity and muscle differentiation capacity. The self-reproducing ability and muscle differentiation ability can be confirmed as markers. In addition, the muscle stem cell may be, for example, a cell showing the expression of Pax7 or Myf5 together with the self-renewal ability.

 The term "somatic cell" in the present invention means a cell constituting an adult with limited ability to differentiate and regenerate. For example, the somatic cells may be somatic cells constituting the skin, hair, fat, blood, etc. of an individual, and may be fibroblasts, but are not limited thereto. The somatic cells may be specifically embryonic period fibroblasts. The somatic cells may be naturally occurring somatic cells or genetically modified somatic cells. The somatic cell may be derived from an animal, including a human, and preferably also from a human or a mouse.

The composition of the present invention comprises the respective proteins of Six1, Eya1, Esrrb and Pax3, or a polynucleotide encoding each of these proteins. The Six1, Eya1, Esrrb, and Pax3 may be human or mouse-derived. The Six1, Eya1, Esrrb, and Pax3 proteins may independently be a protein having a wild-type amino acid sequence, and at least one amino acid in the wild-type amino acid sequence may have a mutation such as a modification, substitution or deletion, All proteins that retain the function of the protein are included.

Each of the polynucleotides encoding each protein of Six1, Eya1, Esrrb and Pax3 may be a nucleic acid sequence encoding a wild-type protein. Specifically, the polynucleotides encoding the respective proteins of Six1, Eya1, Esrrb, and Pax3 are represented by NCBI Reference Sequence NM_009189.3, NCBI Reference Sequence NM_010164.2, NCBI Reference Sequence NM_011934.4, and NCBI Reference Sequence: It may be a nucleic acid sequence corresponding to NM_008781. Also, the nucleic acid sequence encoding the wild-type protein may be mutated by substitution, deletion, insertion, or a combination thereof. The polynucleotides can be isolated from nature or can be prepared using chemical synthesis methods. Each of the polynucleotides having a nucleic acid sequence encoding each protein of Six1, Eya1, Esrrb, and Pax3 described above may be a single-stranded or double-stranded DNA molecule (genomic or cDNA) or an RNA molecule.

Methods of delivering or introducing polynucleotides encoding sixteen, Eya1, Esrrb, and Pax3 proteins or their proteins into somatic cells to the somatic cells are not limited to methods for providing nucleic acid molecules or proteins to cells that are conventionally used in the art . Preferably, the active ingredients contained in the composition of the present invention may be administered to a culture medium of somatic cells or injected directly into somatic cells. The polynucleotide encoding the Six1, Eya1, Esrrb and Pax3 proteins may be introduced into cells as DNA in a vector form, or introduced into cells using liposomes, cationic polymers, or the like. The polynucleotides can be obtained from viruses obtained from packaging cells transformed with viral vectors inserted with respective polynucleotides, messenger RNA produced by in vitro transcription or electroporation . For introduction of the polynucleotide, lentivirus, retrovirus or adenovirus or Sendai virus can be used, and lentivirus can be preferably used. The polynucleotide may also be contained in a vector which has a selectable marker or whose expression can be regulated by a Tet-On system.

The polynucleotides encoding the Six1, Eya1, Esrrb and Pax3 proteins may be independently included in four expression vectors, respectively, and the polynucleotides may be included in one vector, or two or three vectors may be added to the poly Nucleotides may be included in any combination.

Another aspect of the present invention is directed to a composition comprising a first composition comprising a Six1 protein or a polynucleotide encoding the same; And a second composition comprising an Eya1 protein or a polynucleotide encoding it, Esrrb protein or a polynucleotide encoding the same, and a Pax3 protein or a polynucleotide encoding the same, and a kit for producing muscle stem cells from somatic cells .

In the kit of the present invention, the somatic cells and the muscle stem cells are as described above.

In the kit of the present invention, the polynucleotide encoding the Sixl protein contained in the first composition may be included in a vector containing a selectable marker. The screening marker may be an antibiotic or a fluorescent screening marker, preferably an antibiotic marker. In one embodiment of the present invention, puromycin was used as a selection marker.

