JP2006115771A - Skeletal muscle-derived cardiac muscle stem cells - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a technique for isolating stem cells capable of regenerating myocardium by differentiating into myocardial cells, and providing a technique for fundamentally regenerating myocardium using the cells.
A cell suspension is prepared by enzymatic treatment of the collected skeletal muscle tissue, and the cell suspension is subjected to (1) separation of cells by density gradient method and (2) at least one of CD34 and CD105. By selecting cells that are positive for the species, stem cells that can differentiate into cardiomyocytes and regenerate the myocardium are obtained, and myocardial regeneration therapy is performed using this.
[Selection figure] None

Description

  The present invention relates to a skeletal muscle-derived cardiac muscle stem cell, a method for preparing the cardiac muscle stem cell, and a method for treating heart disease using the stem cell.

  Conventionally, heart failure due to mechanical disorders of the heart, myocardial insufficiency, rhythm abnormalities, etc. has been achieved by reducing blood flow with diuretics, enhancing myocardial contractile force with cardiotonic drugs and optimizing the pulse of atrial flutter, vasodilators Symptomatic therapies such as reduction of the heart load due to the symptom. On the other hand, for severe heart failure, the above-mentioned symptomatic therapy cannot provide a sufficient therapeutic effect, and a fundamental therapy by heart transplantation is required. However, heart transplantation has problems such as a shortage of donors and rejection reactions, and is currently not functioning adequately as salvage medicine. Thus, in recent years, attention has been focused on a method of transplanting progenitor cells or stem cells that can differentiate into cardiomyocytes as a fundamental therapy that replaces heart transplantation.

  However, most of the cell transplants reported so far are essentially unable to regenerate myocardial cells, and the improvement effect on the circulation of the microcirculation important for repair of ischemic myocardium and secretion from engrafted donor cells. In most cases, the cardiac function is improved by the secondary myocardial protective effect of the cytokines produced (see, for example, Patent Document 1).

  In addition, stem cells that have differentiated into cardiomyocytes centering on bone marrow-derived hematopoietic cells and mesenchymal stem cells have been searched so far, but in the previously reported cells, the degree of differentiation into cardiomyocytes is extremely low. It is not clinically practical. Conventionally, there has been no report on successful separation of cell groups that differentiate from skeletal muscle tissue into cardiomyocytes.

Against the background of such conventional technology, the myocardial stem cells that can essentially regenerate myocardial cells are isolated and used to transplant the myocardial cells to the damaged myocardial site, thereby fundamentally regenerating myocardium. It is desired to establish a treatment method.
International Publication No. 03/80798 Pamphlet

  The object of the present invention is to solve the above-mentioned problems of the prior art. Specifically, an object of the present invention is to provide a technique for isolating stem cells capable of regenerating myocardium by differentiation into cardiomyocytes, and providing a technique for performing fundamental myocardial regenerative therapy using the cells. .

  The inventors of the present invention diligently studied to solve the above problems, and prepared a cell suspension by enzymatic treatment of the collected skeletal muscle tissue, and using the cell suspension, (1) density It was found that stem cells that can differentiate into cardiomyocytes and regenerate myocardium can be obtained by separating cells by gradient method and (2) selecting cells that are positive for at least one of CD34 and CD105. Furthermore, after the obtained stem cells are cultured and grown in a medium containing fibroblast growth factor and epidermal growth factor, the cells are differentiated into cardiomyocytes by culturing in a medium containing dexamethasone. I found it. The present invention has been completed by further studies based on this finding.

That is, the present invention is the following invention:
Item 1. A stem cell derived from mammalian skeletal muscle tissue, which has the ability to differentiate into cardiomyocytes.
Item 2. Item 8. The stem cell according to Item 1, which is CD34 positive.
Item 3. Item 3. The stem cell according to Item 1 or 2, which is positive for CD105.
Item 4. Item 4. The stem cell according to any one of Items 1 to 3, wherein the mammal is a human.
Item 5. Item 8. The stem cell according to Item 1, which is prepared through the following steps.
(i) collecting skeletal muscle tissue from a mammal and preparing a cell suspension by enzymatic treatment of the obtained skeletal muscle tissue;
(ii) a step of separating skeletal muscle tissue-derived cells from the cell suspension by a density gradient method, and
(iii) A step of selecting and separating cells positive for at least one of CD34 and CD105 from the obtained skeletal muscle tissue-derived cell group.
Item 6. Item 6. The method for preparing a stem cell according to any one of Items 1 to 5, comprising the following steps:
(i) collecting skeletal muscle tissue and preparing a cell suspension by enzymatic treatment of the obtained skeletal muscle tissue;
(ii) a step of separating skeletal muscle tissue-derived cells from the cell suspension by a density gradient method, and
(iii) A step of selecting and separating cells positive for at least one of CD34 and CD105 from the obtained skeletal muscle tissue-derived cell group.
Item 7. Item 6. A method for treating heart disease, comprising transplanting the stem cell according to any one of Items 1 to 5 or a cardiomyocyte differentiated from the stem cell into a heart of a patient having heart disease.
Item 8. Item 8. The method for treating heart disease according to Item 7, comprising the following steps:
(i) collecting skeletal muscle tissue from a patient having heart disease, and preparing a cell suspension by enzymatic treatment of the obtained skeletal muscle tissue;
(ii) a step of separating skeletal muscle tissue-derived cells from the cell suspension by a density gradient method,
(iii) selecting and separating cells positive for at least one of CD34 and CD105 from the obtained skeletal muscle tissue-derived cell group;
(iv) culturing the cells separated in the above step (iii) in a medium containing fibroblast growth factor and epidermal growth factor to proliferate the cells; and
(v) A step of transplanting the cells grown in the step (iv) into the heart of the patient.
Item 9. Item 8. The method for treating heart disease according to Item 7, comprising the following steps:
(i) collecting skeletal muscle tissue from a patient having heart disease, and preparing a cell suspension by enzymatic treatment of the obtained skeletal muscle tissue;
(ii) a step of separating skeletal muscle tissue-derived cells from the cell suspension by a density gradient method,
(iii) selecting and separating cells positive for at least one of CD34 and CD105 from the obtained skeletal muscle tissue-derived cell group;
(iv) a step of proliferating the cells separated in the step (iii) by culturing the cells in a medium containing fibroblast growth factor and epidermal growth factor;
(v) culturing the cells grown in the above step (iv) in a medium containing dexamethasone to induce differentiation into cardiomyocytes, and
(vi) A step of transplanting differentiated cardiomyocytes into the heart of the patient.

