WO2016068257A1 - Method for producing cell and method for producing blood-forming tissue - Google Patents

Abstract

The present invention mainly addresses the problem of providing: a method for producing a cell; a method for producing a blood-forming tissue; a cell or a blood-forming tissue obtained by the respective method; a cell inducing agent; a blood-forming tissue preparing kit; and a screening method of a cell inducing agent. As a means for solving the problem, provided is a method for producing a cell (B) capable of being differentiated into a somatic cell (A), including the following step: culturing a somatic cell (C) which is different from the somatic cell (A) in the presence of a compound represented by general formula (I). [In the formula, R¹, X are as defined in the specification.]

Description

Cell manufacturing method and hematopoietic tissue manufacturing method

[Cross-reference of related applications]
This application claims priority based on Japanese Patent Application No. 2014-223316 filed on October 31, 2014, the entire disclosures of which are incorporated herein by reference.

The present invention mainly relates to a method for producing a cell, a method for producing a hematopoietic tissue, a cell or hematopoietic tissue obtained by the production method, a cell inducer, a kit for producing a hematopoietic tissue, and a method for screening a cell inducer.

In recent years, regenerative medical technology has been attracting attention for the purpose of restoring functions of cells, tissues, organs, etc., which have been lost or abnormal in patients. In addition to establishing induction methods and culture methods for various cells, tissues, organs, etc., the possibility of using iPS cell technology as a source of self-cells has been proposed, and further advancement of the technology is expected .

In regenerative medicine technology, it is important to establish a method for inducing and culturing cells, tissues, organs, etc. that are the purpose of treatment.

In the case of diseases based on abnormalities in hematopoietic tissues typified by leukemia (including hematopoietic stem cells and blood cells derived therefrom), transplantation of hematopoietic tissues such as bone marrow is one of effective treatment methods. However, when transplanting bone marrow tissue derived from a donor different from the patient, it is necessary to match the type of HLA, and there are few donors that can provide bone marrow in the first place, so patients who need treatment Currently, bone marrow transplantation has not been performed for all of these.

Therefore, a technique for producing hematopoietic tissue such as bone marrow tissue derived from its own cells by regenerative medical technology, which does not cause the problem of conformity of HLA type, has been desired. However, at present, a method for producing hematopoietic tissue such as bone marrow tissue by regenerative medical technology has not yet been established.

From the viewpoint of easy acquisition, cells derived from dental pulp tissue that can be acquired in dental treatment are attracting attention as a source of cells used in the regeneration technique (Non-patent Document 1).

The present invention has been made in view of the above-described background art, and includes a method for producing a cell, a method for producing a hematopoietic tissue, a cell or hematopoietic tissue obtained by the production method, a cell inducer, a kit for producing a hematopoietic tissue, and The main object is to provide a screening method for cell inducers.

As a result of intensive studies in order to solve such problems, the present inventor has cultivated somatic cells in the presence of a compound represented by the following chemical formula, thereby dividing them into somatic cells different from the somatic cells. It has been found that cells that can be transformed can be produced. The present invention has been completed based on such findings and further studies.

That is, the present invention includes the following aspects.

Item 1, a method for producing a cell (B) capable of differentiating into a somatic cell (A), comprising the following steps:
A step of culturing a somatic cell (C) different from the somatic cell (A) in the presence of a compound represented by the following chemical formula.

[Wherein R ¹ represents a hydrogen atom (H) or an acetyl group (CH ₃ (C═O) —). X represents a halogen atom. ]

Item 2. The method according to Item 1, wherein the compound is a compound represented by the following chemical formula.

Item 3. The method according to Item 1, wherein the somatic cell (C) is a cell obtained from a dental pulp or a mesenchymal stem cell.

Item 4, a method for producing a hematopoietic tissue, comprising the following steps:
A step of culturing a somatic cell having no hematopoietic function in the presence of a compound represented by the following chemical formula.

Item 5, a method for producing a hematopoietic tissue, comprising the following steps:
(1) culturing a somatic cell having no hematopoietic function in the presence of a compound represented by the following chemical formula;

And (2) a step of transplanting the cultured cells together with a carrier into an animal.

Item 6. The method according to Item 4 or 5, wherein the somatic cell is a cell obtained from a dental pulp or a mesenchymal stem cell.

Item 7. A cell obtained by the method according to any one of Items 7 and 1-3.

A hematopoietic tissue obtained by the method according to any one of Items 8 and 4 to 6.

Item 9. A cell inducer comprising a compound represented by the following chemical formula.

Item 10, (1) a compound represented by the following chemical formula, and

(2) A hematopoietic tissue preparation kit including a carrier.

Item 11. A method for screening a cell inducer comprising the following steps:
(1) a step of culturing somatic cells in the presence of a test substance, and evaluating the cell induction ability of the test substance, and (2) the cell induction ability of the test substance evaluated in the above step, and represented by the following chemical formula: Comparing the cell-inducing ability of the compound.

Item 12, To produce a cell (B) that can differentiate into a somatic cell (A), and for producing a cell (B) from a somatic cell (C) different from the somatic cell (A), Use of a compound represented by a chemical formula.

Item 13. Use of a compound represented by the following chemical formula for producing a hematopoietic tissue.

The present invention provides a novel cell production method, hematopoietic tissue production method, cells or hematopoietic tissue obtained by the production method, cell inducer, hematopoietic tissue preparation kit, and cell inducer screening method. .

