JP5564679B2 - Cardiomyocyte differentiation induction promoter and method of use thereof - Google Patents

Description

  The present invention relates to a cardiomyocyte differentiation induction promoter and a method for using the same.

Mesenchymal stem cells have multipotency and self-proliferation ability to mesenchymal cells such as osteoblasts, bone cells, adipocytes, chondrocytes, muscle cells, stromal cells, tendon cells, etc. Application to regenerative medicine such as cartilage and muscle is expected.
To date, mesenchymal stem cells isolated from endometrium, menstrual blood, umbilical cord blood, or fetal accessory organs have been identified as cells capable of differentiating into cardiomyocytes. These mesenchymal stem cells are known to differentiate into cardiomyocytes by co-culture with feeder cells (see, for example, Patent Document 1).

WO2006 / 078034

  An object of the present invention is to provide a cardiomyocyte differentiation induction promoter and a method for using the same.

  As shown in the Examples below, the present inventors pre-cultured mesenchymal stem cells in the presence of IL-10 or progesterone when co-cultured with feeder cells to induce differentiation into cardiomyocytes. It has been clarified that pretreatment can promote differentiation induction of mesenchymal stem cells into cardiomyocytes, and the present invention has been completed.

  That is, the cardiomyocyte differentiation induction promoter according to the present invention is a promoter that promotes the differentiation induction of mesenchymal stem cells into cardiomyocytes, and is characterized by containing IL-10 or progesterone as an active ingredient. Here, the mesenchymal stem cells are preferably derived from endometrium, amniotic membrane, placenta, umbilical cord blood, menstrual blood.

The method for promoting the induction of cardiomyocyte differentiation according to the present invention is a method for promoting the induction of differentiation of mesenchymal stem cells into cardiomyocytes, comprising mesenchymal stem cells (between human bodies in the presence of IL-10 or progesterone). It is characterized by culturing leaf stem cells) . In this cardiomyocyte differentiation induction promoting method, the mesenchymal stem cells are preferably cultured for 2 weeks or more in the presence of IL-10 or progesterone. The mesenchymal stem cells are preferably derived from endometrium, amniotic membrane, placenta, umbilical cord blood, menstrual blood.

The differentiation induction method according to the present invention is a method for differentiating mesenchymal stem cells into cardiomyocytes, which comprises treating mesenchymal stem cells (excluding mesenchymal stem cells in the human body) with IL-10 or progesterone. And culturing the mesenchymal stem cells treated with IL-10 or progesterone on feeder cells. Here, the feeder cell is preferably a cardiac muscle cell derived from a mammalian fetus. In addition, the mesenchymal stem cells are preferably cultured for 2 weeks or longer in the presence of IL-10 or progesterone. Furthermore, the mesenchymal stem cells are preferably derived from endometrium, amniotic membrane, placenta, umbilical cord blood, menstrual blood.

  The mesenchymal stem cells according to the present invention are mesenchymal stem cells having the ability to differentiate into cardiomyocytes, and are characterized by being treated with IL-10 or progesterone. This treatment with IL-10 or progesterone is preferably carried out by culturing for 2 weeks or more in the presence of IL-10 or progesterone. The mesenchymal stem cells are preferably derived from endometrium, amniotic membrane, placenta, umbilical cord blood, menstrual blood.

  According to the present invention, a cardiomyocyte differentiation induction promoter and a method for using the same can be provided.

It is a graph which shows the number of mesenchymal stem cells per 1 g of each tissue of amniotic membrane, umbilical cord, and placenta, which is an embodiment of the present invention. It is a graph which shows the differentiation induction promotion to the cardiac muscle cell of the human amnion origin mesenchymal stem cell by co-culture with a feeder cell which is one Embodiment of this invention. In one Embodiment of this invention, it is the graph (A) which shows the recovery | restoration of the cardiac function by the transplant to the myocardial infarction foci of an amnion origin mesenchymal stem cell, and the microscope picture (B) of a heart tissue. It is a photograph of the heart tissue 2 weeks after transplantation of GFP-labeled human amnion-derived mesenchymal stem cells, which is an embodiment of the present invention. The expression of HLA-G protein when a mesenchymal stem cell is cultured for 2 weeks in the presence of JEG-3 cell extract, progesterone, IL-10, or INF-γ, which is an embodiment of the present invention is shown. Western blot. It is a graph which shows cardiomyocyte differentiation induction when mesenchymal stem cells are cultured in the presence of progesterone (A) or IL-10 (B), which is an embodiment of the present invention. It is the graph which showed the myocardial differentiated cell rate of the mesenchymal stem cell in the 2nd week after transplantation in the control group, IL-10 treatment group, progesterone treatment group and FK506 treatment group, which is one embodiment of the present invention.

  Hereinafter, embodiments of the present invention completed based on the above knowledge will be described in detail with reference to examples. However, the present invention is not limited to the following examples.

