JP2013202014A - Differentiation accelerator of mesenchymal stem cell, and method for accelerating differentiation by using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel differentiation promoting agent and differentiation promoting method for promoting differentiation of the mesenchymal stem cells with excellent safety.
SOLUTION: As a differentiation promoting agent that promotes differentiation of mesenchymal stem cells, an ultrasonically treated blood fluid component is used. Specifically, differentiation of mesenchymal stem cells can be promoted by culturing mesenchymal stem cells in a medium containing the blood fluid component. Examples of the blood fluid component include those derived from peripheral blood and cord blood. According to the differentiation promoting method of the present invention, differentiation from the mesenchymal stem cells into, for example, osteoblasts or chondrocytes can be promoted.
[Selection] Figure 1

Description

  The present invention relates to a differentiation promoting agent that promotes differentiation of mesenchymal stem cells and a differentiation promoting method using the same.

  Mesenchymal stem cells are pluripotent stem cells having the ability to differentiate into mesenchymal cells such as osteoblasts and chondrocytes, and are present in bone marrow fluid and adipose tissue. Because of this pluripotency, attempts have been made to reconstruct the mesenchymal stem cells into bones, blood vessels, and myocardium and use them in regenerative medicine. A number of the mesenchymal stem cells are required for the reconstruction of the tissue, but the mesenchymal stem cells obtained from a biological sample such as the bone marrow fluid are few. Therefore, at present, development of a method for proliferating the mesenchymal stem cells and differentiating the proliferated mesenchymal stem cells into various cells is expected. The differentiation promoting method includes, for example, fetal bovine serum, trans-retinoic acid, insulin, dexamethasone, IBMX (1-methyl-3- (2-methylpropyl) -7H-purine-2,6-dione; isobutylmethylxanthine ) Or a culture medium containing troglitazone and the like (Patent Document 1), a method using an osteogenesis promoter containing a Rho kinase inhibitor and an osteoinductive factor (Patent Document 2), and bone differentiation containing kaempferol. A method using an inducer (Patent Document 3) has been reported. However, these methods, for example, have a complicated process and may result in high costs. In addition, there are some that use components derived from different types, and there is a concern that safety may be concerned.

International Publication No. 2004/106502 International Publication No. 2001/017562 JP 2009-256350 A

  Therefore, an object of the present invention is to provide a novel differentiation promoting agent and differentiation promoting method for promoting the differentiation of the mesenchymal stem cells with excellent safety.

  In order to achieve the above object, the differentiation promoting agent of the present invention is a differentiation promoting agent that promotes differentiation of mesenchymal stem cells, and is characterized by containing an ultrasonically treated blood fluid component.

The differentiation promoting method of the present invention is a method for promoting the differentiation of mesenchymal stem cells, and has the following step (X).
(X) A step of culturing the mesenchymal stem cells in a medium containing the differentiation promoting agent of the present invention.

  The production method of the present invention is a method for producing the mesenchymal stem cell-derived differentiated cell, and includes a step of promoting the differentiation of the mesenchymal stem cell by the differentiation promotion method of the present invention.

  According to the present invention, for example, differentiation of mesenchymal stem cells can be promoted only by using an ultrasonically treated blood fluid component. Since the active ingredient in the differentiation promoting agent of the present invention is the ultrasonically treated blood fluid component, for example, the origin of the blood fluid component and the origin of the mesenchymal stem cell to be treated are derived from the same species. It can be set, and furthermore, since it can be set from the same individual, it is excellent in safety. Therefore, the present invention can be said to be extremely useful in, for example, regenerative medicine.

FIG. 1 is a stained photograph of cultured cells showing differentiation of mesenchymal stem cells into osteoblasts in an example of the present invention. FIG. 2 is a stained photograph of cultured cells showing the differentiation of mesenchymal stem cells into osteoblasts in the examples of the present invention. FIG. 3 is a stained photograph of cultured cells showing suppression of differentiation of mesenchymal stem cells into adipocytes in Examples of the present invention.

<Differentiation promoter>
As described above, the differentiation promoting agent of the present invention is a differentiation promoting agent that promotes the differentiation of mesenchymal stem cells, and is characterized by containing an ultrasonically treated blood fluid component.

  The differentiation promoting agent of the present invention can promote differentiation from the mesenchymal stem cells to cells belonging to the mesenchymal system. The cells belonging to the mesenchymal system are, for example, cells belonging to the mesenchymal system excluding adipocytes, and specific examples include osteoblasts, chondrocytes, bone cells, cardiomyocytes, tendon cells and the like. Preferred are osteoblasts and chondrocytes.

  In the present invention, the type of blood is not particularly limited, and examples thereof include umbilical cord blood and peripheral blood. As the blood, for example, one of umbilical cord blood and peripheral blood may be used, or both may be used in combination.