The method of the present invention provides a method for providing hematopoietic tissue from somatic cells, and is expected to be a technique that can provide a new treatment method to patients who require treatment such as bone marrow transplantation. The new properties of the compounds found by the present invention are also expected to become a tool for further progress in regenerative medicine technology.

The result of the morphological observation of mouse deciduous dental pulp cells (mDP) is shown. (A) The result of sorting is shown by a plot diagram. The result of the morphological observation of mouse deciduous dental pulp cells (mDP) is shown. (B) A fluorescence microscope image is shown. The result of the morphological observation of mouse deciduous dental pulp cells (mDP) is shown. (C) Quantification results (Relative length (relative length), Relative width (relative width), and elliptical form factor) are shown. The screening result of the drug which induces a dental pulp stem cell like form is shown. (A) Shows the structure of GSK-3 inhibitor IX and BIO. The screening result of the drug which induces a dental pulp stem cell like form is shown. (B) A fluorescence microscope image is shown for the cell morphology. The screening result of the drug which induces a dental pulp stem cell like form is shown. (C) The quantitative result (Relative eliptical form factor) of the form is shown. The effect of BIO processing is shown. (A) Detection of phosphorylation of β-catenin by Western blotting. (B) Quantitative results of phosphorylation of β-catenin are shown. (C) Quantification results of expression of stem cell markers (Oct3 / 4 and Klf-4) are shown. (D) shows the quantification result of the expression of the stem cell marker (Oct3 / 4). The ability of cells cultured in the presence of BIO to differentiate into osteoblasts is shown. (A) An example of the result of von Kossa staining is shown. (B) Quantitative results of the intensity of von Kossa staining (Intensity of von Kossa staining) are shown. The result of the transplantation experiment of mouse bone marrow-derived mesenchymal stem cells cultured in the presence of BIO is shown. (A, B) When mouse bone marrow-derived mesenchymal stem cells (mBM-MSC) are treated. (A) The operation scheme is shown. (B) A microscope image obtained by observing the transplanted site is shown. (C, D) When treating human deciduous dental pulp stem cells (SHED). (C) shows the scheme of operation. (D) shows a microscopic image of the transplanted site observed. The result of the transplantation experiment of human deciduous dental pulp stem cells cultured in the presence of BIO is shown. (A) The result of gating is shown. (B) Detection of human CD11b antigen (hCD11b) expression. (C) Shows external findings of transplanted mice.

1B and 2B also show a monochrome inverted image (Monochrome （inverted image).

1. Cell Production Method The present invention provides the following cell production method:
A method for producing a cell (B) capable of differentiating into a somatic cell (A), comprising the following steps:
A step of culturing a somatic cell (C) different from the somatic cell (A) in the presence of a compound represented by the following general formula (I).

[Wherein R ¹ represents a hydrogen atom (H) or an acetyl group (CH ₃ (C═O) —). X represents a halogen atom. ]

Production Method The production method of the present invention uses somatic cells (C) as a material. The production method of the present invention is characterized in that a somatic cell (C) as a material is different from a cell (A) from which the obtained cell (B) can be differentiated. Here, “different” is understood to mean belonging to different strains in the phylogenetic tree of cell differentiation. For example, cells derived from dental pulp originating from neural crest and mesenchymal stem cells originating from mesenchyme belong to a different lineage from hematopoietic cells originating from mesoderm.

“Can differentiate” means having the ability to differentiate into specific cells under normal physiological conditions or artificially induced differentiation conditions.

Under normal physiological conditions, cell differentiation proceeds in one direction and does not reverse. That is, the differentiated somatic cell does not undergo a special phenomenon such as dedifferentiation, and the property does not change to a cell that can differentiate into another cell. Therefore, it is understood that the present invention is a method that does not include a somatic cell reprogramming process such as a process of producing iPS cells.

∙ Somatic cell initialization, such as in the iPS cell production process, usually involves the introduction of a foreign gene. On the other hand, in the production method of the present invention, cells (B) are obtained using somatic cells (C) as materials by the functions of endogenous genes (in particular, only by the functions of endogenous genes) without introducing foreign genes. Induces differentiation.

Culture The method for producing cells of the present invention includes a step of culturing somatic cells (C) in the presence of the above compound. The culture can be performed according to a normal cell culture method except that the culture is performed in the presence of the above compound.

The concentration of the compound in the culturing step of the present invention is not particularly limited as long as it does not impair the effects of the present invention. For example, the concentration is about 0.01 μM to 100 μM, about 0.1 to 10 μM, about 0.2 to 5 μM, It can be about 1 μM.

Cultivation can be performed efficiently using somatic cells (C). The medium to be used is not particularly limited as long as it is a medium suitable for culturing somatic cells (C). Examples of such a medium include Dulbecco's Modified a Eagle Medium (DMEM), alpha Modified a Eagle Minimum Essential Medium (αMEM), and the like. The culture medium may contain additives that are included when culturing ordinary somatic cells as necessary. Specific examples of the additive include fetal calf serum (fetal （bovine serum), amino acids (for example, L-glutamine), antibiotics (for example, penicillin, streptomycin, and the like). Commercially available products such as DMEM / F12 (manufactured by Life Technologies), to which necessary additives are added in advance, can also be used.

Culturing can be performed in a suitable vessel. Examples of suitable culture techniques include a technique of culturing under conditions of about 37 ° C. and a carbon dioxide concentration of about 5%, but is not limited thereto. Cultivation under the above conditions can be performed using, for example, a known CO ₂ incubator.