In the second composition, the polynucleotide encoding the Eya1 protein, the polynucleotide encoding the Esrrb protein, and the polynucleotide encoding the Pax3 protein may be independently contained in different vectors, Or may be provided in a form that is all contained in one vector. Preferably, the polynucleotide encoding the Eya1 protein, the polynucleotide encoding the Esrrb protein, and the polynucleotide encoding the Pax3 protein may be regulated by a Tet-ON system. The term " Tet-on system "refers to a system in which a target gene can be induced and expressed when tetracycline or a toxicant is present.

The kit may further comprise a substance for screening transformed cells or a substance for regulating the expression of the protein, but the present invention is not limited thereto. Any substance that can be used for the production of muscle stem cells of the present invention . In one embodiment of the present invention, puromycin was used to screen for the introduction of Sixl, and doxycycline was used to control protein expression.

Another aspect of the present invention relates to a method for producing a somatic cell, comprising the steps of: 1) introducing Six1, Eya1, Esrrb and Pax3 protein or a polynucleotide encoding the same into a somatic cell; And 2) culturing a somatic cell into which the protein or polynucleotide has been introduced, in the production of muscle stem cells from somatic cells.

In the method for producing muscle stem cells of the present invention, the somatic cells or the muscle stem cells are as described above.

In the method for producing a stem cell of the present invention, the first step is a step of introducing a Six1 protein or a polynucleotide encoding the Six1 protein into a somatic cell, and then adding the Eya1, Esrrb, and Pax3 proteins to the Sixi- And then introducing the polynucleotide of the present invention. The method of introducing Six1 into the somatic cell and introducing the remaining factors into the cell line transformed with Six1 makes it easier to secure muscle stem cells.

In addition, the method for preparing a muscle stem cell according to the present invention may further comprise the step of introducing the Six protein or a polynucleotide encoding the Six protein into a somatic cell, and then screening the cell into which the Six1 protein or a polynucleotide encoding the polynucleotide encoding the Six protein has been introduced Lt; / RTI > The screening marker may be an antibiotic or a fluorescent screening marker, preferably an antibiotic marker. In one embodiment of the invention, puromycin was used as a selection marker. The marker selected with the selection marker can be subcultured prior to the introduction of Eya1, Esrrb and Pax3 proteins or a polynucleotide encoding the same. Preferably 8 to 10 times. When subculturing within 7 times, transfection and cell sorting may be incomplete.

In the method for producing muscle stem cells of the present invention, the polynucleotides encoding the Eya1, Esrrb and Pax3 proteins may be overexpressed using a Teton system. In this case, the production method may further include the step of introducing polynucleotides encoding Eya1, Esrrb and Pax3 proteins into cells transformed with Sixl and treating them with doxycycline. It is preferred that the doxycycline is treated early after transduction of the transcription factor.

In addition, the method for preparing a muscle stem cell of the present invention may further comprise the step of separating into a single cell using a cell surface marker. This is to increase the purity of the cell line established through transduction. The cell surface markers may include both muscle differentiation markers and stem cell related markers (e.g., mesenchymal stem cell related markers). In one embodiment, CD90.2 negative cells were selected using CD90.2 markers.

In the method for producing a stem cell of the present invention, culturing of the transformed somatic cells in the step 2) is carried out by culturing cells transfected with proteins or polynucleotides of four transcription factors Six1, Eya1, Esrrb, and Pax3 in a muscle cell proliferation medium . The muscle cell proliferation medium includes all media conventionally used in muscle cell culture in the art, and a person skilled in the art can apply appropriate modifications to a known medium. Preferably, the muscle cell proliferation medium may be a medium containing FBS and 6 to 15% of horse serum or a medium containing recombinant murine fibroblast growth factor-basic. In one embodiment, the cells were cultured in DMEM (high glucose) + 10% FBS + 10% horse serum + recombinant mouse fibroblast growth factor-basic (5 ng / ml).

In addition, the method of the present invention may further comprise the step of screening cells having high expression of the early markers of muscle differentiation from the monoclon of the transformed somatic cells secured by the above culture. The initial markers for muscle differentiation may be Pax7, Myf5, MyoD, or a combination thereof. The selected cell line shows higher muscle differentiation ability than the cell line before selection. Specifically, the pre-screening cells show multilamellar bundles from the 10th day after culturing in the differentiation medium, whereas the cells after screening can be observed in the muscle differentiation medium for 3 days to observe multilamellar bundles of muscles, Of the respondents.