  Hereinafter, the present invention will be described in detail.

  The stem cells of the present invention are derived from mammalian skeletal muscle tissue and have the ability to differentiate into cardiomyocytes.

  The mammal from which the stem cells of the present invention are derived is not particularly limited, and examples thereof include mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, pigs, cows, goats, monkeys and humans. When the stem cells of the present invention are used for the treatment of human heart disease, they are preferably derived from humans.

In addition, the stem cells of the present invention may be derived from any body part as long as they are derived from mammalian skeletal muscle tissue, for example, leg, arm, shoulder. The stem cells of the present invention are at least one selected from the group consisting of CD34 positive and CD105 positive as a characteristic of the cell surface antigen. Species are positive. The stem cells of the present invention need only be positive for either CD34 or CD105, but may be positive for both CD34 and CD105. Usually, the stem cells of the present invention showing CD34 positivity show CD105 positivity at a high rate. For example, when the stem cell of the present invention is derived from human, about 90% of CD34 positive cells are CD105 positive, and when derived from mouse, 70 to 80% of CD34 positive cells are CD105 positive. If the stem cell of the present invention is derived from human, it is preferably a CD105 positive cell.

  Since the stem cells of the present invention have the ability to differentiate into cardiomyocytes, in particular, the ability to differentiate into self-pulsating cardiomyocytes, together with proliferative ability, they can function as myocardial stem cells.

A. A method for preparing a stem cell of the present invention, B. A method for inducing differentiation into cardiomyocytes, and C.I. A method for treating heart disease will be described in detail.
A. 1. Preparation method of stem cell of the present invention Preparation of cell suspension First, skeletal muscle tissue is collected from a mammal, and the resulting skeletal muscle tissue is treated with an enzyme to prepare a cell suspension (step (i)).

Here, collection of the skeletal muscle tissue from the mammal is performed by extracting the skeletal muscle tissue by a normal surgical technique. In addition, it is desirable that the extracted skeletal muscle cells are removed from tissues (for example, blood vessels, nerves, tendons, ligaments, bone tissues, etc.) other than skeletal muscle tissues as much as possible prior to the enzyme treatment. In order to increase the efficiency of the enzyme treatment, it is desirable that the collected skeletal muscle tissue is shredded into fragments of about 1 mm ³ or less and then subjected to the enzyme treatment.

  The skeletal muscle tissue is subjected to an enzyme treatment in an appropriate buffer to prepare a cell suspension. Here, the buffer used is not particularly limited as long as it does not adversely affect cells and enzymes. For example, Hanks' Balanced Salt Solution containing 1% by volume of penicillin-streptomycin and 0.0584% by weight of 1-glutamine ( GIBCO).

  The enzyme treatment is performed using an enzyme that is generally used when preparing a cell suspension from a biological tissue piece. Specifically, proteases such as collagenase, trypsin, chymotrypsin, and pepsin are exemplified. Among these, collagenase is preferable. Specific examples of such collagenase include collagenase type 2 (manufactured by Worthington; 205 U / mg). In the present specification, collagenase 1U represents the amount of enzyme capable of releasing 1 μmol of L-leucine from collagen at pH 7.5, 37 ° C. for 5 hours.

Also, the enzyme treatment conditions are not particularly limited, but the following enzyme treatment conditions are exemplified as an example:
Enzyme concentration: For example, when collagenase type 2 (manufactured by Worthington; 205 U / mg) is used, usually 0.2 to 0.6 wt%, preferably about 0.4 wt%; or usually 3075 to 9225 U per 2 g of skeletal muscle tissue The concentration is preferably about 6150 U.
Treatment temperature: A temperature that is usually about 37 ° C can be mentioned.
Treatment time: Usually 30 to 60 minutes, preferably about 45 minutes.