  In the present invention, the ultrasonically treated blood liquid component is hereinafter also referred to as “treated liquid component”. In general, blood can be roughly divided into cell components (cell fraction) and liquid components (liquid fraction). The cell component (cell fraction) is, for example, a blood cell component (blood cell fraction), and examples of the blood cell include red blood cells, white blood cells, and platelets. The liquid component is, for example, serum or plasma. “Plasma” is generally a liquid fraction obtained by removing the blood cells from blood, and “serum” is generally a liquid fraction obtained by removing the blood cells and several blood coagulation factors from blood. . Examples of the blood coagulation factor include fibrinogen (factor I) and prothrombin (factor II).

  The differentiation promoting agent of the present invention only needs to contain the liquid component, and other configurations are not limited at all. For example, the differentiation promoting agent of the present invention may contain, for example, only the liquid component as the blood-derived component, or may further contain other blood-derived components. That is, the differentiation promoting agent of the present invention may contain, for example, only the treated liquid component or may contain sonicated blood itself. Examples of the former include sonicated serum and plasma. The differentiation promoting agent of the present invention is preferably a liquid component from which the cell component has been removed, a liquid component from which the blood coagulation factor has been removed, a liquid component from which the cell component and the blood coagulation factor have been removed. preferable. The removal of the cell components and the removal of the blood coagulation factor are not limited to the complete removal of these.

  In the present invention, the treated liquid component includes, for example, ultrasonically treated blood, a liquid component recovered from the ultrasonically treated blood, and a component obtained by subjecting the liquid component recovered from the blood to ultrasonic treatment. But you can

  In the present invention, the method for preparing the treated liquid component is not particularly limited. The target of the ultrasonic treatment is not particularly limited, and may be, for example, blood itself or a liquid component (eg, serum or plasma) collected from blood.

  In the former case, for example, ultrasonic treatment may be performed on blood. In this case, for example, the sonicated blood may be used as it is as the blood containing the treated liquid component in the differentiation promoting agent of the present invention, or the liquid component may be removed from the sonicated blood. You may collect | recover and use this for the differentiation promoter of this invention as said processed liquid component. When recovering the liquid component from the blood, for example, it is preferable to remove blood coagulation factors such as fibrinogen in addition to blood cells.

  In the latter case, for example, sonication may be performed on a liquid component collected from blood that has not been sonicated. In this case, for example, the ultrasonically treated liquid component can be used as it is for the differentiation promoting agent of the present invention. In the latter case, a liquid component that has not been subjected to ultrasonic treatment and is collected from blood is also referred to as a “liquid component before treatment”. The pre-treatment liquid component is, for example, any of a liquid fraction obtained by removing the blood cell component from blood, a liquid fraction obtained by removing the blood coagulation factor, and a liquid fraction obtained by removing the blood cell component and the blood coagulation factor. In particular, a liquid fraction excluding the blood cell component is preferable. In addition, the pre-treatment liquid component may include, for example, platelets. Examples of the plasma containing platelets include so-called platelet-rich plasma (Platelet-Rich-Plasma). When recovering the liquid component from the blood, for example, it is preferable to remove blood coagulation factors such as fibrinogen in addition to blood cells.

  The conditions for the ultrasonic treatment are not particularly limited. As a specific example, for example, when the ultrasonic wave with respect to the object is measured with a sound pressure meter, the frequency is, for example, 28 to 100 kHz, the distance from the ultrasonic generator under the condition that a sound pressure of 5 mV or more is obtained, For example, the ultrasonic treatment is performed with a treatment time of 5 to 30 cm and a treatment time of, for example, 5 to 60 minutes, preferably 10 to 30 minutes. The ultrasonic waves can be generated using, for example, a normal ultrasonic generator. In this way, the treated liquid component is obtained.

  It is preferable that the blood or the liquid component subjected to the ultrasonic treatment further removes the blood coagulation factor, for example, before and / or after the ultrasonic treatment. Specifically, for example, the blood coagulation factor can be removed from the blood, serum can be collected, and this can be used as the liquid component. When the blood is subjected to ultrasonic treatment, for example, the collected serum can be used as the processed liquid component in the differentiation promoting agent of the present invention, and when the blood is not subjected to ultrasonic treatment, The collected serum is subjected to ultrasonic treatment, and this can be used as the treated liquid component in the differentiation promoting agent of the present invention. Further, when the liquid component is plasma containing the blood coagulation factor, for example, the blood coagulation factor is removed and serum is collected, and this can be used as the liquid component. When the plasma is sonicated, for example, the collected serum can be used as the treated liquid component in the differentiation promoting agent of the present invention, and when the plasma is not sonicated, The collected serum is subjected to ultrasonic treatment, and this can be used as the treated liquid component in the differentiation promoting agent of the present invention.