The culture is preferably cultivated in a passage 7-15. In one embodiment, stable morphology can be observed after transformation in the muscle stem cells cultured in the passage 7-15.

The present invention can provide a method for establishing a cell line having muscle differentiation ability and self-renewing ability by the method for producing muscle stem cells.

Another aspect of the present invention provides a muscle stem cell prepared by the method for producing a muscle stem cell.

The muscle stem cells may express Pax7, Myf5, or MyoD.

In addition, when the muscle stem cells are cultured in an muscle differentiation medium, the expression of myogenin is greatly increased. The muscle differentiation medium includes all of the commonly used muscle differentiation mediums, specifically, a medium containing an FBS-free and a content of horse serum lower than the growth medium of the muscle stem cells. For example, 2% to 5% of horse serum.

In the present invention, the muscle stem cell-like cell line established by selecting four cell surface markers and muscle differentiation markers by introducing four transcription factors has a small adhesive capacity to a cell culture dish. It may be morphologically small in size, rounded in nucleus and cytoplasm, and similar in shape to muscle satellite cells.

In addition, the muscle stem cell line of the present invention is characterized in that it is excellent in the ability to replicate and exhibits self-regenerating ability. The subculture can be continuously performed in the medium described above. In the embodiment of the present invention, the medium can be cultured while maintaining a constant shape up to the passage 50.

The combination of Six1, Eya1, Pax3 and Esrrb of the present invention can provide muscle stem cells having muscle differentiation ability from somatic cells, and thus can be usefully used as a cell therapy agent for congenital and acquired muscle diseases or muscle damage.

FIG. 1 is a schematic diagram of four transcription factor introduction strategies of Six1, Eya1, Esrrb and Pax3 of the present invention.
FIG. 2 shows the results of RT-PCR of overexpressed gene expression by introducing four transcription factors of Six1, Eya1, Esrrb and Pax3 of the present invention.
FIG. 3 shows the result of confirming the muscle differentiation markers by RT-PCR after differentiation of the muscle stem cell-like cells of the present invention.
FIG. 4 shows the results of FACS analysis of expression of various cell surface markers on mesenchymal stem cells and muscle stem cells.
FIG. 5 shows the results of culturing the cells before selection with a cell surface marker in an muscle differentiation medium for 15 days.
FIG. 6 shows the results of selecting muscle stem cell-like cells with a cell surface marker and then culturing the cells in an muscle differentiation medium for 3 days.
FIG. 7 shows the results of confirming the muscle differentiation markers through fluorescence immuno staining of cells after screening of muscle stem cell-like cells with cell surface markers.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited by these examples.

Example  1. Production of muscle stem cells

1.1. Culture of mouse embryonic fibroblasts

The mouse fibroblasts were used as somatic cells for the production of muscle stem cells. The mouse fibroblasts were chopped embryos of C57BL6 mouse at 13.5 days of gestation and single cells were isolated by collagenase treatment. The cells were cultured in a medium consisting of DMEM (high glucose) + 10% FBS (fetal bovine serum) + 1% glutamax, and 1 × 10 ⁵ cells were transferred to 6-well plates And seeded.

1.2. Lentivirus  Produce

Lentiviruses were used for transfection with the fibroblasts prepared in Example 1.1. The lentivirus production method was inoculated with 5x10 < ⁶ > 293FT cell lines. The plasmid was transfected into 293FT cells through a packaging mixture and lipofectamine. After 48 hours, the culture broth was centrifuged at 3000 rpm for 15 minutes. The supernatant was then filtered through a 0.45 mu m filter. The prepared lentiviral solution was used in the following experiment, and the remaining solution was dispensed and stored at 70 ° C.