The culture period is not particularly limited as long as the effects of the present invention are not impaired. For example, it can be about 12 hours to 20 days.

Although not limiting the present invention, it is considered that the effect of inducing the cell (B) by the above compound is achieved in 3 days or less, particularly in about 2 days. If necessary, for example, in order to obtain a sufficient number of cells, culture can be performed for a longer period of time.

In culture, subculture can be performed as necessary. When subculture is performed, the cells are collected before reaching a confluent state, and the cells are seeded in a new medium. In the culture of the present invention, the medium can be changed as appropriate.

However, culture until reaching a confluent state is not limited. For example, in the case of the culture for the purpose of obtaining a sufficient number of cells as described above, the culture may be performed until a confluent state is reached.

Somatic cells (C)
The somatic cell (C) used as a material in the method of the present invention is not particularly limited as long as it is a somatic cell derived from an animal. “Somatic cells” usually refer to differentiated cells and do not have the ability to differentiate into other cells.

The type of somatic cell (C) is not particularly limited.

The origin of somatic cells (C) is not particularly limited, but is preferably derived from mammals such as humans, monkeys, mice, rats, dogs, hamsters, rabbits, cats, cows and the like. When the obtained cell (B) is intended for treatment of a disease, it is preferable that the cell (B) is derived from an individual in need of treatment, but is not limited thereto.

Somatic cells (C) are cells collected from living organisms, cells cultured from cells collected from living organisms (in particular, primary cultured cells), and cells established by continuing subculture using cells collected from living organisms. Any of these may be used.

In one embodiment of the present invention, the somatic cell (C) is a cell derived from the dental pulp. The cells derived from dental pulp include dental pulp cells and dental pulp stem cells. The cells derived from the dental pulp include cells obtained by culturing cells collected from the dental pulp (in particular, primary cultured cells) and cells established by continuing subculture using cells collected from the dental pulp. As the established cell line, those described in Non-Patent Document 1 can be used.

In another embodiment of the present invention, the somatic cell (C) is a mesenchymal stem cell. The origin of mesenchymal stem cells is not particularly limited, and those derived from bone marrow, adipose tissue, and the like can be used. Of these, bone marrow-derived mesenchymal stem cells that are easy to prepare are preferred examples. Mesenchymal stem cells can be prepared according to a known technique. For example, bone marrow-derived mesenchymal stem cells can be collected by centrifuging a bone marrow fluid obtained by bone marrow puncture.

Compound The culture step in the production method of the present invention is characterized in that it is carried out in the presence of a compound represented by the following general formula (I).

[Wherein R ¹ represents a hydrogen atom (H) or an acetyl group (CH ₃ (C═O) —). X represents a halogen atom. ]

The above compound can also be represented by the following general formula (I ′).

[Wherein, R ¹ and X are the same as defined above. ]

The halogen atom represents a fluorine atom (F), a chlorine atom (Cl), a bromine atom (Br) or an iodine atom (I).

In the compound represented by the above general formula (I), the position on the benzene ring to which X is bonded is not particularly limited, but is preferably bonded to the 6-position. That is, a preferred embodiment of the compound of the present invention is represented by the following general formula (II).

[Wherein, R ¹ and X are the same as defined above. ]

The compound represented by the above general formula (I) can be produced by a known production method or a method according to a known production method. For example, it can be produced by a production method described in the literature: J. Med. Chem. 2004, 47, 935-946 or a production method according to the production method described therein.

As a preferred embodiment of the compound of the present invention, a compound represented by the following chemical formula is exemplified.

The above compound can also be represented by the following chemical formula.

The compound is a selective inhibitor of glycogen synthase kinase-3 α isoform (GSK-3α) and β isoform (GSK-3β). The compound includes (2Z, 3E) -6'-Bromo-3- (hydroxyimino)-[2,3'-biindolinylidene] -2'-one, 6-bromoindirubin-3'-oxime, GSK-3 Inhibitor IX, It is known by names such as BIO. The compound is assigned CAS number: 667463-62-9, PubChem® CID: 5284844. The above compound may be referred to as “BIO” hereinafter.

The compound can be produced by a known production method. Commercial products can also be used.

Instead of the above compound, a compound having an action of suppressing the expression and / or function of GSK-3 other than the above compound (both GSK-3α and / or GSK-3β) is used in the method of the present invention. You can also. For example, a low molecular weight compound that functions as an inhibitor or antagonist of GSK-3, a compound that inhibits transcription and / or translation of the gene product of GSK-3 (eg, siRNA, shRNA, miRNA, etc.), specificity for GSK-3 Specific antibodies. However, it is understood that LiCl and LiF are unfavorable examples.

Furthermore, the β isoform of GSK-3 belongs to the Wnt / β-catenin pathway. Therefore, compounds having the same action as BIO on the Wnt / β-catenin pathway can also be used in the present invention.

Production of Cell (B) Thus, the production method of the present invention provides a cell (B) that can differentiate into a somatic cell (A) different from the somatic cell (C).

The somatic cell (A) from which the cell (B) can be differentiated is not particularly limited as long as it is different from the somatic cell (C).

For example, when cells obtained from dental pulp as somatic cells (C) or mesenchymal stem cells are used, the resulting cells (B) can be differentiated into osteoblasts, hematopoietic cells, and the like. Here, “hematopoietic cells” are hematopoietic stem cells and blood cells that can be differentiated from hematopoietic stem cells (specifically, leukocytes (neutrophils, eosinophils, basophils, lymphocytes, monocytes, macrophages, etc. ), Red blood cells, platelets, etc.).