  Unless otherwise stated in the embodiments and examples, J. Sambrook, EF Fritsch & T. Maniatis (Ed.), Molecular cloning, a laboratory manual (3rd edition), Cold Spring Harbor Press, Cold Spring Harbor, New York (2001); FM Ausubel, R. Brent, RE Kingston, DD Moore, JG Seidman, JA Smith, K. Struhl (Ed.), Current Protocols in Molecular Biology, John Wiley & Sons Ltd. The method described in the protocol collection, or a modified or modified method thereof is used. In addition, when using commercially available reagent kits and measuring devices, unless otherwise explained, protocols attached to them are used.

  The objects, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description of the present specification, and those skilled in the art can easily reproduce the present invention from the description of the present specification. it can. The embodiments and specific examples of the invention described below show preferred embodiments of the present invention, and are shown for illustration or explanation. It is not limited. It will be apparent to those skilled in the art that various modifications can be made based on the description of the present specification within the spirit and scope of the present invention disclosed herein.

(1) Pharmacological action When mesenchymal stem cells are cultured in the presence of IL-10 or progesterone and the cultured mesenchymal stem cells are co-cultured with mammalian fetal cardiomyocytes as feeder cells, the mesenchymal stem cells Differentiation into cardiomyocytes can be promoted. From this, it is considered that IL-10 or progesterone acts on the mesenchymal stem cells before differentiation induction to advance the differentiation state of the mesenchymal stem cells toward the cardiomyocytes.

(2) Usefulness of drugs containing IL-10 or progesterone As described above, IL-10 and progesterone act on mesenchymal stem cells before differentiation induction, thereby inducing differentiation of mesenchymal stem cells into cardiomyocytes. Can be promoted. Therefore, a drug containing IL-10 or progesterone as an active ingredient is useful as a cardiomyocyte differentiation induction promoter that promotes differentiation induction of mesenchymal stem cells into cardiomyocytes. The cardiomyocyte differentiation induction promoter according to the present invention may contain an effective amount of either IL-10 or progesterone, or may contain both.

  Mesenchymal stem cells themselves have self-renewal ability, self-proliferation ability, and pluripotency. Therefore, by using these drugs or preparations, a large amount of mesenchymal stem cells can be obtained by culturing in vitro. It becomes possible to produce cardiomyocytes. Currently, in the development of new drugs, there is a demand for the supply of cardiomyocytes that can be used in drug screening tests, toxicity tests, safety pharmacology tests, and the like. It is possible to secure a sufficient amount of cardiomyocytes in accordance with the implementation of such tests.

  In addition, by treating mesenchymal stem cells with the agent according to the present invention, cells that can be transplanted to another family can be obtained. The cells thus obtained have high utility value in the field of regenerative medicine. For example, by transplanting a necessary amount of mesenchymal stem cells treated with the drug according to the present invention into damaged or dysfunctional heart tissue, it promotes new cardiomyocyte differentiation at the transplantation site, tissue regeneration and Can improve heart function. In this case, the mesenchymal stem cell-derived animal species (donor) and transplanted animal species (recipient) are preferably the same, but the mesenchymal cells treated with IL-10 or progesterone are preferred. Stem cells have high immunological tolerance and are not particularly limited. For example, human mesenchymal stem cells can be transplanted into the heart of a mouse. The preparation method for transplantation of mesenchymal stem cells having the ability to differentiate into myocardium is not particularly limited and can be appropriately selected by those skilled in the art. For example, even if prepared by suspending in a medium or buffer, A cell sheet may be prepared. Here, the disease of the recipient is not particularly limited as long as the disease is expected to be improved by regeneration of the myocardium, and examples thereof include myocardial infarction.

(3) Production of drug according to the present invention IL-10 may be isolated from a living body or may be a recombinant produced using a gene recombination technique. The animal from which IL-10 is derived is not particularly limited as long as it has a cardiomyocyte differentiation-inducing function, but is preferably the same as the animal species from which the mesenchymal stem cells are derived. For example, human-derived mesenchymal stem cells It is preferable to use human-derived IL-10. The concentration of IL-10 contained in this drug can be appropriately determined by those skilled in the art in consideration of the final concentration required for the differentiation induction method described below.

  The progesterone according to the present invention may be natural progesterone (chemical name 4-prehnene-3,20-dione) isolated and purified from a living body, or artificial progesterone (hydroxyprogesterone caproate, chemical name 17-hydroxy). -4-pregnene-3,20-dione hexanoate), and is not limited as long as it has a cardiomyocyte differentiation inducing function of mesenchymal stem cells. The animal used for isolation from the living body is not particularly limited as long as progesterone has a cardiomyocyte differentiation inducing function of mesenchymal stem cells, preferably the same as the animal species from which the mesenchymal stem cells are derived, For example, when culturing human-derived mesenchymal stem cells, it is preferable to use human-derived progesterone. These natural and artificial progesterones may be commercially available as reagents and pharmaceuticals. Examples of commercially available drugs for natural progesterone include Progehormon (Mochida Pharmaceutical Co., Ltd.). Moreover, as a commercially available pharmaceutical product of artificial progesterone, there is, for example, Progedevo (Mochida Pharmaceutical Co., Ltd.) Any pharmaceutical may contain additives other than progesterone as necessary for formulation. The progesterone concentration contained in this drug can be appropriately determined by those skilled in the art in consideration of the final concentration required for the following cardiomyocyte differentiation inducing method.