  The cell suspension thus obtained is preferably subjected to enzyme treatment, centrifuged to remove the supernatant, and a medium suitable for cell growth is added. As a medium suitable for cell growth, for example, Dulbecco's modified Eagle medium containing 10% by volume fetal bovine serum (FBS) and 1% by volume penicillin-streptomycin (a mixture of 5000 U / ml penicillin and 5000 μg / ml streptomycin sulfate) ( DMEM) medium is exemplified.

2. Separation of skeletal muscle tissue-derived cell group Next, the skeletal muscle tissue-derived cell group is separated from the cell suspension by a density gradient method (step (ii)).

  In this step, the skeletal muscle tissue-derived cell group can be separated by a density gradient method usually employed for cell separation. As an example of a preferred embodiment of the separation of skeletal muscle tissue-derived cells, a method of separating skeletal muscle tissue-derived cells by percoll density gradient centrifugation is exemplified. The Percoll density gradient centrifugation method is a known method of performing centrifugation using Percoll, which is a kind of silica gel. Since Percoll is used in layers, it can be separated without disrupting cells by centrifugal force.

  In order to separate skeletal muscle tissue-derived cells from the cell suspension by Percoll density gradient centrifugation, for example, the cell suspension is made up of a discontinuous density comprising 40% and 70% Percoll solutions by volume ratio. The target skeletal muscle tissue-derived cell group can be obtained at the interface between the 40% percoll solution and the 70% percoll solution by centrifugation at 1000 G for 20 minutes at a gradient.

3. Separation of CD34 and / or CD105 positive cells Next, cells positive for at least one of CD34 and CD105 are selected from the skeletal muscle tissue-derived cell group obtained above (step (iii)).

  Selection and isolation of cells positive for at least one of CD34 and CD105 can be performed by a known method using an antibody that recognizes CD34 or CD105. For example, a method of selectively separating CD34 positive or CD105 positive cells by a flow cytometer having a sorting function using an antibody that recognizes CD34 or CD105 bound to a marker such as a fluorescent dye or biotin. For example, cells that are positive for CD34 or CD105 can be selectively separated by using an antibody that recognizes CD34 or CD105 bound to magnetic beads. A suitable method includes a method using a flow cytometer having a sorting function.

  Thus, by selecting and separating cells positive for at least one of CD34 and CD105, stem cells (skeletal muscle-derived cardiac stem cells) having the ability to differentiate into cardiomyocytes as well as proliferative ability can be obtained.

  In this step, when cells that are positive for both CD34 and CD105 are selected, stem cells that exhibit a more uniform differentiated morphology can be obtained.

B. 3. Differentiation induction into cardiomyocytes Proliferation of myocardial stem cells The stem cells can be grown by culturing the stem cells in a medium containing an epidermal growth factor (EGF) and a fibroblast growth factor (FGF). (Step (iv)).

  Regarding the ratio of epidermal growth factor and fibroblast growth factor added to the medium used in this step, for example, epidermal growth factor is about 20 ng / ml and fibroblast growth factor is about 10 ng / ml A ratio of

  Moreover, the culture medium used at this process should just add the epidermal growth factor and the fibroblast growth factor to the culture medium used for normal cell culture. Suitable examples of the medium include a medium in which the epidermal growth factor and fibroblast growth factor are added to a DMEM / F12HAM medium containing human serum or bovine serum albumin. The medium used in this step contains leukemia inhibitory factor (LIF; 10 ng / ml); antibiotics such as streptomycin, kanamycin, penicillin; HEPES (5 mM), etc., as necessary. May be.

By culturing the stem cells (cells positive for at least one of CD34 and CD105) using the medium at 37 ° C. and 5% CO ₂ for 5 to 10 days, preferably 7 days, Proliferation of the stem cells is observed.

5. Differentiation induction of cardiomyocyte stem cells into cardiomyocytes The stem cells thus proliferated are cultured in a medium containing dexamethasone to induce differentiation into cardiomyocytes (step (v)).

  In this step, after removing the medium used for the proliferation of the stem cells, the stem cells are cultured by adding a medium containing dexamethasone, whereby the stem cells can be induced to differentiate into cardiomyocytes at a certain ratio. .

In this step, the ratio of dexamethasone added to the medium is not particularly limited as long as differentiation into cardiomyocytes can be induced, but usually, dexamethasone is contained in the medium at a rate of about 1 × 10 ⁻⁸ mol / l. It only has to be included.

  In this step, the type of the medium to be used is not particularly limited, but a medium in which dexamethasone is added to a MEM medium (minimum essential medium, manufactured by GIBCO) is exemplified as a suitable medium. Moreover, the culture medium used in this step may contain antibiotics such as streptomycin, kanamycin, and penicillin; HEPES (5 mM) and the like, if necessary, in the same manner as the medium used for the proliferation of myocardial stem cells. .

The stem cells are differentiated into cardiomyocytes at a certain rate by culturing the stem cells using the medium at 37 ° C. and 5% CO ₂ for usually 7 to 21 days, preferably 14 to 21 days. Can be guided.

C. Method of treating heart disease The heart disease is treated by transplanting the above-mentioned myocardial stem cells (cells positive for at least one of CD34 and CD105) or cardiomyocytes differentiated from the stem cells into the heart of a patient with heart disease. be able to.