It can be confirmed by those skilled in the art that the obtained cells (B) can differentiate into somatic cells (A). One example is shown in the examples described later. Moreover, it can also confirm by the manufacturing method of the hematopoietic tissue mentioned later.

In a preferred embodiment of the present invention, the cell (B) obtained by the method of the present invention is a stem cell. A stem cell has the ability to differentiate into another cell (differentiation ability) with self-replication ability. In the present specification, confirmation that a cell is a stem cell can also be confirmed by expression of a stem cell marker such as Oct3 / 4, Klf-4, etc., instead of confirming that it has self-renewal ability and differentiation ability. . In the case where the cell (B) is a stem cell, this point is also different from the somatic cell (C).

The present invention also includes a cell (B) obtained by the method for producing the cell.

The cells (B) obtained by the production method of the present invention can be used, for example, for the production of somatic cells (A) and tissues containing them. An example of the method for producing a hematopoietic tissue will be described in detail below.

2. Method for Producing Hematopoietic Tissue The present invention also provides the following method for producing hematopoietic tissue:
A step of culturing a somatic cell having no hematopoietic function in the presence of a compound represented by the following general formula (I).

[Wherein, R ¹ and X are the same as defined above. ]

In a preferred embodiment of the method for producing a hematopoietic tissue of the present invention,
(1) a step of culturing a somatic cell having no hematopoietic function in the presence of the compound, and (2) a step of transplanting the cultured cell together with a carrier into an animal.

In this specification, “hematopoietic tissue” mainly refers to a tissue having a function of hematopoiesis, that is, a function capable of generating various blood cells. It is preferably a tissue containing hematopoietic stem cells or stem cells having a similar function. A typical example of a hematopoietic tissue in vivo is bone marrow, and the hematopoietic tissue obtained by the production method of the present invention preferably has a function similar to that of bone marrow at least in terms of hematopoietic function. Therefore, the resulting hematopoietic tissue can be rephrased as “bone marrow” or “myeloid tissue”.

Culturing The culturing step in the method for producing a hematopoietic tissue of the present invention can be performed in the same manner as the culturing step described in the above “1.” column, except that somatic cells having no hematopoietic function are used.

Somatic cells having no hematopoietic function In the culturing step in the method for producing a hematopoietic tissue of the present invention, somatic cells having no hematopoietic function are cultured.

Somatic cells that do not have a hematopoietic function include blood cells (specifically, leukocytes (neutrophils, eosinophils, basophils, lymphocytes, monocytes, macrophages, etc.), erythrocytes, platelets, etc. If it is a somatic cell except the cell which produces | generates), it will not specifically limit. Also, blood cells are not applicable.

The origin of somatic cells having no hematopoietic function is not particularly limited, but is preferably derived from mammals such as humans, monkeys, mice, rats, dogs, hamsters, rabbits, cats and cows. In the case where the resulting hematopoietic tissue is intended for treatment of a disease, it is preferably derived from an individual in need of treatment, but is not limited thereto.

Somatic cells that do not have hematopoietic functions include cells collected from living organisms, cells cultured from cells collected from living organisms (especially primary cultured cells), and subculture using cells collected from living organisms. It may be any of the cells that have been turned into cells.

The type of somatic cell that does not have a hematopoietic function is not particularly limited.

In one embodiment of the present invention, the somatic cell having no hematopoietic function is a cell obtained from the dental pulp. Cells that can be obtained from dental pulp include dental pulp cells and dental pulp stem cells. The cells obtained from the dental pulp include cells obtained by culturing cells obtained from the dental pulp (in particular, primary cultured cells) and cells obtained by continuing subculture using cells collected from the dental pulp. As the established cell line, those described in Non-Patent Document 1 can be used. The dental pulp may be derived from any tooth such as an incisor, a canine or a molar.

In another embodiment of the present invention, the somatic cell having no hematopoietic function is a mesenchymal stem cell. Of these, bone marrow-derived mesenchymal stem cells that are easy to prepare are preferred examples. Mesenchymal stem cells can be prepared according to a known technique. For example, bone marrow-derived mesenchymal stem cells can be collected by centrifuging a bone marrow fluid obtained by bone marrow puncture.

Transplantation A preferred embodiment of the method for producing a hematopoietic tissue of the present invention includes a step of transplanting cultured cells into an animal together with a carrier.

The animal to be transplanted is not particularly limited. For example, preferred examples include mammals such as humans, monkeys, mice, rats, dogs, hamsters, rabbits, cats, and cows. However, it is desirable to comply with laws and regulations, ethics, informed consent, etc. when selecting transplant targets. Moreover, the target animal can also be made into a non-human animal as needed.

The animal to be transplanted may be the same or different from an animal from which a somatic cell having no hematopoietic function is derived. In the case of the same species, the animal to be transplanted may be the same or different from the individual from which the somatic cell having no hematopoietic function is derived.

The cell transplant site is not particularly limited. It is preferably a non-humoral animal part capable of supporting the hematopoietic tissue produced. For example, subcutaneous, intramuscular, bone cavity and the like. Although the technique to transplant is not specifically limited, Since transplantation by injection is easy, it is mentioned as a preferable example.

It is preferable to remove the compound represented by the general formula (I) (for example, BIO) by means such as washing before transplantation.