  Furthermore, the cardiomyocyte differentiation induction promoter according to the present invention is a substance other than IL-10 and progesterone, and one or more substances that act on mesenchymal cells and promote differentiation induction into cardiomyocytes. It may contain seeds. Examples of this substance include, but are not limited to, angiotensin II receptor drugs such as telmisartan and valsartan, and pioglitazone.

  In addition, this drug is formulated by containing a pharmaceutically acceptable carrier other than the above active ingredients as necessary. The pharmaceutically acceptable carrier used here can be appropriately selected from commonly used carriers according to the form of the pharmaceutical composition to be prepared. For example, when the pharmaceutical composition is prepared in solution form, purified water (sterile water) or physiological buffer, injectable organic esters such as glycol, glycerol, olive oil, etc. can be used as a carrier. . The pharmaceutical composition may contain conventionally used stabilizers, excipients and the like.

(4) Method for Inducing Differentiation of Mesenchymal Stem Cells into Cardiomyocytes The method for promoting differentiation induction of mesenchymal stem cells into cardiomyocytes according to the present invention treats mesenchymal stem cells in the presence of IL-10 or progesterone. It is characterized by that. Hereinafter, the method for inducing differentiation of mesenchymal stem cells into cardiomyocytes will be described in detail.

(I) Preparation and pretreatment of mesenchymal stem cells The mesenchymal stem cells may be mesenchymal stem cells having the ability to differentiate into cardiomyocytes, such as endometrium, amniotic membrane, placenta, umbilical cord, umbilical cord blood, It can be taken from menstrual blood. Since these mesenchymal stem cell materials can be easily secured in a sufficient amount during menstruation or childbirth, there is no shortage of donors unlike normal organ transplantation. In addition, in the case of autologous transplantation, a waiting time for pre-transplantation treatment is necessary to collect transplant cells and tissues as raw materials after the necessity arises. It is possible to prepare for transplantation by performing necessary processing.

  When preparing mesenchymal stem cells from these materials, appropriate pretreatment is performed on the materials to be used. For example, in the case of a cell mass involving mesenchymal stem cells, in order to dissociate the contained cells, the material is subjected to physical treatment such as pipetting or chemical treatment such as enzymes. Just do it. Examples of the enzyme include enzymes conventionally used, such as trypsin and collagenase. For example, when preparing mesenchymal stem cells from blood such as umbilical cord blood or menstrual blood, it is preferable to hemolyze erythrocytes by treatment with a hypotonic solution (for example, water).

The mesenchymal stem cells thus obtained are centrifuged (500 to 2000 rpm, 3 to 10 minutes), a medium is added to the precipitated cells, and a cell suspension is prepared. The plate is seeded at 1,000 / cm ² and cultured in a 37 ° C., 5% CO ₂ incubator for 5 days or longer, preferably 2 weeks or longer, most preferably 2 weeks.

The medium used here is not particularly limited as long as it can culture mesenchymal stem cells. For example, α-MEM (α-minimum essential medium), DMEM (Dulbecco's modified Eagle's medium), IMDM (Isocove's modified) Dulbecco's medium). The medium preferably contains 5 to 10% fetal calf serum (FCS), and may contain antibiotics such as penicillin and streptomycin. In addition, when the cells are later co-cultured with feeder cells to induce differentiation into cardiomyocytes, a myocardial differentiation induction promoter containing IL-10 or progesterone is added to the medium in order to promote the differentiation. The concentration of IL-10 to be added is, for example, preferably 1 to 100 ng / mL, and particularly preferably 10 ng / mL. The concentration of progesterone is preferably 1 to 100 ng / mL, and particularly preferably 10 ng / mL. As a result of culturing under such conditions, mesenchymal stem cells become mesenchymal stem cells with enhanced ability to differentiate into cardiac muscle.

  When culturing mesenchymal stem cells in the presence of IL-10 or progesterone, other differentiation induction promoting methods may be combined. For example, co-culture with feeder cells (for example, cardiomyocytes), 5-azacytidine , DMSO, or oxytocin. In addition, mesenchymal stem cells may be cultured in the presence of both IL-10 and progesterone.

(Ii) Preparation of feeder cells The feeder cells are not particularly limited as long as they can induce differentiation of mesenchymal stem cells, and examples include primary cultured cells of cardiomyocytes. In addition, when using a cultured cell as a feeder cell, in order to prevent the proliferation of a cell, it is preferable to perform the process by a gamma ray irradiation, mitomycin, etc. with respect to the cultured cell to be used beforehand. Here, an example of a method for preparing feeder cells using mammalian fetal cardiomyocytes will be described (for details, see The Journal of Gene Medicine, 6, 833-845, 2004).

  First, the fetal heart of a mammal (for example, mouse etc.) of embryonic day 14-16 is excised and placed in a DMEM medium containing 5-10% FCS or a DMEM / F-12 medium containing 5-10% FCS, and ophthalmology Finely cut using scissors. Next, in order to dissociate cardiomyocytes from the myocardial tissue, an enzyme such as trypsin that digests the stroma of the tissue is placed in the culture dish and incubated at 37 ° C. for 5 minutes.