  The blood coagulation factor removal treatment is not particularly limited, and examples thereof include a method in which the blood coagulation factor is denatured and removed as an insoluble fraction. For example, fibrinogen can be formed by forming fibrin by denaturation and removing it as the insoluble fraction. Inactivating the blood coagulation factor by denaturation is referred to as “inactivation”. The denaturing method is not particularly limited, and examples thereof include thermal denaturation. Examples of the thermal denaturation method include a method of subjecting blood or the liquid component to heat treatment. The conditions for the heat treatment are not particularly limited. As a specific example, for example, the heating temperature is preferably 50 to 60 ° C., and the heating time is preferably 10 to 120 minutes, more preferably 30 to 60 minutes. The insoluble fraction can be removed by, for example, subjecting the blood or liquid component after the heat treatment to standing, centrifugation or filter filtration. The liquid fraction obtained by removing the insoluble fraction can be used as the liquid component.

  The method for removing the blood coagulation factor is, for example, a method using an activation mechanism of the blood coagulation system such as addition of a coagulation factor such as calcium chloride and thrombin to the blood or the liquid component in addition to the heat treatment. can give. The specific conditions are not limited at all, and conventionally known conditions can be adopted.

  According to the heat treatment, for example, the protein is not limited to the blood coagulation factor, and other proteins such as complement can be denatured and can be similarly removed by a subsequent removal treatment.

  The origin of the blood is not particularly limited, and examples thereof include humans and non-human mammals such as rodents, livestock, and primates excluding humans.

  The blood is usually collected from a living body using a blood collection device. Examples of the blood collection device include a device containing an anticoagulant, a device containing a coagulant, and a device not containing any of them. Since the device containing the anticoagulant contains the anticoagulant, for example, the collected blood is in a state where coagulation is suppressed. Since the device containing the coagulant contains the coagulant, coagulation is promoted in the collected blood, for example. In the case of using a device that does not include any of them, for example, blood collected is promoted to coagulate due to contact with the inner wall of the device.

  Hereinafter, an example of a method for preparing serum as the treated liquid component from blood whose coagulation is promoted by a coagulant, etc., from the blood whose coagulation is suppressed by an anticoagulant or the like, the treated liquid property An example of a method for preparing serum as a component will be shown. The present invention is not limited to these examples.

(1) Preparation from blood whose coagulation is promoted First, the blood is subjected to centrifugation to separate it into serum and blood clot. Centrifugation conditions are not particularly limited, and are, for example, centrifugal acceleration 2940 to 39200 m / s ² (300 to 4000 × g), temperature 1 to 20 ° C., and time 3 to 15 minutes.

Next, the serum is heat-treated to inactivate it. The inactivated serum is subjected to centrifugation, and the supernatant serum is collected as a liquid fraction. The conditions for the heat treatment are as described above, for example. The conditions for the centrifugation are not particularly limited, and are, for example, centrifugal acceleration 2940 to 39200 m / s ² (300 to 4000 × g), temperature 1 to 20 ° C., and time 3 to 15 minutes.

  Then, the serum is subjected to ultrasonic treatment to obtain a treated serum. The ultrasonic treatment conditions are, for example, as described above.

  The treated serum may be further filtered, for example. The filter used for the filter filtration is not particularly limited. The filter has a pore size of, for example, 0.1 to 0.3 μm, and the material is, for example, polyethersulfone (PES), polyvinylidene fluoride (PVDF), cellulose mixed ester (MCE), or polytetrafluoroethylene (PTFE). And polycarbonate (PC). Specific examples include a filter made of polyethersulfone (PES) having a pore diameter of 0.22 μm.

  The obtained treated serum can be used as the treated liquid component in the differentiation promoting agent of the present invention. The differentiation promoting agent of the present invention may contain, for example, only the treated liquid component, or may contain other components in addition to the treated liquid component. The other components are not particularly limited, and examples thereof include differentiation inducing factors, cytokines, antibiotics, salts, vitamins and the like. The differentiation inducing factor can be appropriately set according to, for example, the type of target cell to be differentiated. As a specific example, for differentiation into osteoblasts, for example, dexamethasone, ascorbic acid, β-glycerophosphoric acid and the like can be used as the differentiation-inducing factor, and these may be used alone or in combination of two or more. Or all of them may be used together.

(2) Preparation from blood in which coagulation is suppressed The blood in which coagulation is suppressed normally contains the anticoagulant as described above. Examples of the anticoagulant include heparin, protamine, citric acid, CPD solution (Citrate-Phosphate-Dextrose solution), ACD-A solution (Acid-Citrate-Dextrose-A solution) and the like. Since umbilical cord blood is usually collected in a blood collection bag containing an anticoagulant, for example, the method exemplified here is suitable for preparation from umbilical cord blood.

  Therefore, first, an erythrocyte sedimentation agent is added to the blood and left to stand to separate into a precipitate fraction of erythrocytes and a supernatant fraction containing plasma, platelets and leukocytes. Examples of the erythrocyte sedimenting agent include HES (Hydroxy Ethyl Starch). The addition amount of the erythrocyte sedimenting agent is not particularly limited, and is, for example, 1 to 50 mg / mL. The conditions for separation are not particularly limited, and for example, the temperature is 15 to 25 ° C.