1.3. Six1  Introduction of genes

Six1 and puromycin resistance genes were used to produce lentivirus. When mouse embryonic fibroblasts were in the second passage, 1 x 10 < ⁵ > cells were inoculated into 6 well plates. Lentivirus solution and polybrene were mixed and cultured at 37 ° C in 5% CO2 for 24 hours. DMEM (high glucose) + 10% FBS + 10% horse serum + recombinant mouse fibroblast growth factor-basic (5 ng / ml) + 1 % Penicillin / streptomycin medium and cultured. The cells were selected with puromycin (5 / ml) when cells in 6 well plates accounted for 80% to 90% of the plate bottom area. Following the selection with puromycin, the majority of cells die and some puromycin-resistant cells survive. The cells selected by puromycin are cultured until passage 8.

1.4. Eya1 , Esrrb  And Pax3  Gene introduction

Transfected Sixl transformed cell lines were inoculated into 6 well plates at a cell number of 1x10 < ⁵ > cells. 18 hours after inoculation, three kinds of lentivirus solutions prepared by the method described in 1.2 using three kinds of plasmids including Eya1, Esrrb, and Pax3 were put into a 15 ml conical tube in 600 ul each, and polybrene (10 mg / ml) is added and mixed well by vortexing. A mixture of three types of lentivirus and polybrene was placed in a culture vessel inoculated with Sixs cell line of passage 8. After that, the cells were cultured at 37 ° C in 5% CO ₂ for 24 hours and cultured in DMEM (high glucose) + 10% FBS + 10% horse serum + recombinant myline fibroblast growth factor-basic (5 ng / ml) + 1% penicillin / streptomycin And then cultured. At this time, doxycycline was added to the medium to overexpress transduced Eya1, Esrrb and Pax3. Subsequently, subculture was performed and the expression of muscle differentiation markers was observed in passage 7.

Example  2. Analysis of prepared muscle stem cell line

Examination of the muscle differentiation ability of the cells transfected with the four transcription factors Six1, Eya1, Esrrb and Pax3 prepared in Example 1 The muscle differentiation markers were confirmed. In addition, muscle differentiation markers for muscle stem cells into which some of the four transcription factors were introduced were also identified (see FIG. 3). To confirm the markers of muscle differentiation, each cell line established by passage 7 was inoculated into a 6-well plate. When cells grew to about 90% of the 6-well plate, RNA was isolated using tripol and cDNA was synthesized using this. Then, PCR was performed on the muscle differentiation markers Pax7, Myf5, MyoD, Myogenin, and Desmin, and electrophoresis was performed on the 1.5% agarose gel. The results were as follows: 1) cells transfected with Six1, 2) cells transfected with Six1 and Eya, 3) cells transfected with Six1, Eya1 and Pax3, Pax7, Myf5, MyoD, Myogenin and Desmin Myx 5, MyoD, Myogenin, and Desmin were highly expressed in the cells transfected with the four transcription factors Six1, Eya1, Esrrb, and Pax3, respectively ).

In order to identify the differentiation markers at the protein level, immunofluorescent staining was performed on Pax7, Myf5, MyoD, Myogenin and Myosin heavy chain2. Cells with the four factors introduced were inoculated into 4 well chamber slides and fixed with 4% paraformalin at 80% of the slide area. Methanol was added to the chamber slide after the water wash, and then allowed to stand at -20 ° C for 15 minutes, followed by washing with water. Myofilin, MyoD, Myosin, and Myosin heavy chain2 antibody were diluted 1: 200 and then incubated for 1 hour at room temperature. The cells were incubated for 1 hour at room temperature with 5% BSA (Bovine serum albumin) Lt; / RTI > After washing with PBS / T, the secondary antibody conjugated with FITC and TRITC was diluted 1: 500 and added to the chamber slides. This was reacted at room temperature for 1 hour and then washed with PBS / T. Then, DAPI was applied for 10 minutes to stain the nuclei. The slides were covered with cover slip and fluorescence was observed with a confocal laser microscope. Thus, it can be confirmed that the cell line of the present invention has the ability to express the muscle differentiation markers Pax7, Myf5, MyoD, Myogenin, and Myosin heavy chain2 at the protein level and to differentiate into muscles (Fig. 7).