After enzyme treatment, the cells prepared by the above method are centrifuged (500 to 2000 rpm, 3 to 10 minutes), and a medium such as DMEM is added to the precipitate to prepare a cell suspension. The medium preferably contains 5 to 10% FCS, and may contain antibiotics such as penicillin and streptomycin. The cell suspension is placed in a culture dish and cultured in a 37 ° C., 5% CO ₂ incubator. The culture dish is preferably coated with gelatin or the like in advance. After culture, the culture supernatant is removed by aspiration, and the cells attached to the culture dish are used as feeder cells for the following experiments.

(Iii) Differentiation induction of mesenchymal stem cells into cardiomyocytes In order to induce differentiation of the obtained mesenchymal stem cells into cardiomyocytes, a concentration of 1 × 10 ¹ to 1 × 10 ² cells / cm ² is provided on the feeder cells. Mesenchymal stem cells are added and cultured at 37 ° C. in a 5% CO ₂ incubator. At this time, IL-10 or progesterone may or may not be added to the medium, and both IL-10 and progesterone may be added.

  The feeder cells used here are preferably prepared from the same animal species as the mesenchymal stem cells, but are not limited thereto, for example, mice that are relatively easily available for co-culture with human mesenchymal stem cells. Or rat cardiomyocytes can be used.

  In order to distinguish between feeder cells and mesenchymal stem cells, the feeder cells may be labeled in advance. Examples of the labeling method include preparing feeder cells from a transgenic animal that expresses the green fluorescent protein (GFP) gene throughout the body, introducing a GFP gene into a feeder cell for labeling, or a pigment that is harmless to cells. And the like by microinjection (see Takeda et al., The Journal of Gene Medicine, 6, 833-845, 2004).

(Iv) Identification of differentiation-induced cardiomyocytes Differentiation-induced cardiomyocytes pulsate so that the edges of each cell gather toward the center of the cell, and the cytoplasm becomes thicker during contraction. If the leaf stem cells are fluorescently labeled with GFP or the like in advance, the cardiomyocytes can be identified at the cell level, and the cardiomyocytes can be easily observed. In addition, the pulsation of a plurality of cells may be synchronized, and the pulsation may occur with a group of cells. Differentiated cardiomyocytes have such characteristics and can be easily identified under a microscope.

  Alternatively, the number of cardiomyocytes induced to differentiate may be measured using a marker specific for cardiomyocytes. For example, the expression of mRNA and protein specific for cardiomyocytes can be detected by in situ hybridization or immunostaining. Examples of markers specific to cardiomyocytes include Nkx2.5 / Csx, GATA4, TEF-1, MEF-2C, MEF-2D, MEF-2A, atrial natriuretic peptide (ANP), brain natriuretic peptide ( BNP), α-myosin heavy chain (α-MHC), β-myosin heavy chain (β-MHC), myosin light chain-2a (MLC-2a), myosin light chain-2v (MLC-2v), α-myocardium Examples include, but are not limited to, actin, myocardial Troponin T, myocardial Troponin I, Connexin 43 (Cx43) (see J. Clin. Invest., 103, 697-705, 1999).

[experimental method]
== Antibody ==
Primary antibodies include anti-human myocardial Troponin I mouse IgG antibody (Monoclonal mouse anti-cardiac Troponin I for human (Cat. No. 4T21, Lot 98 / 10-T21-C2, HyTest)), anti-Sarcomeric-alpha actinin mouse IgG antibody ( Catalog number A7811, Sigma-aldrich), anti-Connexin 43 rabbit IgG antibody (catalog number C6219, Sigma-aldrich), and anti-HLA-G mouse IgG antibody (Ab7758, Abcam) were used. Troponin I labels the myocardium. Sarcomeric-alpha actinin is an actin-binding protein, and the localization of actin can be detected by an antibody against it. Connexin 43 is a protein present in the heart gap junction, and is considered to be involved in the differentiation and development of the heart.

  As secondary antibodies for immunohistochemical staining, TRITC-labeled anti-mouse IgG antibody (Sigma) and Cy5-labeled anti-rabbit IgG antibody (Sigma) were appropriately used.

== Preparation of GFP-labeled mesenchymal stem cells ==
Amniotic membrane, placenta, or umbilical cord was collected at the time of childbirth from volunteers who gave informed consent according to the pro-call approved by Keio University Hospital Ethics Committee. Each tissue was transferred to a 50 ml polypropylene tube containing 20 ml of DMEM medium (containing 1% FCS, 100 U / ml penicillin, 100 ng / ml streptomycin, and 500 U / L heparin). The tissue was gently washed in the tube and then transferred to a 10 cm culture dish. Using ophthalmic scissors, the tissue was cut into approximately 1 to 5 mm ³ pieces, allowed to stand for several minutes, and then the supernatant containing blood cells was removed and washed again with DMEM medium. This operation is repeated 3 to 4 times. Finally, each cut tissue is placed in α-MEM medium containing 10% FCS, and the tissue pieces are placed in a culture dish so that the concentration of the tissue pieces is 3 to 5 pieces / cm ^2. It was allowed to stand and cultured in a 37 ° C., 5% CO ₂ incubator. Then, according to the method of Takeda et al. (J. Gene Med., 6, 833-845, 2004), mesenchymal stem cells that have migrated naturally from the tissue are infected with adenovirus expressing GFP gene, and mesenchymal stem cells Was labeled with GFP. After further culturing for 4 days, the expression of GFP in the mesenchymal stem cells was confirmed using a confocal fluorescence microscope (FV1000, Olympus).

