JP2019146492A - Method for inducing differentiation of induced pluripotent stem cells - Google Patents

Abstract

To provide differentiation induction methods capable of efficiently inducing differentiation of induced pluripotent stem cells into endoderm cells.SOLUTION: Provided is a method for inducing differentiation of induced pluripotent stem cells, comprising coating at least the culture surface of a culture vessel in which at least the culture surface is composed of an alicyclic structure-containing polymer, with a coating agent with a vitronectin concentration of 0.50 to 1.20 μg/ml; culturing induced pluripotent stem cells in the culture vessel subjected to the coating; and adhering the induced pluripotent stem cells to the culture surface to induce differentiation into endoderm cells.SELECTED DRAWING: None

Description

  The present invention relates to a method for inducing differentiation of induced pluripotent stem cells (iPS cells), and particularly to a method for inducing differentiation from induced pluripotent stem cells to endoderm cells.

  In the induction of differentiation of induced pluripotent stem cells, there is a method of forming an embryoid body (EB) that is a three-dimensional cell cluster that can differentiate into all three germ layers by suspension culture of induced pluripotent stem cells. It is common.

International Publication No.2015 / 199117 International Publication No. 2017/115706 JP-A-2017-018137

Kazutoshi Takahashi et al., `` Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors '', 2006, Cell, Volume 126, Issue 4, p663-676 Kazutoshi Takahashi et al., `` Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors '', 2007, Cell, Volume 131, Issue 5, p861-872

  However, in this method, since the embryoid body can be differentiated into all germ layers (Non-Patent Documents 1 and 2), it is necessary to control the differentiation into the target germ layer.

  In Patent Documents 1 and 2, as a method for inducing differentiation without forming an embryoid body, differentiation induction is performed when the undifferentiated cells come into contact with the alicyclic structure-containing polymer molded product during differentiation induction culture of undifferentiated cells. It is described that it is promoted.

  By the way, in order to proliferate induced pluripotent stem cells while maintaining undifferentiated ability, a solution-like coating agent containing an extracellular matrix such as gelatin, fibronectin, vitronectin, laminin, or a partial sequence thereof is used as a culture container. It is common to allow the artificial pluripotent stem cells to easily adhere to the culture container by allowing them to stand for a certain period of time and physically adsorbing them to the inner surface of the culture container (for example, Patent Document 3).

  The present invention has been made in view of the above-described circumstances, and an object thereof is to provide a differentiation induction method capable of efficiently inducing differentiation of induced pluripotent stem cells into endoderm cells.

  Under such a conventional technique, the present inventors have conducted induction studies on differentiation of induced pluripotent stem cells in a culture vessel composed of an alicyclic structure-containing polymer to obtain endoderm cells with high efficiency. When the concentration of the vitronectin agent is within a certain range, artificial pluripotent stem cells can be attached to a culture vessel composed of a polymer containing an alicyclic structure, and as a result, high differentiation induction into endoderm cells is promoted The inventors have found that an effect can be obtained and have completed the present invention.

The present invention has been made on the basis of the above findings, and is intended to advantageously solve the above problems. One aspect of the present invention is that at least the culture surface is an alicyclic structure-containing polymer. A coating container having a vitronectin concentration of 0.50 to 1.20 μg / ml is applied to the configured culture container, and at least the culture surface is coated, and artificial pluripotency is applied in the culture container to which the coating is applied. It is a method for inducing differentiation of induced pluripotent stem cells, in which stem cells are cultured, and induced pluripotent stem cells are adhered to the culture surface to induce differentiation into endoderm cells.
Induction of differentiation of induced pluripotent stem cells into endoderm cells efficiently by using a coating agent having a vitronectin concentration of 0.50 to 1.20 μg / ml to a culture vessel composed of a polymer containing an alicyclic structure can do.

  Moreover, in the said aspect, it is preferable that the said alicyclic structure containing polymer does not have a polar group. This is because, when the alicyclic structure-containing polymer does not have a polar group, it is easy to adjust the adhesion of iPS cells by controlling the concentration of vitronectin.

  According to the present invention, induced pluripotent stem cells can be efficiently induced to differentiate into endoderm cells.

FIG. 1 shows that artificial pluripotent stem cells are placed on a culture vessel made of a ring-opening polymer hydride of norbornene monomer surface-treated with a coating agent having a vitronectin concentration of 0.15 to 5.00 μg / ml. It is a microscope observation image at the time of sowing. FIG. 2 shows that artificial pluripotent stem cells are placed on a culture vessel composed of a ring-opening polymer hydride of a norbornene monomer surface-treated with a coating agent having a vitronectin concentration of 0.50 to 5.00 μg / ml. The amount of transcription of the endoderm marker gene when seeded and induced to differentiate into endoderm cells with ACTIVINA A and BMP4 is quantified.