The cells were applied to the muscle differentiation medium to observe the muscle differentiation ability in the muscle differentiation medium. For this purpose, the cells were inoculated into 6-well plates at ⁵ × 10 ⁵ cells / ml. When the cells were stabilized after 24 hours, the medium was replaced with H-DMEM containing 5% horse serum and 1% penicillin-streptomycin as the differentiation medium. The medium was changed every 24 hours. After incubation for 15 days, RNA was isolated and the cDNA was synthesized and PCR was performed on Pax7, Myf5, MyoD and Myogenin. The results were as follows: 1) cells transfected with Six1, 2) cells transfected with Six1 and Eya, and 3) cells transfected with Six1, Eya1, and Pax3 transfected with increasing amounts of Pax7, Myf5, MyoD, and Myogenin However, it was confirmed that the cells in which all four factors of the present invention were introduced showed a specific increase in the expression of Myogenin, a late differentiation marker, when cultured in an muscle differentiation medium for 15 days (FIG. 3). This is a result of demonstrating the muscle differentiation ability in the muscle differentiation medium of the muscle stem cells according to the present invention. In addition, in addition, the cells in which all four factors of the present invention were introduced were observed to differentiate into myotubes having polynuclear differentiation in the muscle differentiation medium. These multinucleated muscle bundles were visible from the 5th day of application of the differentiation medium and became clearer at the 10th day.

Claims (15)

Six1 (Sine oculis homeobox homolog 1) protein or a polynucleotide encoding the same;
Eya1 (Eyes absent homolog 1) protein or a polynucleotide encoding the same;
Esrrb (Estrogen-related receptor beta) protein or a polynucleotide encoding the same; And
A composition for producing muscle stem cells from somatic cells, comprising Pax3 (Paired box 3) protein or a polynucleotide encoding the same. The composition of claim 1, wherein the polynucleotide encoding the Six1 protein, the polynucleotide encoding the Eya1 protein, the polynucleotide encoding the Esrrb protein, and the polynucleotide encoding the Pax3 protein are each independently included in the vector. The composition according to claim 1, wherein the polynucleotide encoding the Eya1 protein, the polynucleotide encoding the Esrrb protein, and the polynucleotide encoding the Pax3 protein are contained in a single vector. A first composition comprising a Sixl protein or a polynucleotide encoding the same; And
A kit for producing muscle stem cells from somatic cells, comprising a second composition comprising an Eya1 protein or a polynucleotide encoding the same, an Esrrb protein or a polynucleotide encoding the same, and a Pax3 protein or a polynucleotide encoding the same. 5. The kit of claim 4, wherein the polynucleotide encoding the Sixl protein is included in a vector comprising a selectable marker. 5. The kit according to claim 4, wherein the polynucleotide encoding the Eya1 protein, the polynucleotide encoding the Esrrb protein, and the polynucleotide encoding the Pax3 protein are contained in a vector whose expression is regulated by the Tet-on system. 5. The kit according to claim 4, wherein the somatic cells are fibroblasts. 1) introducing Six1, Eya1, Esrrb and Pax3 proteins or polynucleotides encoding the same into somatic cells; And
2) culturing the cells into which the protein or polynucleotide has been introduced. [Claim 8] The method according to claim 8, wherein the first step comprises introducing a Six1 protein or a polynucleotide encoding the same into a somatic cell, and then introducing each protein of Eya1, Esrrb and Pax3 or a polynucleotide encoding the same, ≪ / RTI > [Claim 9] The method according to claim 9, further comprising the step of screening the cells into which the Six1 protein or the polynucleotide encoding the polynucleotide encoding the proteins Eia1, Esrrb, and Pax3, or the polynucleotide encoding the same, A method for producing muscle stem cells from somatic cells. 9. The method of claim 8, wherein the polynucleotide encoding the Eya1, Esrrb, and Pax3 proteins is over-expressed using a Teton system. [Claim 9] The method according to claim 8, wherein the culturing of the cells in the step 2) comprises culturing the cells into which the protein or polynucleotide has been introduced, in a muscle cell proliferation medium. 9. The method of claim 8, further comprising separating into a single cell using the cell surface marker prior to step 2). A muscle stem cell produced by the method according to any one of claims 8 to 12. 14. The stem cell according to claim 13, wherein the muscle stem cell expresses Pax7, Myf5 or MyoD.

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