Carrier In the transplantation step, the cultured cells are transplanted together with the carrier into an animal. The carrier is used to support the cells that are induced into the hematopoietic tissue and to prevent diffusion or absorption by surrounding cells.

The carrier is not particularly limited as long as it can achieve the above purpose. Preferred examples of the carrier include hydroxyapatite (HA), calcium triphosphate (β-TCP), calcium octaphosphate (OPC) and the like.

There are no particular limitations on the mixing ratio of the cells and the carrier at the time of transplantation. For example, for 1.0 × 10 ⁷ cells, the carrier can be about 10 to 200 mg, about 20 to 80 mg, and about 40 mg.

After the transplantation, manage the transplanted animals for an appropriate period. If it is a non-human animal, it is preferable to manage it under normal breeding conditions. The period until the hematopoietic tissue is produced after transplantation can be appropriately set by those skilled in the art. For example, when the animal is a mouse, the period can be about 2 weeks to June, about January to March, or about February.

Production of hematopoietic tissue Thus, a hematopoietic tissue is produced.

Production of hematopoietic tissue can be confirmed by evaluating that it has a function of generating blood cells. The generation of blood cells can be confirmed by a method known to those skilled in the art, and an example thereof is shown in the examples described later.

The present invention also includes a hematopoietic tissue obtained by the method for producing the hematopoietic tissue.

The hematopoietic tissue obtained by the production method of the present invention can be used for the treatment of diseases based on abnormalities of hematopoietic tissue such as leukemia. Even if hematopoietic tissue is produced and treated in the body of a diseased animal (such as a patient), the hematopoietic tissue produced in the body of an animal other than the patient and transplanted to the animal having the disease Can also be treated.

3. Cell Inducing Agent The present invention also provides a cell inducing agent that can be used in the above-described cell production method and hematopoietic tissue production method. The cell inducer of the present invention uses somatic cells (eg, somatic cells (C) described in the column “1.”) as raw materials, and cells that can differentiate into somatic cells different from the somatic cells of the raw materials (“ The cell (B) described in the column “1.” is exemplified.)

As the active ingredient of the cell inducer, the compounds described in the column “1.” are used. As a compound, the above-mentioned BIO is mentioned as a preferred example. Accordingly, the present invention also provides the use of the compound for inducing cells.

The cell inducer may contain a normal solvent, a base material, and the like as long as the effect of the above compound is not inhibited depending on the form to be provided.

4). Hematopoietic tissue preparation kit The present invention also provides
There is also provided a kit for preparing a hematopoietic tissue, comprising (1) a compound, and (2) a carrier.

As the compound and carrier contained in the kit of the present invention, those described in the above “2.” column are used. As a compound, the above-mentioned BIO is mentioned as a preferred example.

Moreover, the kit of the present invention can contain other components as required. Examples of other components include, but are not limited to, a tool for transplanting cells (for example, a syringe), a positive control sample, a negative control sample, and the like. It can also include a document in which a procedure for performing the above method for producing a hematopoietic tissue is written.

The kit of the present invention can be suitably used in the method for producing a hematopoietic tissue described in the above “2.” column.

5). Cell Inducing Agent Screening Method The present invention also provides a cell inducing agent screening method comprising the following steps:
(1) a step of culturing somatic cells in the presence of a test substance, and evaluating the cell induction ability of the test substance; and (2) the cell induction ability of the test substance evaluated in the above step, and the following general formula (I): A step of comparing the cell inducibility of a compound represented by (for example, BIO).

[Wherein, R ¹ and X are the same as defined above. ]

Cell Inducing Agent Screening Method The cell inducing agent selected by the screening method of the present invention is a somatic cell (the somatic cell (C) described in the column “1.”) is used as a raw material. It is a cell inducer capable of inducing cells that can differentiate into somatic cells different from cells (the cells (B) described in the column “1.” are exemplified).

Process (1)
In the first step of the screening method of the present invention, somatic cells are cultured in the presence of a test substance, and the cell induction ability of the test substance is evaluated.

In this step, somatic cells are first cultured in the presence of a test substance. Somatic cell culture can be performed according to the culture process described in the column “1.” above.

There are no particular limitations on the type of somatic cells .

The origin of somatic cells is not particularly limited, but is preferably derived from mammals such as humans, monkeys, mice, rats, dogs, hamsters, rabbits, cats and cows.

Somatic cells are cells collected from living organisms, cells cultured from cells collected from living organisms (especially primary cultured cells), and cells established by continuing subculture using cells collected from living organisms. There may be.

In one embodiment of the screening method of the present invention, the somatic cell is a cell derived from the dental pulp. The cells derived from dental pulp include dental pulp cells and dental pulp stem cells. The cells derived from the dental pulp include cells obtained by culturing cells collected from the dental pulp (in particular, primary cultured cells) and cells established by continuing subculture using cells collected from the dental pulp. As the established cell line, those described in Non-Patent Document 1 can be used.

In another embodiment of the present invention, the somatic cell is a mesenchymal stem cell. The origin of mesenchymal stem cells is not particularly limited, and those derived from bone marrow, adipose tissue, and the like can be used. Of these, bone marrow-derived mesenchymal stem cells that are easy to prepare are preferred examples. Mesenchymal stem cells can be prepared according to a known technique. For example, bone marrow-derived mesenchymal stem cells can be collected by centrifuging a bone marrow fluid obtained by bone marrow puncture.

Test substance The test substance is not particularly limited, and any compound can be used. In a preferred embodiment, the compound is confirmed to be safe for living organisms.