== Preparation of feeder cells ==
As feeder cells, mouse (BALB / C) fetal cardiomyocytes were used. The preparation was performed as follows.

  First, the hearts of embryonic day 14 to 16 were excised and cut into pieces in a DMEM medium containing 10% FCS using scissors for ophthalmology. To dissociate cardiomyocytes from myocardial tissue, proteolytic enzymes that digest tissue stroma (PBS containing 0.05% trypsin and 0.25 mmol / L EDTA) were added to the medium and incubated at 37 ° C. for 5 minutes. .

After enzyme treatment, the mixture was centrifuged (1500 rpm, 3 minutes), the precipitated cells were suspended in 10% FCS-containing DMEM medium to prepare a cell suspension, seeded on a culture dish, and incubated at 37 ° C. After 30 minutes to 3 hours, the culture supernatant was removed by aspiration and seeded on a culture dish to a concentration of 5 × 10 ³ cells / cm ² . This was used as a feeder cell in each Example.

== Evaluation method of cardiomyocyte differentiation induction ==
In this example, Troponin I expression was used as a differentiation marker for cardiomyocytes, and the frequency of Troponin I positive cells relative to the number of GFP-labeled mesenchymal stem cells was calculated (Exp. Cell Res., 15, 313 (12), 2550 -62, 2007), the induction of differentiation of mesenchymal stem cells into cardiomyocytes was evaluated.

<In vitro differentiation induction evaluation>
One week after the start of co-culture of mesenchymal stem cells and cardiomyocytes as feeder cells, the cells present in the culture dish were immunized with an antibody against Troponin I, a protein specific for human cardiomyocytes. Staining was performed.

  First, a 10-fold diluted Poly-L-Lysine solution (P8920, Sigma) was overlaid on a slide glass washed with 70% ethanol containing 1% HCL, left at room temperature for 5 minutes, and then the Poly-L-Lysine solution. The slide glass to be immunostained was lysine-coated by removing the sample and sufficiently drying the slide glass. Next, the cells after the co-culture of the mesenchymal stem cells and the cardiomyocytes as feeder cells were peeled off with PBS containing 0.25 mmol / L EDTA, centrifuged (1500 rpm, 3 minutes), and the precipitated cells were treated with 10% FCS. A cell suspension was prepared in the DMEM medium. This cell suspension is placed on a lysine-coated slide glass and allowed to stand for 1 hour. After the cells are allowed to adhere to the slide glass, the cells are fixed using a 4% formaldehyde solution, and 0.02% Triton-X. The cell membrane was disrupted by soaking in a PBS solution containing 20 minutes. As a primary antibody, an anti-Troponin I antibody diluted with PBS (400-fold dilution) is overlaid, reacted in a refrigerator for 1 day while moisturizing, washed with PBS, and then as a secondary antibody, a TRITC labeled anti-antibody Mouse IgG antibody (diluted 200 times) was overlaid and allowed to react for 30 minutes at room temperature while moisturizing. The cells were washed with PBS and then observed using a confocal laser microscope (FV1000, Olympus).

<In vivo differentiation induction evaluation>
The heart tissue extracted from the rat was fixed with 4% formaldehyde, and then a frozen heart short-axis section was prepared. The slice was overlaid with anti-Troponin I antibody (diluted 400 times) diluted with PBS, reacted at 4 ° C. for 12 hours, and then washed with PBS. TRITC-labeled anti-mouse IgG antibody (diluted 100 times) diluted in PBS as a secondary antibody was overlayered, reacted at 20 ° C. for 1 hour, washed with PBS, and confocal laser microscope (FV1000, Olympus) Was observed.

== Myocardial infarction model rat production ==
Rats (Wister Rat, 6-8 weeks old, male and female) were anesthetized with 2% isoflurane and fitted with a ventilator. The chest of this rat was incised, and the coronary arteriovenous vein was ligated with silk thread to induce cardiovascular injury. Thereafter, the chest was sutured, and after the rat recovered, breeding was continued. Myocardial infarction is usually caused in tissues where blood flow is lost due to this cardiovascular disorder.

== Stock solution of mesenchymal stem cell treatment reagent ==
IL-10 (I9276, Sigma-aldrich), progesterone (p7556, Sigma-aldrich), JEG-3 cell extract (GTX14841, GeneTex), INF-γ (R & D systems), FK506 (immunosuppressant, F4679, Sigma-aldrich), 0.1 mM stock solutions were prepared, and it was confirmed that this solvent had no effect on the induction of differentiation of mesenchymal stem cells into cardiomyocytes.