  The target heart disease is a heart disease in which the myocardium or coronary artery is damaged and contractile force is reduced. Specifically, myocardial infarction, dilated cardiomyopathy, ischemic heart disease, congestive heart failure, etc. Is mentioned.

  Examples of the method for transplanting myocardial stem cells include a method of injecting the myocardial stem cells using a catheter into a heart site to be treated, or a method of injecting the myocardial stem cells directly into the myocardium after thoracotomy.

  Moreover, as a method for transplanting cardiomyocytes differentiated from the above-mentioned myocardial stem cells, for example, a method in which cardiomyocytes are carried on a sheet-like bioabsorbable material and affixed to a myocardial injury site can be exemplified.

  In the treatment method of the present invention, it is desirable to transplant myocardial stem cells derived from the skeletal muscle tissue of a patient having heart disease or cardiomyocytes differentiated from the stem cells from the viewpoint of suppressing rejection.

  The present invention provides stem cells derived from skeletal muscle tissue and capable of regenerating myocardium by differentiation into cardiomyocytes. Therefore, according to the stem cell of the present invention, a new heart disease treatment method such as transplantation of myocardial stem cells or cardiomyocytes becomes possible. In particular, according to the stem cells of the present invention, since cardiomyocytes can be prepared easily and in large quantities, it is possible to provide a new treatment method by cell transplantation for patients with severe heart failure who must rely on heart transplantation, and heart diseases that replace heart transplantation It is useful as a method of treatment.

Hereinafter, the present invention will be described in detail based on examples and the like, but the present invention is not limited thereto.
Example 1 Acquisition of mouse-derived cardiomyocyte stem cells and induction of differentiation of the stem cells into cardiomyocytes
(1) Preparation of cell suspension
6-8 week old female C57Bl / 6J mice (manufactured by Shimizu Experimental Materials Co., Ltd.) were manually euthanized by cervical dislocation under diethyl ether anesthesia, and the whole body was disinfected by soaking in 70 vol% ethyl alcohol aqueous solution. . Using sharp tweezers and scissors that had been pre-sterilized with high-pressure steam, the skin of both lower limbs below the waist was peeled off. In order to avoid mixing blood cell components due to bleeding as much as possible, the femoral artery exposed to the groin was ligated with grasping forceps, and the artery below the ligature was peeled within the visible range. Only the muscles were carefully dissected so that other blood vessels, nerves, tendons, ligaments, and bone tissues were not mixed. In the Hanks' Balanced Salt Solution (manufactured by GIBCO) (hereinafter referred to as Buffer 1), the blood components were rinsed until they were sufficiently removed and then stored in fresh Buffer 1. Furthermore, tissue other than muscle is removed as much as possible while crushing the tissue in the buffer 1 with sharp tweezers. The crushed muscle fragments using sterilized scissors were shredded into pieces of about 1 mm ³ or less until they became muddy sand. The muscle fragments were collected in a 50 ml conical tube together with Buffer 1 and centrifuged once to remove the supernatant. Subsequently, 15 ml of 0.4% collagenase type 2 (manufactured by Worthington), which had been kept at 37 ° C. in advance, was added to about 4 g of muscle tissue and shaken in a 37 ° C. constant temperature bath for 45 minutes for enzyme treatment. After enzyme treatment, add 20 ml of Buffer 1 per tube, stir well, and centrifuge to remove the supernatant. After adding 10 ml of 10 volume% FBS (fetal calf serum) (Hyclone) and 1 volume% penicillin-streptomycin DMEM (GIBCO) medium per tube to prepare a cell-containing solution, 40 μm cell It filtered with the strainer (made by FALCON). Then, after filtration, the cell-containing solution was centrifuged to remove the supernatant, and 3 ml of Buffer 1 was added per tube and suspended well. Thus, a cell suspension was made.

(2) Separation of skeletal muscle tissue-derived cells by percoll density gradient centrifugation Percoll stock solution (Amersham Biosciences): 10 × PBS (-) (GIBCO) = 9: 1 (volume ratio) The solution was used as percoll stock. Percoll stock was diluted with 1 × PBS (−) (manufactured by GIBCO) to prepare a solution having a volume ratio of 40% and 70% with respect to Percoll stock. The 40% percoll solution was colored by adding 0.1% by volume of phenol red (manufactured by SIGMA). First, 3 ml of 40% percoll solution was poured into a 15 ml conical tube, and then 70% percoll solution was carefully added to the lower layer of 40% percoll solution using an electric pipettor. Subsequently, 3 ml of the above cell suspension was carefully layered on top of the 40% Percoll solution. Centrifugation was performed at room temperature and 1000G for 20 minutes with acceleration and deceleration as slow as possible. After centrifugation, the target cell population was confirmed to be distributed at the interface of 40% -70% percoll solution. In addition, it was confirmed that blood cell components were distributed at the bottom, and cell debris was mainly distributed in the upper layer of 40% percoll. First, cell debris was removed with a pipette, and then the target cell population present at the interface was collected with another pipette into a conical tube having a volume of 50 ml. 20 ml of Buffer 1 was added to the conical tube, and after stirring sufficiently, the supernatant was removed by centrifugation. The precipitate was sufficiently suspended using an appropriate amount of MACS buffer [1 × PBS (−) (GIBCO), 0.5 vol% bovine serum albumin (SIGMA)], and the number of cells was counted with a hemocytometer.