Next, the supernatant fraction is subjected to centrifugation, and separated into a leukocyte precipitate fraction and a supernatant fraction containing plasma and platelets. The conditions for the centrifugation are not particularly limited, and are, for example, centrifugal acceleration 2940 to 39200 m / s ² (300 to 4000 × g), temperature 1 to 20 ° C., and time 3 to 15 minutes.

The plasma is then sonicated. The conditions for the ultrasonic treatment are not particularly limited, and are as described above, for example. Subsequently, the treated plasma is subjected to heat treatment and centrifuged to separate the insoluble fraction and the supernatant fraction of the treated serum. The conditions for the heat treatment are not particularly limited, and are as described above, for example. The conditions for the centrifugation are not particularly limited, and are, for example, centrifugal acceleration 2940 to 39200 m / s ² (300 to 4000 × g), temperature 1 to 20 ° C., and time 3 to 15 minutes.

  The treated serum may be further filtered, for example. The treated serum is ultrafiltered, for example, and the obtained filtrate is further filtered. The ultrafiltration conditions are not particularly limited. For example, the lower limit of the molecular weight cut-off is, for example, 3 to 100 k, and preferably 10 k. The filter used for filter filtration is not particularly limited and is the same as described above.

  The obtained treated serum can be used in the differentiation promoting agent of the present invention as a treated liquid component. The differentiation promoting agent of the present invention may contain, for example, only the treated liquid component, or may contain other components in addition to the treated liquid component. The other components are not particularly limited, and are as described above, for example.

  In the above (1) and (2), the method for preparing the treated serum from blood was exemplified. For example, the separation method of various fractions, the presence and condition of heat treatment, the presence and condition of filtration, etc. It is not limited. Examples of the separation method include, for example, precipitation, membrane separation, and adsorption in addition to centrifugation and filtration.

  The differentiation promoting agent of the present invention may be a medium, for example, a medium containing the treated liquid component. The medium can be prepared, for example, by containing the treated liquid component in a basic medium. The basal medium is not particularly limited, and for example, those described in the differentiation promoting method described later can be used.

  The present inventors have also clarified that peripheral blood and serum thereof suppress differentiation from mesenchymal stem cells to cells belonging to the mesenchymal system by performing inactivation treatment such as heat treatment. . However, in the present invention, the mechanism is unclear by applying sonication, but regardless of the presence or absence of inactivation, for example, the inactivated blood and its serum can be differentiated from mesenchymal stem cells. Shows ability to promote. This is a finding that the present inventors have found for the first time. Therefore, the method of sonicating blood, plasma or serum after inactivation can be said to be, for example, a method for expressing differentiation promoting ability of humoral components of inactivated peripheral blood. Here, the cells belonging to the mesenchymal system are cells belonging to the mesenchymal system excluding, for example, adipocytes, and specific examples include, for example, osteoblasts, chondrocytes, bone cells, cardiomyocytes, tendons Examples include cells.

  The differentiation promoting agent of the present invention is used, for example, in combination with a blood fluid component that has not been subjected to ultrasonic treatment and has been subjected to inactivation treatment, thereby enabling, for example, a switch for inducing differentiation of mesenchymal stem cells to be turned ON / OFF. it can.

  In addition, according to the differentiation promoting agent of the present invention, for example, differentiation from a mesenchymal stem cell to a cell belonging to a mesenchymal system other than an adipocyte is promoted, and differentiation from a mesenchymal stem cell into an adipocyte is performed. Can be suppressed. For this reason, according to the present invention, for example, differentiation into cells belonging to the mesenchymal system other than adipocytes, particularly osteoblasts or chondrocytes, can be promoted with high selectivity.

<Differentiation promotion method>
The differentiation promoting method of the present invention is a method for promoting the differentiation of mesenchymal stem cells as described above,
It has the following (X) process, It is characterized by the above-mentioned.
(X) A step of culturing the mesenchymal stem cells in a medium containing the differentiation promoting agent of the present invention.

  The differentiation promoting method of the present invention is characterized by using the differentiation promoting agent of the present invention, and the other steps and conditions are not particularly limited. The differentiation promoting agent of the present invention is as described above. According to the differentiation promoting method of the present invention, in the step (X), differentiation of the mesenchymal stem cells can be promoted by culturing the mesenchymal stem cells in a medium containing the differentiation promoting agent of the present invention. .

  According to the present invention, differentiation from mesenchymal stem cells into differentiated cells can be promoted. The differentiated cells formed by the differentiation of the mesenchymal stem cells are not particularly limited and are cells belonging to the mesenchymal system, for example, cells belonging to the mesenchymal system excluding adipocytes, specifically, bone Examples include blast cells, chondrocytes, bone cells, cardiomyocytes, and tendon cells.