[Example 1]
In this example, it is shown that mesenchymal stem cells are contained in the pregnancy appendage.

  First, amnion, placenta, and umbilical cord were obtained from volunteers, and GFP-labeled mesenchymal stem cells were prepared. The number of GFP-labeled mesenchymal stem cells obtained per gram of each tissue is shown in FIG.

  All of the amniotic membrane, placenta, and umbilical cord contain mesenchymal stem cells. Among them, the amnion contained a significantly high proportion. This result shows that mesenchymal stem cells can be collected from pregnancy appendages such as amniotic membrane, placenta, and umbilical cord, but particularly efficiently from amniotic membrane.

[Example 2]
In this example, differentiation induction of human amnion-derived mesenchymal stem cells into cardiomyocytes by co-culture with cardiomyocytes is shown.

== Induction of differentiation into cardiomyocytes ==
GFP-labeled human amnion-derived mesenchymal stem cells were prepared in a DMEM medium containing 10% FCS so as to have a concentration of 5 × 10 ⁴ cells / cm ² . The mesenchymal stem cells were layered on feeder cells and cultured in a 37 ° C., 5% CO ₂ incubator (see J. Gene Med., 6, 833-845, 2004). The mesenchymal stem cells of the control group were cultured under the same conditions without using feeder cells. One week after the start of co-culture, differentiation induction of cultured mesenchymal stem cells into cardiomyocytes was evaluated.

  FIG. 2 shows the number of cells differentiated into cardiomyocytes relative to the number of GFP-labeled mesenchymal stem cells (the number of Troponin I positive cells). The cardiomyocyte rate in the amnion-derived mesenchymal stem cells co-cultured with the feeder cells was 33%, which was significantly higher than the cardiomyocyte rate in the mesenchymal stem cells not co-cultured (0%) (p < 0.05, t test).

  Thus, co-culture with feeder cells promotes differentiation induction of mesenchymal stem cells into cardiomyocytes.

[Example 3]
This example shows that cardiac function is improved by transplanting mesenchymal stem cells into myocardial infarction.

== Mesenchymal stem cell transplantation ==
Two weeks after the infarct preparation operation for preparing a myocardial infarction model rat, the rat's chest was incised again. Here, GFP-labeled human amnion-derived mesenchymal stem cells were suspended in DMEM-H (high glucose DMEM) at a concentration of 2 to 4 × 10 ⁷ cells / mL, and transplanted to myocardial infarction. After the transplantation, the chest of the rat was sutured again and continued for another 2 weeks (transplant group, N = 11).

  In this example, as a control group, a group in which sham operation was performed for both infarction and transplantation (sham group, N = 6), and a group in which an infarct was produced but subjected to sham transplant operation (sham transplant group, N = 8) Using.

== Evaluation of improvement of cardiac function ==
In this example, left ventricular diameter shortening rate (LVFS) indicating left ventricular contractility was used as an evaluation method for improving cardiac function. Echocardiograms of each rat were obtained 2 weeks after myocardial infarction preparation surgery (before transplantation) and 2 weeks after transplantation, and LVFS was calculated from these images (clinical echocardiography, Junichi Yoshikawa, Spectroscopy Do, see).

  In addition, in the rats of the experimental group and the sham transplant group, the heart was removed 2 weeks after the transplantation. The heart tissue was fixed with 4% formaldehyde, and then a heart short axis frozen section was prepared. This was stained with Masson, and the heart tissue was observed under an optical microscope.

  As shown in FIG. 3A, in the mock transplant group (white circle), PVSF decreased after infarct formation. On the other hand, in the group transplanted with mesenchymal stem cells after preparation of myocardial infarction (black circle), LVSF increased by 2 weeks after transplantation, and was significantly higher than that in the sham transplant group (p <0.05, ANOVA method, Bonferroni comparison method). FIG. 3B also shows that the myocardium (arrow) is thickened in the heart tissue of the transplant group compared to the heart tissue of the sham transplant group.

  Thus, the transplantation of mesenchymal stem cells promotes new cardiomyocyte differentiation and cardiac function improvement in the myocardial infarction lesion.

[Example 4]
This example shows that amnion-derived mesenchymal stem cells have immunological tolerance.

  GFP-labeled human amnion-derived mesenchymal stem cells were transplanted into model rats two weeks after the preparation of myocardial infarction. The rats were kept for another 2 weeks or 2 months, and then heart tissue was collected for histological analysis.

  The heart tissue was fixed with 4% formaldehyde, and then a heart short axis frozen section was prepared. This section was double-labeled with an anti-Sarcomeric-alpha actinin antibody (300-fold dilution, 4 ° C. overnight) and an anti-Connexin 43 antibody (300-fold dilution, 4 ° C. overnight), and TRITC-labeled anti-mouse IgG antibody (100 And a Cy5-labeled anti-rabbit IgG antibody (100-fold dilution) were used as secondary antibodies for visualization. Alternatively, it was labeled with anti-Troponin I antibody (300-fold dilution, 4 ° C. overnight), and TRITC-labeled anti-mouse IgG antibody (100-fold dilution) was used as the secondary antibody. Further, each section was counterstained with DAPI (Wako) and observed under a fluorescence microscope.