Evaluation of cell-inducing ability Next, the cell-inducing ability of the test substance is evaluated based on the cells obtained by culturing.

“Cell-inducing ability” means an action that allows the test substance to induce somatic cells of a raw material into cells that can differentiate into somatic cells different from the somatic cells of the raw material. Therefore, evaluation of the cell inducing ability of the test substance evaluates the degree of such action.

The method for evaluating the “cell inducing ability” can be appropriately set by those skilled in the art.

As an example, when somatic cells are derived from dental pulp, as described in Examples below, the morphology of cells obtained by culture, acquisition of the properties of stem cells (acquisition of self-replication ability and differentiation ability; acquisition of stem cell markers) And the like can be evaluated. Further, as the differentiation ability, the acquisition of differentiation ability to osteoblasts by von Kassa staining exemplified in the examples, and the acquisition of differentiation ability to hematopoietic tissues exemplified in the examples should be used as indices. You can also.

As another example, when the somatic cell is a mesenchymal stem cell, the acquisition of differentiation ability into a hematopoietic tissue exemplified in the examples can be used as an index.

Process (2)
In the second step of the screening method of the present invention, the cell inducing ability of the test substance evaluated in the above step is compared with the cell inducing ability of the compound (for example, BIO).

Cell induction ability of the compound The cell induction ability of the compound (eg, BIO) means the cell induction ability when the somatic cells used in step (1) are cultured in the presence of the compound (eg, BIO). . The cell inducing ability of the compound (for example, BIO) is preferably performed in the same manner as the evaluation performed in step (1) in order to appropriately compare.

Selection of cell inducer The cell inducer is selected based on the result of the comparison in step (2).

Specifically, a test substance that meets a preset standard is selected as a cell inducer. The preset standard can be set to the same level or higher than the cell inducing ability of the above compound (for example, BIO), for example. Even when the cell inducing ability of the test substance is equal to or less than the cell inducing ability of the compound (for example, BIO), selection of the test substance as a cell inducer is not prevented. In this case, for example, when the test substance has an advantageous effect compared with the compound (for example, BIO) from the viewpoint of safety and economy, it can be considered.

Thus, the cell inducer is screened. The cell inducer selected by screening can be suitably used in place of BIO in the method for producing cells in the column “1.” and the method for producing hematopoietic tissue described in the column “2.”.

6). Use The present invention is also for producing a cell (B) that can differentiate into a somatic cell (A), and for producing a cell (B) from a somatic cell (C) different from the somatic cell (A). Of the above-mentioned general formula (I) (for example, BIO) is also provided.

[Wherein, R ¹ and X are the same as defined above. ]

Also provided is the use of the above compound for producing hematopoietic tissue.

Specific examples of usage include the method for producing cells in the column “1.” and the method for producing hematopoietic tissues described in the column “2.”.

Hereinafter, examples are given to describe the present invention in more detail, but the present invention is not limited thereto.

Reference Example 1: Observation of morphology of mouse deciduous tooth-derived dental pulp stem cell The morphology of mouse deciduous tooth-derived dental pulp cell (mDP) prepared in Non-Patent Document 1 was observed.

<Method and results>
The mouse deciduous dental pulp stem cell (mDP) cell line prepared in Non-Patent Document 1 was stained with two colors of dyes, Hoechst 33342 (Hoechst Blue, blue) and Hoechst33258 (Hoechst Red, red), according to a conventional method. By the flow cytometry method, the cells that had been discharged of the dye and weakly stained in two colors were sorted to select dental pulp stem cells (mDP-SP). Stem cells are thought to have improved pigment discharge capacity.

The result of sorting is shown in Fig. 1A. Similar to the presence of 0.5 to 1.0% of dental pulp stem cells in the dental pulp, it was confirmed that cells having stem cell properties were also present in the mouse deciduous dental pulp cell (mDP) cell line.

Next, the morphology of the selected dental pulp stem cells (mDP-SP) and dental pulp cells (mDP-MP) was observed. Each cell was stained with Phalloidin-Alexa594 (actin staining, red) and DAPI (nuclear staining, blue) according to a conventional method, and observed with a fluorescence microscope.

The microscope image is shown in FIG. 1B. Furthermore, the quantitative determination results (Relative length (relative length), Relataive width (relative width), and elliptical length factor) are shown in FIG. 1C. “Elliptical form factor” is a value obtained by the formula Relative length / Relative breadth, and is an index indicating the degree of slenderness of the cell of interest.

Dental pulp stem cells have a spindle-shaped form. On the other hand, pulp cells have a flat form.

Example 1: Screening of drugs that induce dental pulp stem cell-like morphology Agents that can induce dental pulp stem cell-like morphology were screened by adding to dental pulp cells.

<Method and results>
Add 2 μM (final concentration) of each drug of FDA Approved Drug Screen-Well Library to DMEM / F12 medium (Life Technologies) supplemented with 10% FCS (fetal calf serum), and culture mDP cells for 2 days did. Thereafter, for the cells stained with actin with Phalloidin-Alexa594, the induction of dental pulp stem cell-like morphology was evaluated by microscopic observation. Each drug is provided as a solution in DMSO.

As a result, GSK-3 inhibitor IX and BIO were selected as drugs that can induce dental pulp stem cell-like morphology.

The results are shown in FIG. FIG. 2A shows the structure of GSK-3 inhibitor IX, BIO and the structure of MeBIO (control compound) that does not have similar activity. FIG. 2B shows the morphology of mDP cells cultured in the presence of BIO. FIG. 2C shows the quantitative results of the morphology.