The thus-separated skeletal muscle tissue-derived cell group was subjected to FACS analysis, and the CD34 positive fraction (the amount corresponding to 20-30% of the skeletal muscle tissue-derived cell group) was sorted. Sorted cells were treated with mouse expansion medium [DMEM / F12Ham (GIBCO), 20% FBS, 1% penicillin-streptomycin, 10ng / ml mouse LIF (CHEMICON), 10ng / ml recombinant human basic FGF (Promega And 20 ng / ml mouse EGF (manufactured by SIGMA)] on a fibronectin-coat cell culture dish (Becton Dickinson) at 37 ° C., 5% CO ₂ Incubated for 5 days. Various cell surface antigens (Sca-1, CD34, CD105, CD90, CD45) of cultured skeletal muscle tissue-derived cells were analyzed. As a result, it was confirmed that about 80% of the skeletal muscle tissue-derived cell group was Sca-1 positive, and about 60% of them was CD34 positive (see FIG. 1A). Further, about 60% or more of the skeletal muscle tissue-derived cell group is positive for CD105 (see FIG. 1B), and about 90% of the skeletal muscle tissue-derived cell group is CD90 positive (C in FIG. 1). (See the figure) Further, in the skeletal muscle tissue-derived cell group, CD34-positive cells and CD105-positive cells are negative for CD45 (see FIGS. 1D and 1E), confirming that these cells are not derived from bone marrow or peripheral blood. It was.

(3) Isolation of CD34-positive cells The skeletal muscle tissue-derived cell suspension obtained in (2) above is equally distributed in a 5 ml round bottom tube within the range of 5-10 × 10 ⁵ cells. One of them was used as a control, and the other was used as a sorting tube. After removing the supernatant by centrifugation, add 100 μl of MACS buffer to each tube and suspend well. Biotinylated rat IgG2a κ isotype control (Pharmingen) is used for the control tube, and biotin-labeled for the other tubes. 1 μl each of anti-mouse CD34 (Pharmingen) antibody was added and stirred well, and allowed to stand on ice for 15 minutes. Thereafter, 1 ml of MACS buffer was added to each tube and stirred sufficiently, followed by centrifugation to remove the supernatant, and 100 μl of MACS buffer was again added to each tube and well suspended. 1 μl of streptavidin-PE was added to each tube, stirred well, and allowed to stand on ice for 10 minutes. Next, similarly, add 1 ml of MACS buffer to each tube, stir well, and centrifuge to remove the supernatant. The precipitate in each tube was suspended in 500 μl of MACS buffer, and the filtrate passed through a 40 μm cell strainer was applied to a cell sorter (FACSAria cell sorter, Becton Dickinson). The details of using the cell sorter were in accordance with the manufacturer's manual. Thus, CD34 positive cells were obtained, and skeletal muscle tissue-derived cardiac muscle stem cells were obtained.

Sorted CD34 positive cells (myocardial stem cells) are mouse expansion medium [DMEM / F12Ham (GIBCO), 20% FBS, 1% penicillin-streptomycin, 10ng / ml mouse LIF (CHEMICON), 10ng / ml recombinant human basic FGF (Promega) and 20ng / ml mouse EGF (SIGMA) included] on a fibronectin-coat cell culture dish (Becton Dickinson) at 37 ° C Then, the cells were cultured for 7 days under 5% CO ₂ to proliferate CD34 positive cells (myocardial stem cells).

(4) Confirmation of cardiomyocyte differentiation The CD34-positive cells grown in (3) above were collected by centrifugation, and the cells were ^treated with 1 × 10 ⁻⁸ mol / l dexamethasone and 1% by volume penicillin-streptomycin. The MEM medium (manufactured by GIBCO) contained was cultured for 21 days at 37 ° C. and 5% CO ₂ . This culture confirmed that the CD34-positive cells differentiated into cardiomyocytes. The differentiation into cardiomyocytes was confirmed based on the following analysis results.
<Analysis by microscope>
When the cells after 21 days of culture were observed with a microscope, the presence of pulsatile skeletal muscle cells formed into sheet-like multinucleated (3 to 5) cells (see FIG. 2A), and the number of intracellular nuclei was 1 or The presence of two pulsatile cardiomyocytes (see FIG. 2C) was confirmed. Furthermore, the presence of skeletal muscle cells (see FIG. 2B) is confirmed by actin staining of the cultured cells, and the presence of cardiomyocytes by staining with myocardial specific troponin-I (D in FIG. 2). (See figure).

<Analysis by RT-PCR>
By RT-PCR, various markers of cultured cells (cardiomyocyte-specific troponin-I (cTnI), α-MHC (α-myosin heavy chain) at the start of differentiation induction, 7 days after the start of differentiation induction, and 21 days after the start of differentiation induction , Flt-1, α-cardiac actin, Oct-4, β-actin) were analyzed. The obtained results are shown in FIG. As a result, it was confirmed that the expression of cTnI, α-MHC and α-cardiac actin, which are markers for cardiomyocytes, increased with time. A strong enhancement over time was also observed in Flt-1, which is a vascular endothelial marker. On the other hand, although Oct-4 expressed by undifferentiated stem cells was observed at the start of differentiation induction, it was confirmed that the expression decreased with the progress of differentiation induction time. In addition, with respect to β-actin, which is a marker used as an internal control, all bands are of the same level, and it can be confirmed that the same amount of cDNA is used for analysis in any sample.