  FIG. 4 is a photograph of heart tissue 2 weeks after transplantation of GFP-labeled human amnion-derived mesenchymal stem cells. FIG. 4A shows the localization of Troponin I (Trop I, gray) and GFP (EGFP, arrowhead). FIG. 4B is a microscopic image showing only the localization of GFP in the same section as A. The localization of GFP indicates the localization of the transplanted labeled mesenchymal stem cells. In general, it is well known among those skilled in the art that immunological rejection is completed within 2 weeks after transplantation in the Wister Rat used in this example. Many mesenchymal stem cells remain.

  The white parts (*) seen on the left and right of the photograph in FIG. 4C indicate the localization of GFP, and the white dots scattered throughout the tissue indicate the localization of Connexin 43 (Cx43, arrowhead). The GFP-unlabeled cell is a host cell, but expresses Connexin43 protein particularly deeply at the junction between the GFP-labeled cell and the host cell (arrow).

  FIG. 4D shows the localization of cardiac muscle (Trop I, gray) and GFP positive transplanted mesenchymal stem cells (EGFP, *). The transplanted cells labeled with GFP have distinct striated labels labeled with Troponin I, indicating that the mesenchymal stem cells have differentiated into cardiomyocytes.

  Thus, amnion-derived mesenchymal stem cells have an excellent differentiation ability into cardiomyocytes in vivo, and many remain even two weeks after transplantation, that is, have excellent immune tolerance.

[Example 5]
HLA-G (human leukocyte antigen-G) is known to be involved in immune tolerance to the maternal fetus during pregnancy (Hunt et al., FASEB Journal, vol. 19, 681-693, 2005). In this example, changes in the expression level of HLA-G protein were analyzed as an index of immune tolerance of mesenchymal stem cells cultured under various conditions.

== Mesenchymal stem cell treatment ==
GFP-labeled human amnion-derived mesenchymal stem cells were prepared in a DMEM medium containing 10% FCS so as to have a concentration of 5 × 10 ⁴ cells / cm ² . To this, each stock solution of IL-10, progesterone, JEG-3 cell extract, and INF-γ was added to a final concentration of 10 ng / mL, and cultured in a 37 ° C., 5% CO ₂ incubator for 2 weeks. The control group was cultured in the same manner without adding any of the above reagents.

== Western blotting ==
50 mM cell lysis buffer (Tris-HCl (pH 8.0), 1% NP40, 250 mM NaCl, 50 mM NaF, 1 mM NaVO4, 1 mM protease inhibitor (PMSF, aprotinin, leupeptin), 1 mM DDT) Dissolved in and centrifuged. The supernatant obtained here was electrophoresed on a denaturing SDS-polyacrylamide gel and blotted onto a PVDF membrane. This membrane was labeled with an anti-HLA-G antibody (200-fold dilution, 4 ° C. overnight), and Goat f (ab ′) 2 anti-mouse Ig's, HRP conjugate (8000-fold dilution, room temperature, 30 minutes) as a secondary antibody. It was visualized using catalog number AMI4404, BIOSOURCE. The specific band obtained here was quantified using NIH-Image (American National Institutes of Health), and the expression of HLA-G protein under each condition was compared. Β-actin was used to correct the amount of protein migrated to the internal standard and gel.

  FIG. 5A shows the Western blot results. The result of quantifying this band is shown in FIG. 5B. The expression level of HLA-G protein compared to the control group is 100 times when cultured in the presence of JEG-3 cell extract, 1000 times in the presence of progesterone, 10,000 times in the presence of IL-10, INF-γ Then it increased 15 times.

  Thus, by culturing human amnion-derived mesenchymal stem cells in the presence of IL-10 or progesterone, the expression of HLA-G protein is significantly increased, that is, the immune tolerance of mesenchymal stem cells is improved.

[Example 6]
In this example, in vitro differentiation induction efficiency of mesenchymal stem cells into cardiomyocytes by IL-10 and progesterone is examined.

GFP-labeled human amnion-derived mesenchymal stem cells were prepared in a DMEM medium containing 10% FCS so as to have a concentration of 5 × 10 ⁴ cells / cm ² . The mesenchymal stem cells were layered on feeder cells and cultured in a 37 ° C., 5% CO ₂ incubator (see J. Gene Med., 6, 833-845, 2004). At this time, IL-10 (final concentration 10 ng / mL) or progesterone (final concentration 10 ng / mL) was added to the medium from 48 hours before the start of co-culture with feeder cells to the start of co-culture, and mesenchymal stem cells were added. Processed. Four days after culturing, differentiation from mesenchymal stem cells to cardiomyocytes was detected.

  As shown in FIG. 6A, the cardiomyocyte differentiation induction efficiency was significantly higher when treated with progesterone for 2 weeks before the start of co-culture with feeder cells compared to the case where no progesterone treatment was performed (p <0.05). , ANOVA method, Bonferroni comparison method). In addition, as shown in FIG. 6B, for IL-10, the mesenchymal stem cells treated with IL-10 were treated with the untreated group regardless of the conditions before coculture, after the start of coculture, or both. Compared with the cardiomyocyte differentiation induction rate, the cardiomyocyte differentiation induction rate was higher in the group treated with IL-10 before co-culture (p <0.05).