  The origin of the mesenchymal stem cells is not particularly limited, and examples thereof include humans and non-human mammals such as rodents, livestock, and primates excluding humans. The type of the mesenchymal stem cell is not particularly limited, and examples thereof include bone marrow-derived, dental pulp-derived, and adipose tissue-derived.

  In the differentiation promoting method of the present invention, the mesenchymal stem cells and the humoral component in the differentiation promoting agent are preferably derived from, for example, the same species, and more preferably from the same individual. The individual is not particularly limited, and examples thereof include humans and non-human mammals, and preferably humans.

  In addition to the differentiation promoting agent, the medium further contains the differentiation inducing factor, for example. The differentiation-inducing factor can be appropriately set according to, for example, the type of target cell to be differentiated, and is as described above. The type of the differentiation-inducing factor and the amount added to the medium are not particularly limited, and known conditions can be referred to.

  The medium may contain the differentiation promoting agent and the differentiation inducing factor, and other configurations and conditions are not limited at all. Examples of the medium include a medium that can be used for culturing the mesenchymal stem cells. Specifically, for example, α-Minimal essential medium medium (αMEM medium), Dulbecco's modified easy medium medium (DMEM medium). ) Etc. can be used. The concentration of the differentiation promoting agent in the medium is not particularly limited. For example, the treated liquid component is 0.01 to 20 v / v%, preferably 1 to 15 v / v%, more preferably. Is 5-10 v / v%. The number of mesenchymal stem cells per 1 mL of the medium is, for example, 1000 to 100,000. The medium may further contain, for example, an additive. Examples of the additive include cytokines, antibiotics, salts, vitamins and the like.

  The culture conditions are not particularly limited, and the temperature is, for example, 37 ° C., and the time is, for example, 2 to 30 days. The medium is preferably replaced with a new medium every 1 to 5 days, for example. The method for replacing the medium is not limited at all, and examples thereof include a method for exchanging the medium every predetermined time and a method for supplying a new medium continuously or intermittently. In the latter case, for example, it is preferable to discard a part of the old medium continuously or intermittently in accordance with the supply of the new medium.

<Method for producing mesenchymal stem cell-derived differentiated cells>
The production method of the present invention is a method for producing mesenchymal stem cell-derived differentiated cells, and includes a step of promoting differentiation of the mesenchymal stem cells by the differentiation promotion method of the present invention.

  The production method of the present invention is characterized in that the mesenchymal stem cells are cultured using the differentiation promoting agent of the present invention, and other steps and conditions are not limited at all. For example, the description of the differentiation promoting agent and the differentiation promoting method of the present invention can be used for the production method of the present invention.

  Next, examples of the present invention will be described. However, the present invention is not limited by the following examples.

[Example 1]
Sonicated serum was prepared from human peripheral blood and human umbilical cord blood, and the effect on differentiation from mesenchymal stem cells to osteoblasts was confirmed.

(1) Preparation of treated serum from peripheral blood Peripheral blood sample collected with a centrifuge tube (trade name: 50 mL centrifuge tube, Corning) containing 2 glass beads (particle size: 4 mm, manufactured by Bright Label Co., Ltd.) as a coagulant Centrifugation (2330 × g, 4 ° C., 10 minutes) was performed on 50 mL of blood and separated into a serum supernatant fraction and a clot precipitation fraction. The supernatant fraction was heat-treated at 56 ° C. for 30 minutes, and then centrifuged (2330 × g, 4 ° C., 10 minutes) to recover the serum supernatant fraction. 10 mL of the serum was placed in a PVC bag having a size of 5 × 10 cm and sonicated. The ultrasonic treatment uses an ultrasonic generator (trade name: Ultrasonic Multi Cleaner, manufactured by Honda Electronics Co., Ltd.), and the ultrasonic wave with respect to the object is measured with a sound pressure meter, and a sound pressure of 5 mV is obtained. The frequency was set to 45 kHz, the distance from the ultrasonic generator to the back surface was set to 15 cm, and the treatment was performed for 30 minutes. And the said blood serum after ultrasonication was filtered with the filter (0.22 micrometer, brand name micro filter unit, the product made by Millipore), and the filtrate was collect | recovered. The filtrate was used as sonicated serum. This is hereinafter referred to as peripheral blood serum (+).

  As a comparative example, serum obtained by centrifugation after the heat treatment was filtered with a filter (trade name: Microfilter Unit, manufactured by Millipore), and the filtrate was collected. The filtrate was used as sonicated serum. This is hereinafter referred to as peripheral blood serum (−).