Hereinafter, embodiments of the present invention will be described in detail.
In the method of the present invention, when an induced pluripotent stem cell is differentiated into an endoderm cell via a mesendoderm cell, a coating agent containing a predetermined concentration of vitronectin is used to contact a cell or a medium (hereinafter referred to as “culture surface” Is characterized by using a culture vessel composed of an alicyclic structure-containing polymer surface-treated.
By performing this surface treatment, the induced pluripotent stem cells can be adhered to the culture vessel, and the effect of improving differentiation induction efficiency obtained by contact with the alicyclic structure-containing polymer can be obtained. According to the above method, since the induced pluripotent stem cells are not cultured in a suspended state, an embryoid body that can differentiate into any of the three germ layers is not formed.

  As used herein, an induced pluripotent stem cell refers to a cell that has artificially acquired differentiation pluripotency by causing a reprogramming factor to act on a somatic cell.

  In this specification, vitronectin is a glycoprotein consisting of 478 amino acid residues having adhesion activity to cells having a vitronectin receptor. When animal cells are cultured, anchorage-dependent cells are cultured on the culture surface (exclusively inside the container). It is the bottom surface, but is not limited to this.) It is used to adhere and extend to promote proliferation. The vitronectin may have a partial sequence deleted as long as it functions as a cell adhesion molecule. Specifically, recombinant vitronectin in which the signal domain consisting of 20 amino acids on the N-terminal side is deleted; recombinant vitronectin in which the C-terminal part (399th amino acid to 478th amino acid sequence part) is deleted; Recombinant vitronectin.

  A coating agent containing a predetermined concentration of vitronectin is usually prepared by dissolving the aforementioned vitronectin in a buffer solution having a pH buffering property equal to the osmotic pressure of a body fluid such as phosphate buffered saline (PBS). The concentration of vitronectin dissolved in the buffer is 0.50 to 1.20 μg / ml, preferably 0.50 to 1.00 μg / ml, more preferably 0.50 μg / ml to 0.83 μg / ml, particularly preferably 0.50 μg / ml to 0.70 μg / ml. If the concentration is too low, vitronectin will not be sufficiently coated on the culture surface and artificial pluripotent stem cells will not adhere. On the other hand, if it is too high, sufficient differentiation induction promoting effect will not be obtained, and the coating operability will be poor. Or

The culture surface is coated with vitronectin by surface-treating the culture surface of the culture vessel made of the alicyclic structure-containing polymer using the coating agent.
The surface treatment method of the culture surface is the same as the method of coating a general cell substrate on a culture container. Usually, the above-mentioned coating agent is put in the culture container and the temperature is approximately 10 minutes to 5 hours at a temperature near the culture temperature. Preferably, a method of removing the coating agent after allowing it to stand for 30 minutes to 2 hours and bringing the coating agent into contact with the culture surface is employed. If the contact time is too short, the vitronectin coat will be insufficient. On the other hand, the protein adsorptivity to the surface of a molded body made of an alicyclic structure-containing polymer is low, and multilayer adsorption is not performed as in polystyrene, so that even if the contact time is increased, the amount of adsorption does not increase. Therefore, it is not necessary to lengthen the contact time.

In addition, it is desirable to add a medium immediately after removing the coating agent in order to prevent drying of vitronectin.
The amount of the coating agent added to the culture vessel is 1. more than the amount of the coating agent added to a general polystyrene cell culture vessel because the surface of the culture vessel composed of the alicyclic structure-containing polymer easily repels the aqueous solution. It is desirable to add about 5 to 3 times more, specifically, 0.15 to 0.25 ml is preferably added to 1 cm ^{2 of the} bottom area of the culture vessel.

  In the present invention, in order to induce differentiation of induced pluripotent stem cells into endoderm cells, it is first necessary to induce differentiation into mesendoderm cells. Differentiation induction into mesendoderm cells may be performed using a known differentiation induction medium for mesendoderm cells and culture conditions suitable for the adopted differentiation induction medium. Thereafter, in order to induce differentiation into endoderm cells, the medium may be changed to a known differentiation induction medium for endoderm cells and cultured under culture conditions suitable for the adopted differentiation induction medium.

An additive can also be mix | blended with a liquid culture medium. Examples of additives include minerals, metals, and vitamin components.
These additives can be used alone or in combination of two or more.

  The culture vessel composed of the alicyclic structure-containing polymer used in the present invention is formed by molding the alicyclic structure-containing polymer into an arbitrary shape that can be used as a culture vessel. Of the culture containers, at least the culture surface in contact with the cells and the culture solution may be composed of an alicyclic structure-containing polymer. Alternatively, the entire culture vessel may be made of an alicyclic structure-containing polymer. For example, in the case of a bag or the like, it may be a laminate of films made of different polymer materials, and the innermost layer (bag inner surface) may be a layer formed of a film made of an alicyclic structure-containing polymer. Moreover, if it is a culture dish, an additive may be added as needed and an alicyclic structure containing polymer may be shape | molded, and the whole container can also be comprised with an alicyclic structure containing polymer.