<Analysis by real-time RT-PCR>
The cells at the start of culture and after 21 days of culture were analyzed for expression of various markers (GATA4, Nkx-2.5, α-MHC, β-MHC, cTnI) by real-time RT-PCR. In addition, as a comparison, CD34 positive cells grown in (2) above were cultured under the same conditions except that a medium containing 3 μM demethylating agent (5-azacytidine) was used instead of dexamethasone. The expression of various markers was analyzed as described above. The obtained results are shown in FIG. As can be seen from FIG. 4, Nkx-2.5, α-MHC and cTnI, which are markers of cardiomyocytes, are expressed by culturing in the presence of 5-azacytidine or dexamethasone. It was confirmed that it was differentiated. For mice, the response to 5-azacytidine was better than the response to dexamethasone.

(5) Observation of cell morphology in each step The cells after the enzyme treatment in (1) above were treated with GMEM (GIBCO) containing 15% by volume of KSR (Knock Out Serum Replacement) (GIBCO). The cells were cultured at 37 ° C. and 5% CO ₂ for 1 week. When the cultured cells were observed under a microscope, cells that formed clusters and proliferated like colonies were observed (see FIG. 5A).

The CD34-positive cell obtained in (2) above is made into a single cell, and this is used for 7 days at 37 ° C. and 5% CO ₂ using complete methylcellulose medium (Methocult GF, M3534, manufactured by Stem Cell Technologies). When cultured, formation of clones was confirmed (see FIG. 5B).

  Skeletal muscle-derived cells isolated from mice expressing green fluorescent chromoprotein (GFP) (purchased from Jackson Laboratory) were cultured in medium [DMEM / F12 (GIBCO), B-27 Supplement (50 ×), 20 ng / ml mouse] EGF (manufactured by SIGMA) containing 40 ng / ml recombinant human FGF (manufactured by Promega)] was cultured for 3 days, and a clone consisting of 3 to 5 stem cells was confirmed (see C and D diagrams in FIG. 5). ). Further, mouse skeletal muscle-derived cells were cultured for 7 and 16 days in the same manner, and observed cell clones are shown in FIGS. 5E and 5F.

Example 2 Transplantation of mouse-derived myocardial stem cells The CD34 positive cells (myocardial stem cells) obtained in Example 1 above were transferred to mouse expansion medium (DMEM / F12Ham (GIBCO), 20 vol% FBS, 1 vol% penicillin-streptomycin, The cells were grown in 10 ng / ml mouse LIF (manufactured by CHEMICON), 10 ng / ml recombinant human basic FGF (Promega), and 20 ng / ml mouse EGF (SIGMA). Next, the proliferated CD34 positive cells (about 1 × 10 ⁶ cells) were suspended in 15 μl of PBS (−) (GIBCO), and this was suspended using a BD Ultra Fine II lancet (Becton Dickinson). They were transplanted into infarcted myocardium created in 10-12 week old female C57Bl / 6J mice (manufactured by Shimizu Experimental Materials Co., Ltd.). Twenty-one days after transplantation of myocardial stem cells, the heart was removed from the mouse. With respect to the isolated heart myocardium, engraftment in the host myocardium of CD34 positive cells that developed green fluorescence (GFP) was confirmed (see FIG. 6A). Further, cTnI staining (recognized as red) was performed in the same visual field as FIG. 6A (see FIG. 6B). When the A and B diagrams in FIG. 6 are superimposed, the presence of CD34 positive cells (green) and the presence of cTnI expression (red) overlap (see FIG. 6C), and CD34 positive derived from transplanted skeletal muscle It was confirmed that the cells were differentiated into cardiomyocytes.

Example 3 Acquisition of human-derived myocardial stem cells and induction of differentiation of the stem cells into myocardial cells Patients with lower leg amputation due to obstructive arteriosclerosis or diabetic gangrene (50-85 years old, 2 males, 1 female, gender unknown 3 Human), and patients (19-77 years old, 2 males, 5 females, 1 gender unknown) who had undergone extensive resection of the lower limb or upper limb with osteosarcoma or soft tissue tumor. Using the skeletal muscle tissue collected from the subject, “(1) Preparation of cell suspension” and “(2) Separation of skeletal muscle tissue-derived cells by percoll density gradient centrifugation described in Example 1 above. The human skeletal muscle-derived cells were obtained and proliferated according to the method. A micrograph of the resulting human skeletal muscle-derived cells forming a clone is shown in FIG. 7A.

  When FACS analysis was performed on the cell group obtained by collagenase treatment of skeletal muscle tissue and analyzed for cell surface antigen (CD56), CD34 positive cells do not express CD56 that recognizes skeletal myoblasts Was confirmed (see FIG. 7B). As a result of FACS analysis, it was confirmed that the CD34 positive cells were negative for CD45 and were not contaminated with bone marrow or peripheral blood (see FIG. 7C). In addition, the obtained human skeletal muscle-derived cells were proliferated by culturing for 7 days. As a result of FACS analysis on the expression of CD105 in the obtained cells, human skeletal muscle-derived cells were almost recognized as CD105 positive cells. (See FIG. 7D).