(2) Preparation of treated serum from umbilical cord blood Cord blood collected with a blood collection bag (trade name: JMS blood bag 200 (with CPD), manufactured by JMS) containing an anticoagulant solution (CPD solution) 1/5 volume HES40 (trade name HES40, manufactured by Nipro Co., Ltd.) was added and allowed to stand at room temperature for 60 minutes. By standing, it was separated into a supernatant fraction containing plasma, platelets and white blood cells and a precipitate fraction containing red blood cells. The supernatant fraction was collected and centrifuged (400 × g, 4 ° C., 5 minutes), and separated into a supernatant fraction containing plasma and platelets and a leukocyte precipitate fraction. The supernatant fraction was centrifuged (2330 × g, 4 ° C., 10 minutes), and separated into a plasma supernatant fraction and a platelet precipitate fraction. 10 mL of the plasma was placed in a PVC bag having a size of 5 × 10 cm, and the plasma in the bag was sonicated. The ultrasonic treatment uses the ultrasonic generator, and when the ultrasonic wave with respect to the object (that is, the bag) is measured with a sound pressure meter, a sound pressure of 5 mV is obtained under the condition that a sound pressure of 5 mV is obtained. The distance from the generator to the back surface was set to 15 cm, and this was performed for 30 minutes. The plasma after sonication was inactivated by heat treatment at 56 ° C. for 30 minutes, and then centrifuged (2330 × g, 4 ° C., 10 minutes) to collect a serum supernatant fraction. The serum was subjected to ultrafiltration (fractionated molecular weight: 10 k), and the obtained filtrate was further filtered through a filter (0.22 μm, trade name: Microfilter Unit, manufactured by Millipore) to collect the filtrate. The filtrate was used as sonicated serum. This is hereinafter referred to as cord blood serum (+).

  As a comparative example, the plasma was similarly subjected to heat treatment, centrifugation, ultrafiltration and filter filtration, except that the ultrasonic treatment was not performed, and the filtrate was collected. The filtrate was used as sonicated serum. This is hereinafter referred to as cord blood serum (−).

(3) Culture of Mesenchymal Stem Cells Bone marrow-derived mesenchymal stem cells BM-MSC (Lonza) were used as mesenchymal stem cells. The medium is added with the peripheral blood serum (+), the peripheral blood serum (−), the umbilical cord blood serum (+) or the umbilical cord blood serum (−) as a serum, and dexamethasone, ascorbic acid as a differentiation inducing factor. In addition, a serum-added differentiation induction medium having the following composition using αMEM as a basic medium and supplemented with β-glycerophosphate was used.

(Serum-added differentiation induction medium)
αMEM basic medium Serum 10% (v / v)
Dexamethasone 100 nmol / L
Ascorbic acid 50μg / ml
β-glycerophosphoric acid 10mmol / L

  And using a well plate, it seed | inoculated so that the said mesenchymal stem cell might become 5000 cells per well in the said serum addition differentiation-inducing medium, and it culture | cultivated at 37 degreeC for 21 days (n = 2 about each medium). The cultured cells were stained with Alizanin Red S (trade name, manufactured by Wako Pure Chemical Industries, Ltd.) to confirm the presence of osteoblasts. By the staining, osteoblasts are stained red.

  These results are shown in FIG. FIG. 1 is a photograph showing staining of cells after culture using each serum-added differentiation-inducing medium (n = 1). In the figure, the red stained portion is represented by dark gray. As shown in the figure, as a result of using a medium containing sonicated peripheral blood serum (+) and umbilical cord blood serum (+), the cultured cells were stained in red. It was confirmed that the cells were differentiated into osteoblasts. In contrast, peripheral blood serum (−) and umbilical cord blood serum (−) that had not been subjected to ultrasonic treatment were not able to confirm the staining of cultured cells, so that mesenchymal stem cells differentiated into osteoblasts. It wasn't. From these results, it was found that differentiation of mesenchymal stem cells can be promoted by sonication even with the same serum.

[Example 2]
Serum was treated under different ultrasonic conditions, and the effect on the differentiation of mesenchymal stem cells was confirmed for the serum after the ultrasonic treatment.

(1) Preparation from fetal bovine serum Fetal bovine serum (FBS, Gibco) was used as a serum to be subjected to ultrasonic treatment, and a blood bag having a volume of 50 mL (PVC blood bag having a size of 5 × 10 cm) as in Example 1 above. ). Then, the serum in the blood bag was subjected to ultrasonic treatment using an ultrasonic generator (trade name: Ultrasonic Multi Cleaner, manufactured by Honda Electronics Co., Ltd.). In the ultrasonic treatment, when the ultrasonic wave with respect to the object (the blood bag) is measured with a sound pressure meter, the distance from the ultrasonic generator to the back surface is set to 15 cm under the condition that a sound pressure of 5 mV is obtained. The maximum output was set at a predetermined frequency (45 kHz, 100 kHz) and processing time (30 minutes, 60 minutes). The serum after sonication is deactivated by a heat treatment at 56 ° C. for 30 minutes, centrifuged (2330 × g, 15 minutes) to remove fibrin clots, and a filter (0.22 μm, trade name microfilter) The filtrate was recovered by filtration through a unit (Millipore). The filtrate was used as sonicated serum. This is hereinafter referred to as serum (+).