The morphology of mDP cells cultured in the presence of BIO was similar to that of mDP-SP cells. Such an effect was not observed when cultured in the presence of control DMSO alone and in the presence of MeBIO. As described above, BIO was selected as a drug capable of inducing dental pulp stem cell-like morphology.

Example 2: Verification of involvement of GSK-3β and Wnt-β catenin pathway Since BIO is an inhibitor of GSK-3β belonging to the Wnt-β-catenin pathway, GSK-3β and Wnt-β- The involvement of the catenin pathway was verified.

<Method and results>
Calyculin-A, an inhibitor of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A-C), activates the Wnt signal and enhances phosphorylation of β-catenin It has been known. The upper part of FIG. 3A shows β-catenin phosphorus detected with an antibody against phosphorylated β-catenin in mDP cells cultured for 0, 30 or 60 minutes in the presence of 0 to 20 nM Calyculin-A. Indicates the degree of oxidation.

In the same manner as in Example 1, mDP cells cultured in the presence of BIO were cultured in the presence of 20 nM Calyculin-A after washing and medium exchange, and the degree of phosphorylation of β-catenin was detected. Controls were mDP cells cultured in the presence of BIO in the presence of DMSO.

The results are shown in the lower part of FIG. 3A and FIG. 3B. In mDP cells cultured in the presence of BIO, the enhanced phosphorylation of β-catenin observed in the control was not observed. This result indicates that the function of GSK-3β is suppressed by culturing in the presence of BIO.

Example 3 Evaluation of Expression of Stem Cell Marker Expression of stem cell marker was detected in cells cultured in the presence of BIO.

<Method and results>
In the same manner as in Example 1, the relative gene expression levels of stem cell markers Oct3 / 4 and Klf-4 in mDP cells cultured in the presence of BIO were measured by real-time RT-PCR.

In addition, using the primary cultured cells collected from the dental pulp of the mouse incisor and 1st molar (1st molar), similarly, in the presence of BIO, the relative expression level of Oct3 / 4 (Relative gene expression) was measured.

Results are shown in FIGS. 3C and D. In mDP cells cultured in the presence of BIO and cells derived from mouse incisors and first molars, expression of stem cell markers was increased. This indicates that culture as a stem cell was induced by culturing in the presence of BIO.

Example 4: Evaluation of differentiation ability into osteoblasts The differentiation ability of cells cultured in the presence of BIO into osteoblasts was evaluated.

<Method and results>
As in Example 1, mDP cells were cultured in the presence of BIO. Thereafter, washing and medium exchange were performed, followed by culturing for 7 days in an osteoinduction medium or an osteoinduction medium further supplemented with 100 ng / ml of BMP2 protein.

The bone induction medium is the literature: Nakamura T, Naruse M, Chiba Y, Komori T, Sasaki K, Iwamoto M, Fukumoto S: Novel Hedgehog Agonists Promote Osteoblast Differentiation in Mesenchymal S : 10.1002 / jcp.24823. [Epub ahead of print]

Next, the calcified product was stained by von Kossa staining, and the induction into osteoblasts was evaluated. von Kossa staining was performed according to Non-Patent Document 1. A group similar to that except that only DMSO was added to the addition medium or MeBIO was added to the medium was used as a control.

The results are shown in FIG. FIG. 4A shows an example of the result of von Kossa staining. FIG. 4B shows the quantitative results of the intensity of von Kossa staining (Intensitytensof von Kossatainingstaining).

In cells cultured in the presence of BIO, strong staining of von Kossa staining was observed, and it was revealed that the cells had acquired the ability to differentiate into osteoblasts.

Example 5: Production of bone marrow-like tissue from mouse bone marrow-derived mesenchymal stem cells Using mouse bone marrow-derived mesenchymal stem cells (mBM-MSC) treated with BIO, bone marrow-like tissue was produced in the mouse body.

<Method and results>
Mouse bone marrow-derived mesenchymal stem cells (mBM-MSC) were prepared according to the literature: Yamaza T et al. PLoS One. 2008 Jul 9; 3 (7): e2615. The obtained mBM-MSC was cultured in the presence of BIO for 12 days in the same manner as in Example 1. The medium was replaced with DMEM / F12 medium supplemented with 10% FCS (fetal calf serum), and αMEM medium supplemented with 20% FCS was used. Cells cultured without the addition of BIO served as controls.

After culturing, the resulting cells were removed and washed with 1xPBS buffer BIO, a mixture of 1.0x 10 ⁷ cells and hydroxyapatite (HA) (40mg), immunodeficient mice (NOD / ShiJic-scid / jcl mice ) Was implanted by subcutaneous injection.

After transplanted mice were reared normally for 8 weeks, the transplanted site was excised and stained with hematoxylin and eosin (HE staining) according to a conventional method.

Results are shown in FIGS. 5A and 5B. FIG. 5A shows the scheme of operation. FIG. 5B shows a microscopic image of the transplanted site observed (hematoxylin / eosin staining (HE staining)).

In mice transplanted with cells cultured without addition of control BIO (transplant without BIO), a bone-like hard tissue could be confirmed around HA. On the other hand, in mice transplanted with cells cultured in the presence of BIO (transplant with BIO), HA was completely absorbed, and formation of a large amount of bone marrow-like tissue was confirmed along with bone tissue.