  Subsequently, differentiation of the proliferated human skeletal muscle-derived CD105-positive cells into cardiomyocytes was performed according to the method of “(4) Confirmation of differentiation into cardiomyocytes” described in Example 1 above. Thus, it was confirmed that CD105 positive cells derived from human skeletal muscle differentiate into cardiomyocytes. The differentiation into cardiomyocytes was confirmed based on the following analysis results.

<Analysis by microscope>
When human skeletal muscle-derived CD105-positive cells were cultured for 1 week for differentiation induction, the cells were observed under a microscope. As a result, muscle tubes were observed on one side (see FIG. 8A). Furthermore, the presence of skeletal muscle cells was confirmed by actin staining of the cultured cells, all of which were multinucleated cells, and showed typical skeletal muscle cell characteristics (see FIG. 8B). Further, it was estimated that the observed cells were mixed with 1 or 2 nuclei and were cardiomyocytes (see FIG. 8C). Furthermore, the presence of cardiomyocytes was revealed by staining the cultured cells with myocardial specific troponin-I (see FIG. 8D).

<Analysis by real-time RT-PCR>
The expression of various markers (GATA4, Nkx-2.5, α-MHC, β-MHC, cTnI) was analyzed by real-time RT-PCR for the cells at the start of differentiation induction and 21 days after the start of differentiation induction. The obtained results are shown in FIG. As can be seen from FIG. 9, the various markers were expressed by culturing in the presence of dexamethasone, and it was confirmed that the human skeletal muscle-derived CD105-positive cells differentiated into cardiomyocytes.

Example 4 Transplantation of human-derived myocardial stem cells Human skeletal muscle-derived CD105-positive cells (myocardial stem cells) obtained in Example 3 above were human expansion medium [DMEM / F12Ham (GIBCO), 20% by volume FBS, 1 volume] Incubation was carried out with% penicillin-streptomycin, 10 ng / ml human LIF (manufactured by SIGMA), 10 ng / ml recombinant human basic FGF (manufactured by Promega), and 20 ng / ml human EGF (manufactured by SIGMA)]. Subsequently, the proliferated human skeletal muscle-derived CD105 positive cells (about 1 × 10 ⁶ cells) were transplanted into mouse infarcted myocardium in the same manner as in Example 2 above. Twenty-one days after transplantation of myocardial stem cells, the heart was removed from the mouse. The isolated cardiac muscle is stained blue with DAPI (4'6-diamino-2-pheny1indo1e) and the human cardiomyocytes are red with human myocardial specific topolonin-I. Stained. As a result, it was confirmed that human skeletal muscle-derived cells transplanted into the thinned infarct were migrated and engrafted, and that new cardiomyocytes were regenerated mainly on the endocardium side (FIG. 10). reference).