(2) Culture of Mesenchymal Stem Cells Bone marrow-derived mesenchymal stem cells BM-MSC (Lonza) were used as mesenchymal stem cells. As the medium, the serum-added differentiation-inducing medium of Example 1 using αMEM as a basic medium with the serum (+) added as serum was used. As a comparative example, instead of the serum (+), αMEM basal medium supplemented with ultrasound-untreated fetal bovine serum (FBS, Gibco) so as to be 10% (v / v) was used. For each serum, as a comparative example, an αMEM basal medium to which serum was similarly added and the differentiation-inducing factor was not added was used.

  And using a well plate, it seed | inoculated so that the said mesenchymal stem cell might become 3000 cells per well to the said culture medium, and it culture | cultivated at 37 degreeC for 21 days (n = 3 about each culture medium). The cultured cells were stained with Alizanin Red S (trade name, manufactured by Wako Pure Chemical Industries, Ltd.) to confirm the presence of osteoblasts. By the staining, osteoblasts are stained red.

  These results are shown in FIG. FIG. 2 is a photograph showing staining of cells after culture (n = 2). In FIG. 2, the comparative example is the result of ultrasonically untreated (−) FBS, and 45 kHZ 30 min, 45 kHZ 60 min, and 100 kHZ 60 min are the results of serum (+) sonicated under these conditions, respectively. The two columns from the left are the results using the medium containing the differentiation-inducing factor, and the one column on the right are the results using the medium not containing the differentiation-inducing factor. The portion stained in red is represented in dark gray in FIG. As shown in FIG. 2, as a result of using a medium containing sonicated serum (+), the cultured cells were stained in red regardless of the treatment conditions. It was confirmed that they were differentiated. As a specific example, differentiation can be promoted as the treatment time of the ultrasonic treatment is long, and differentiation can be promoted in a short treatment time as the frequency is low.

[Example 3]
Sonicated serum was prepared and the effect on differentiation from mesenchymal stem cells to adipocytes was confirmed.

(1) Preparation of serum from cord blood (1-1) Cord blood serum (-)
A blood bag (PVC blood bag having a size of 5 × 10 cm) containing 20 ml of umbilical cord blood (n = 1) collected from a human umbilical cord and containing two glass beads (particle size: 4 mm, manufactured by Bright Label Co., Ltd.) as a coagulant. ). The blood collection bag was lightly shaken 2 to 3 times, allowed to stand at room temperature for 1 hour, and then stored at 4 ° C.

  The umbilical cord blood was allowed to stand and separated into a supernatant fraction containing plasma, platelets and leukocytes and a precipitate fraction containing red blood cells. The supernatant fraction is collected and centrifuged (400 × g, 4 ° C., 5 minutes) to obtain a supernatant fraction containing plasma and platelets (platelet rich plasma: PRP) and a leukocyte precipitate fraction. separated. The supernatant fraction (PRP) was centrifuged (2330 × g, 4 ° C., 10 minutes) to separate into a plasma supernatant fraction (PPP) and a platelet precipitate fraction. The supernatant fraction (PPP) was inactivated by heat treatment at 56 ° C. for 30 minutes, and then centrifuged (2330 × g, 4 ° C., 10 minutes) to remove fibrin clots and remove serum supernatant. Fractions were collected. This was designated as cord blood serum (−) which was not sonicated.

(1-2) Cord blood serum (+)
The supernatant fraction (PRP), which is platelet-rich plasma, was placed in a 50 mL blood bag (PVC blood bag having a size of 5 × 10 cm) without centrifugation. Then, the supernatant fraction in the blood bag was sonicated using an ultrasonic generator (trade name: Ultrasonic Multi Cleaner, manufactured by Honda Electronics Co., Ltd.). The ultrasonic treatment was performed in the same manner as in Example 1. Then, after the ultrasonic treatment, the supernatant fraction was deactivated by heat treatment at 56 ° C. for 30 minutes, and then centrifuged (2330 × g, 4 ° C., 10 minutes) to remove the fibrin clot, Serum supernatant fractions were collected. This was designated as umbilical cord blood serum (+) subjected to ultrasonic treatment.

(2) Preparation of serum from peripheral blood (2-1) Peripheral blood serum (-)
Serum fractions were prepared from adult peripheral blood (n = 3) using a blood component separation bag (trade name Cell Aid, manufactured by JMS). The serum fraction was inactivated by heat treatment at 56 ° C. for 30 minutes, and then centrifuged (2330 × g, 4 ° C., 10 minutes) to remove fibrin clots and remove the serum supernatant fraction. Was recovered. This was defined as peripheral blood serum (−) that was not sonicated.