  This result shows that the induction of cardiomyocyte differentiation is promoted by treating mesenchymal stem cells with IL-10 or progesterone before co-culture with feeder cells for cardiomyocyte induction.

[Example 7]
In this example, it is shown that the mesenchymal stem cell rate after transplantation into heart tissue is improved by treating mesenchymal stem cells with IL-10 or progesterone.

GFP-labeled human amnion-derived mesenchymal stem cells were prepared in a DMEM medium containing 10% FCS so as to have a concentration of 5 × 10 ⁴ cells / cm ² , where IL-10 (final concentration 10 ng / mL) or progesterone ( (Final concentration 10 ng / mL) was added and cultured for 2 weeks. The mesenchymal stem cells of the control group were cultured in the same manner without adding any of these reagents. These cells were suspended in DMEM-H at a concentration of 2-4 × 10 ⁷ cells / mL.

  Two weeks after the infarct preparation operation for preparing a myocardial infarction model rat, the rat's chest was dissected again, and this mesenchymal stem cell solution was transplanted into the myocardial infarction. After transplantation, the chest of the rat was sutured again and continued for another 2 weeks (each N = 6).

  In addition, FK506 (1 mg / kg / day) was administered by intramuscular injection over 2 weeks after transplantation in rats treated in the same manner as in the control group (N = 6), and this was used as the FK506 treatment group.

  Two weeks later, the heart was removed from the rat. A 1 mm thick frozen section was prepared according to the above-described in vivo differentiation induction evaluation method, and the differentiation of transplanted mesenchymal stem cells into myocardial cells in the myocardial infarct was detected.

  The myocardial differentiated cell ratio (%) was calculated as the ratio (%) of the number of Troponin I positive cells per mesenchymal stem cell transplanted to the myocardial infarction.

  The number of Troponin I positive cells observed in the section was A, and the formula Ax1000 / 30 was used. This formula takes into account the frequency of cardiomyocytes appearing in the section, calculated from the standard length and diameter of the myocardium (see Cric. Res., 68, 1501-1526, 1991).

  FIG. 7 shows the myocardial differentiated cell rate of the mesenchymal stem cells subjected to each treatment at 2 weeks after transplantation. Compared with the control group and the FK506-treated group, the mesenchymal stem cells treated with IL-10 or progesterone had a significantly higher myocardial differentiated cell rate (p <0.05, ANOVA method, Bonferroni comparison method).

  Thus, when transplanting mesenchymal stem cells for the purpose of myocardial regeneration, treatment with IL-10 or progesterone prior to transplantation can promote cardiomyocyte differentiation of the transplanted cells.

Claims (12)

A promoter that promotes differentiation induction of mesenchymal stem cells into cardiomyocytes,
A cardiomyocyte differentiation induction promoter characterized by containing IL-10 or progesterone as an active ingredient.   The promoter according to claim 1, wherein the mesenchymal stem cells are derived from endometrium, amniotic membrane, placenta, umbilical cord blood, menstrual blood. A method of promoting differentiation induction of mesenchymal stem cells into cardiomyocytes,
A method for promoting cardiomyocyte differentiation induction, comprising culturing mesenchymal stem cells (excluding mesenchymal stem cells in a human body) in the presence of IL-10 or progesterone.   The method for promoting the induction of cardiomyocyte differentiation according to claim 3, wherein the mesenchymal stem cells are cultured for 2 weeks or more in the presence of IL-10 or progesterone.   5. The method for promoting the induction of cardiomyocyte differentiation according to claim 3 or 4, wherein the mesenchymal stem cells are derived from endometrium, amniotic membrane, placenta, umbilical cord blood, menstrual blood. A method for inducing differentiation of mesenchymal stem cells into cardiomyocytes, wherein the mesenchymal stem cells (excluding mesenchymal stem cells in the human body) are treated with IL-10 or progesterone;
A step of culturing the mesenchymal stem cells treated with IL-10 or progesterone on feeder cells.   The differentiation induction method according to claim 6, wherein the feeder cell is a cardiac muscle cell derived from a mammalian fetus.   The method for inducing cardiomyocyte differentiation according to claim 6 or 7, wherein the mesenchymal stem cells are cultured for 2 weeks or more in the presence of the IL-10 or progesterone.   The method for inducing cardiomyocyte differentiation according to any one of claims 6 to 8, wherein the mesenchymal stem cells are derived from endometrium, amniotic membrane, placenta, umbilical cord blood, menstrual blood. Mesenchymal stem cells having the ability to differentiate into cardiomyocytes,
A mesenchymal stem cell treated with IL-10 or progesterone.   The mesenchymal stem cell according to claim 10, which has been cultured in the presence of IL-10 or progesterone for 2 weeks or more.   The mesenchymal stem cell according to claim 10 or 11, wherein the mesenchymal stem cell is derived from endometrium, amniotic membrane, placenta, umbilical cord blood, menstrual blood.