It is a figure which shows the various cell surface antigen characteristics of the skeletal muscle tissue origin cell group derived from the mouse | mouth isolate | separated by the density gradient centrifugation method of Percoll and sorted by surface antigen CD34. FIG. A shows the detection results for Sca-1 and CD34. FIG. B shows the detection results for Sca-1 and CD105. Figure C shows the results detected for CD90. FIG. D shows the detection results for CD34 and CD45. FIG. E shows the detection results for CD105 and CD45. It is a figure which shows the result of having culture | cultivated the CD34 positive cell in the dexamethasone containing medium, and observing the obtained cell under the microscope. FIG. A shows pulsatile skeletal muscle cells formed into multi-nuclei (3 to 5) differentiated into a sheet. FIG. B shows skeletal muscle cells observed by actin staining. C shows pulsatile cardiomyocytes with one or two intracellular nuclei. FIG. D shows cardiomyocytes observed by myocardial specific troponin-1 staining. For cells cultured in a dexamethasone-containing medium for CD34 positive cells, various markers (cardiomyocyte-specific troponin-I (cTnI), α-) were observed at the start of differentiation induction, 7 days after the start of differentiation induction, and 21 days after the start of differentiation induction. It is a figure which shows the result of having analyzed the expression of MHC ((alpha) -myosin heavy chain), Flt-1, (alpha) -cardiac actin, Oct-4, (beta) -actin) by RT-PCR. Regarding cells cultured in a dexamethasone-containing medium with CD34 positive cells, various markers [GATA4 (Fig. A), Nkx-2.5 (Fig. B), α-MHC (Fig. C), It is a figure which shows the result of having analyzed the expression of (beta-MHC (D figure), cTnI (E figure)] by real-time RT-PCR. In FIG. 4, the numerical values on the vertical axis represent values corrected by GAPDH. It is a figure which shows the form of the cell observed by culturing the mouse | mouth skeletal muscle origin cell used for various processes. FIG. A is a photomicrograph of cells showing a colony-like growth form by culturing cells obtained by collagenase treatment of muscle tissue cut from a mouse. FIG. B is a photomicrograph of cells formed by culturing mouse CD34 positive cells derived from skeletal muscle as single cells. FIG. C is a photomicrograph of a clone composed of 3 to 5 stem cells observed by culturing mouse skeletal muscle-derived cells expressing a green fluorescent dye for 3 days. FIG. D is a photomicrograph showing the findings of green emission in FIG. Fig. E is a photomicrograph of a clone observed by culturing mouse skeletal muscle-derived cells expressing a green fluorescent dye for 7 days, and Fig. F is a group of cells observed by culturing the cells for 16 days. FIG. It is a figure which shows the engraftment state in the cardiac muscle of the mouse | mouth of the mouse | mouth which transplanted the CD34 positive cell (myocardial stem cell) obtained in Example 1 to the mouse infarcted cardiac muscle, and was observed. Figure A shows engraftment of CD34 positive cells (green) in the host myocardium. FIG. B shows the result of cTnI staining (showing red) in the same visual field as FIG. The C diagram is a superposition of the above A and B diagrams. It is a figure which shows the microphotograph of a human-derived cardiac muscle stem cell, and the surface antigen characteristic of this cell. FIG. A is a photomicrograph of a state in which human skeletal muscle tissue-derived CD105 positive cells form a clone. FIG. B shows the detection results for CD56 and CD34. FIG. C shows the results detected for CD34 and CD45. FIG. D shows the results detected for CD56 and CD105. It is a figure which shows the result of having observed the human skeletal muscle origin CD105 positive cell in the dexamethasone containing culture medium, and having observed the microscope of the obtained cell. FIG. A shows cells differentiated into muscle tubes. FIG. B shows skeletal muscle cells observed by actin staining. FIG. C shows that one or two nuclei are mixed. FIG. D shows cardiomyocytes observed by myocardial specific troponin-I staining. For cells obtained by culturing human skeletal muscle-derived CD105 positive cells in a medium containing dexamethasone, various markers [GATA4 (Fig. A), Nkx-2.5 (Fig. B), α-MHC ( (C diagram), β-MHC (D diagram), cTnI (E diagram)] is a diagram showing the results of analysis by real-time RT-PCR. In FIG. 9, the numerical values on the vertical axis represent values corrected by GAPDH. It is a figure which shows the engraftment state in the cardiac muscle of the mouse | mouth which transplanted the human skeletal muscle tissue origin cell (cardiac muscle stem cell) obtained in Example 3 to the mouse infarcted cardiac muscle, and was observed. In the figure, the isolated myocardium is stained in nuclei in cells in blue, and human-derived cardiomyocytes are stained in red. FIG. B is an enlarged view of FIG.

Claims (9)

  A stem cell derived from mammalian skeletal muscle tissue, which has the ability to differentiate into cardiomyocytes.   The stem cell according to claim 1, which is CD34 positive.   The stem cell according to claim 1 or 2, which is CD105 positive.   The stem cell according to any one of claims 1 to 3, wherein the mammal is a human. The stem cell according to claim 1, which is prepared through the following steps.
(i) collecting skeletal muscle tissue from a mammal and preparing a cell suspension by enzymatic treatment of the obtained skeletal muscle tissue;
(ii) a step of separating skeletal muscle tissue-derived cells from the cell suspension by a density gradient method, and
(iii) A step of selecting and separating cells positive for at least one of CD34 and CD105 from the obtained skeletal muscle tissue-derived cell group. A method for preparing a stem cell according to any one of claims 1 to 5, comprising the following steps:
(i) collecting skeletal muscle tissue and preparing a cell suspension by enzymatic treatment of the obtained skeletal muscle tissue;
(ii) a step of separating skeletal muscle tissue-derived cells from the cell suspension by a density gradient method, and
(iii) A step of selecting and separating cells positive for at least one of CD34 and CD105 from the obtained skeletal muscle tissue-derived cell group.   A method for treating heart disease, comprising transplanting the stem cell according to any one of claims 1 to 5 or a cardiomyocyte differentiated from the stem cell into a heart of a patient having heart disease. The method for treating a heart disease according to claim 7, comprising the following steps:
(i) collecting skeletal muscle tissue from a patient having heart disease, and preparing a cell suspension by enzymatic treatment of the obtained skeletal muscle tissue;
(ii) a step of separating skeletal muscle tissue-derived cells from the cell suspension by a density gradient method,
(iii) selecting and separating cells positive for at least one of CD34 and CD105 from the obtained skeletal muscle tissue-derived cell group;
(iv) culturing the cells separated in the above step (iii) in a medium containing fibroblast growth factor and epidermal growth factor to proliferate the cells; and
(v) A step of transplanting the cells grown in the step (iv) into the heart of the patient. The method for treating a heart disease according to claim 7, comprising the following steps:
(i) collecting skeletal muscle tissue from a patient having heart disease, and preparing a cell suspension by enzymatic treatment of the obtained skeletal muscle tissue;
(ii) a step of separating skeletal muscle tissue-derived cells from the cell suspension by a density gradient method,
(iii) selecting and separating cells positive for at least one of CD34 and CD105 from the obtained skeletal muscle tissue-derived cell group;
(iv) a step of proliferating the cells separated in the step (iii) by culturing the cells in a medium containing fibroblast growth factor and epidermal growth factor;
(v) culturing the cells grown in the above step (iv) in a medium containing dexamethasone to induce differentiation into cardiomyocytes, and
(vi) A step of transplanting differentiated cardiomyocytes into the heart of the patient.

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