(2-2) Peripheral blood serum (+)
The peripheral serum is put into a 50 mL capacity blood bag (PVC blood bag having a size of 5 × 10 cm), and the serum in the blood bag is converted into an ultrasonic generator (trade name: Ultrasonic Multi Cleaner, manufactured by Honda Electronics Co., Ltd.). ) Was sonicated. The ultrasonic treatment was performed in the same manner as in Example 1. Then, after the ultrasonic treatment, the supernatant fraction was deactivated by heat treatment at 56 ° C. for 30 minutes, and then centrifuged (2330 × g, 4 ° C., 10 minutes) to remove the fibrin clot, Serum supernatant fractions were collected. This was sonicated peripheral blood (+).

(3) Culture of Mesenchymal Stem Cells Bone marrow-derived mesenchymal stem cells BM-MSC (Lonza) were used as mesenchymal stem cells. As the culture medium, a serum-added differentiation-inducing medium having the following composition and using DMEM as a basic medium, to which serum, a differentiation-inducing factor (dexamethasone, indomethacin, and IBMX) and a growth inhibitor (insulin) were added was used. As the serum, umbilical cord blood serum (−), umbilical cord blood serum (+), peripheral blood serum (−), and peripheral blood serum (+) were used. As a comparative example, a DMEM basic medium supplemented with ultrasound-untreated fetal bovine serum (FBS, Gibco) so as to be 10% (v / v) was used instead of the serum. In addition, for each serum, a DMEM basal medium in which the serum was added in the same manner and the differentiation-inducing factor was not added was used as a control.

(Serum-added differentiation induction medium)
DMEM basal medium Serum 10% (v / v)
Dexamethasone 1μmol / L
Indomethacin 0.2mmol / L
IBMX 0.5 mol / L
Insulin 10 μg / ml

  And using the well plate, it seed | inoculated so that the said mesenchymal stem cell might become 8000 cells per well in the said serum addition differentiation induction medium, and it culture | cultivated at 37 degreeC for 21 days. Then, the cultured cells were stained with OilRed (trade name, manufactured by Wako Pure Chemical Industries, Ltd.) to confirm the presence of adipocytes.

  These results are shown in FIG. FIG. 3 is a photograph showing staining of cells after culturing using each serum-added medium. FIG. 3 shows the results of using umbilical cord blood-derived serum (n = 1), peripheral blood-derived serum (n = 3) and comparative FBS. As shown in FIG. 3, differentiation into adipocytes was confirmed by the addition of FBS not treated with sonication. In contrast, differentiation into adipocytes was suppressed by the addition of sonicated cord blood serum (+) or peripheral blood serum (+).

  From these results, it was confirmed that sonicated serum suppresses differentiation of mesenchymal stem cells into adipocytes. For this reason, by comparing with the results of Examples 1 and 2, it can be said that differentiation into osteoblasts can be induced with excellent selectivity without differentiation from mesenchymal stem cells into adipocytes.

  Thus, according to the present invention, for example, differentiation of mesenchymal stem cells can be promoted only by using an ultrasonically treated blood fluid component. Since the active ingredient in the differentiation promoting agent of the present invention is the ultrasonically treated blood fluid component, for example, the origin of the blood fluid component and the origin of the mesenchymal stem cell to be treated are derived from the same species. It is possible to set, and it is also possible to set it from the same individual. Thus, according to the present invention, it is possible to use components of the same species, and thus the safety is excellent. Therefore, the present invention can be said to be extremely useful in, for example, regenerative medicine.

Claims (12)

A differentiation promoting agent that promotes differentiation of mesenchymal stem cells, comprising an ultrasonically treated blood fluid component. The differentiation promoting agent according to claim 1, wherein the blood is umbilical cord blood or peripheral blood. The differentiation promoting agent according to claim 1 or 2, wherein the liquid component is serum or plasma. The differentiation promoting agent according to any one of claims 1 to 3, wherein the blood is mammalian blood. The differentiation promoting agent according to claim 4, wherein the mammal is a human. A method for promoting the differentiation of mesenchymal stem cells,
A differentiation promoting method comprising the following step (X):
(X) A step of culturing the mesenchymal stem cell in a medium containing the differentiation promoting agent according to any one of claims 1 to 5. The differentiation promoting method according to claim 6, wherein the mesenchymal stem cell and the humoral component in the differentiation promoting agent are derived from the same individual. The differentiation promoting method according to claim 7, wherein the individual is a mammal. The method of promoting differentiation according to claim 8, wherein the mammal is a human. The differentiation promoting method according to any one of claims 6 to 9, wherein the differentiation from the mesenchymal stem cells to at least one of osteoblasts and chondrocytes is promoted. A method for producing mesenchymal stem cell-derived differentiated cells,
A production method comprising the step of promoting the differentiation of the mesenchymal stem cells by the differentiation promoting method according to any one of claims 6 to 10. The production method according to claim 11, wherein the differentiated cells are at least one of osteoblasts and chondrocytes.