Example 6: Production of bone marrow-like tissue from human deciduous dental pulp stem cells Using BIO-treated human deciduous dental pulp stem cells (SHED), bone marrow-like tissue was produced in the body of a mouse.

<Method and results>
The cells were treated with immunodeficient mice (NOD / MSD) in the same manner as in Example 5 except that stem cells from Human Exfoliated Deciduous teeth (SHED) were used instead of mouse bone marrow-derived mesenchymal stem cells (mBM-MSC). ShiJic-scid / jcl mice).

The results are shown in FIGS. 5C and D. FIG. 5C shows the scheme of operation. FIG. 5D shows a microscopic image of the transplanted site observed (hematoxylin and eosin staining (HE staining)).

Although HA was not completely absorbed, bone formation was promoted in mice transplanted with cells cultured in the presence of BIO (transplant with BIO) compared to the control group, and mouse bone marrow-derived mesenchymal stem cells The formation of bone marrow-like tissue was confirmed as in the case of using.

Example 7: Detection of peripheral blood cell formation In the mouse obtained in Example 6, the formation of human-derived peripheral blood cells was confirmed.

<Method and results>
In mice obtained according to the method of Example 6 (transplant with BIO), blood was collected from mice 8 weeks after transplantation. First, monocytes (high FSC, low SSC; R1 group) and granulocytes (high FSC, high SSC; R2 group) were gated on the FSC / SSC plot from blood components by flow cytometry. For each of the gated R1 group and R2 group, the expression of human CD11b antigen (hCD11b) was detected. Two independent cases were tested.

Samples derived from blood collected from humans (human blood cells), mice not transplanted (non-transplant), and mice transplanted with cells cultured without the addition of BIO (transplant® without BIO) were used as controls.

The results are shown in FIG. FIG. 6A shows an example of the results of gating by flow cytometry (in the case of human blood cells). FIG. 6B shows detection of human CD11b antigen (hCD11b) expression.

In human mice transplanted with cells cultured in the presence of BIO, positive cells of human CD11b antigen (hCD11b) could be detected specifically. This indicates that human functional blood cells were generated from the transplanted cells, and the transplanted cells formed hematopoietic tissue.

Example 8: External findings of transplanted mice The external findings of the mice obtained in Example 6 were observed.

<Result>
The result is shown in FIG. 6C.

In the mouse transplanted with human milk tooth-derived stem cells (SHED) cultured for 2 days in the presence of BIO, skin symptoms (inflammatory findings) were observed around the eyes and in the limbs. Thereafter, the mice die about 3 to 6 months after transplantation.

Although this is not a limitation of the present invention, it is considered that this result is that human blood cells are generated from the transplanted cells and cause graft-versus-host disease (GVHD). That is, it shows that human functional blood cells were generated from the transplanted cells, and the transplanted cells formed hematopoietic tissue.

<Discussion>
Although the mechanism of action by which the method for producing a hematopoietic tissue of the present invention can form a hematopoietic tissue is not clear enough, by culturing in the presence of BIO, (1) somatic cells have a function of hematopoietic stem cells or the like It is considered that the ability to differentiate into stem cells is acquired, and (2) induction into hematopoietic stem cells or stem cells having a function similar to that after transplantation is induced by stimulation from around the transplant site. However, such an action mechanism is only an estimation and does not restrict the present invention.

Claims (13)

1. A method for producing a cell (B) capable of differentiating into a somatic cell (A), comprising the following steps:
   A step of culturing a somatic cell (C) different from the somatic cell (A) in the presence of a compound represented by the following general formula (I).
   

   

   [Wherein R ¹ represents a hydrogen atom (H) or an acetyl group (CH ₃ (C═O) —). X represents a halogen atom. ]
2. The method according to claim 1, wherein the compound is a compound represented by the following chemical formula.
   

   
3. The method according to claim 1 or 2, wherein the somatic cells (C) are cells obtained from dental pulp or mesenchymal stem cells.
4. A method for producing a hematopoietic tissue comprising the following steps:
   A step of culturing a somatic cell having no hematopoietic function in the presence of a compound represented by the following chemical formula.
   

   
5. A method for producing a hematopoietic tissue comprising the following steps:
   (1) culturing a somatic cell having no hematopoietic function in the presence of a compound represented by the following chemical formula;
   

   

   And (2) a step of transplanting the cultured cells together with a carrier into an animal.
6. The method according to claim 4 or 5, wherein the somatic cells are cells obtained from dental pulp or mesenchymal stem cells.
7. A cell obtained by the method according to any one of claims 1 to 3.
8. A hematopoietic tissue obtained by the method according to any one of claims 4 to 6.
9. A cell inducer comprising a compound represented by the following chemical formula:
   

   
10. (1) a compound represented by the following chemical formula, and
    

    

    (2) A hematopoietic tissue preparation kit including a carrier.
11. A method for screening a cell inducer comprising the following steps:
    (1) a step of culturing somatic cells in the presence of a test substance, and evaluating the cell induction ability of the test substance, and (2) the cell induction ability of the test substance evaluated in the above step, and represented by the following chemical formula: Comparing the cell-inducing ability of the compound.
    

    
12. In order to produce a cell (B) that can differentiate into a somatic cell (A), the cell is represented by the following chemical formula for producing a cell (B) from a somatic cell (C) different from the somatic cell (A). Use of the compounds to be made.
    

    
13. Use of a compound represented by the following chemical formula for producing a hematopoietic tissue.
    

    

Images