  The method for forming the culture vessel can be arbitrarily selected according to the shape of the culture vessel. Specific examples of molding methods include injection molding, extrusion molding, cast molding, inflation molding, blow molding, vacuum molding, press molding, compression molding, rotational molding, calendar molding, rolling molding. Method, cutting molding method, spinning, and the like. These molding methods can be combined, or post-treatment such as stretching can be performed as necessary after molding.

  The alicyclic structure-containing polymer is a resin having an alicyclic structure in the main chain and / or side chain, and preferably contains an alicyclic structure in the main chain from the viewpoint of mechanical strength, heat resistance, etc. From the viewpoint of induction efficiency, those having no polar group are more preferable. Here, the polar group refers to a polar atomic group. Examples of the polar group include an amino group, a carboxyl group, a hydroxyl group, and an acid anhydride group.

  Examples of the alicyclic structure include a saturated cyclic hydrocarbon (cycloalkane) structure and an unsaturated cyclic hydrocarbon (cycloalkene) structure. From the viewpoint of mechanical strength and heat resistance, the cycloalkane structure and the cycloalkene structure are exemplified. A structure is preferable, and a structure having a cycloalkane structure is most preferable.

  The number of carbon atoms constituting the alicyclic structure is not particularly limited, but is usually 4 to 30, preferably 5 to 20, and more preferably 5 to 15. When the number of carbon atoms constituting the alicyclic structure is within this range, the mechanical strength, heat resistance, and moldability characteristics are highly balanced, which is preferable.

  The proportion of the repeating unit having an alicyclic structure in the alicyclic structure-containing polymer may be appropriately selected according to the purpose of use, but is usually 30% by weight or more, preferably 50% by weight or more, more preferably 70% by weight. % Or more. If the proportion of the repeating unit having an alicyclic structure in the alicyclic structure-containing polymer is excessively small, the heat resistance is inferior, which is not preferable. The remainder other than the repeating unit having an alicyclic structure in the alicyclic structure-containing polymer is not particularly limited and is appropriately selected according to the purpose of use.

  Specific examples of the alicyclic structure-containing polymer include (1) norbornene polymer, (2) monocyclic olefin polymer, (3) cyclic conjugated diene polymer, and (4) vinyl alicyclic carbonization. Examples thereof include hydrogen polymers and hydrides of (1) to (4). Among these, norbornene-based polymers and hydrides thereof are preferable from the viewpoints of heat resistance, mechanical strength, and the like.

(1) Norbornene-based polymer The norbornene-based polymer is obtained by polymerizing a norbornene-based monomer that is a monomer having a norbornene skeleton, and is obtained by ring-opening polymerization or by addition polymerization. Broadly divided into things.

  Examples of the ring-opening polymer obtained by ring-opening polymerization include ring-opening polymers of norbornene monomers, ring-opening polymers of norbornene monomers and other monomers capable of ring-opening copolymerization, and these A hydride etc. are mentioned. Examples of those obtained by addition polymerization include addition polymers of norbornene monomers and addition polymers of norbornene monomers and other monomers copolymerizable therewith. Among these, a ring-opening polymer hydride of a norbornene-based monomer is preferable from the viewpoint of heat resistance, mechanical strength, and the like.

Examples of norbornene monomers that can be used for the synthesis of norbornene polymers include bicyclo [2.2.1] hept-2-ene (commonly used norbornene) and 5-methyl-bicyclo [2.2.1] hepta. 2-ene, 5,5-dimethyl-bicyclo [2.2.1] hept-2-ene, 5-ethyl-bicyclo [2.2.1] hept-2-ene, 5-ethylidene-bicyclo [2 2.1] hept-2-ene, 5-vinyl-bicyclo [2.2.1] hept-2-ene, 5-propenylbicyclo [2.2.1] hept-2-ene, 5-methoxycarbonyl -Bicyclo [2.2.1] hept-2-ene, 5-cyanobicyclo [2.2.1] hept-2-ene, 5-methyl-5-methoxycarbonyl-bicyclo [2.2.1] hepta Bicyclic monomers such as 2-ene;
Tricyclo [4.3.0 ^1,6 . 1 ^2,5 ] deca-3,7-diene (common name dicyclopentadiene), 2-methyldicyclopentadiene, 2,3-dimethyldicyclopentadiene, 2,3-dihydroxydicyclopentadiene, etc. Mer;
Tetracyclo [4.4.0.1 ^2,5 . 1 ^7,10 ] -3-dodecene (tetracyclododecene), tetracyclo [4.4.0.1 ^2,5 . 1 ^7,10 ] -3-dodecene, 8-methyltetracyclo [4.4.0.1 ^2,5 . 1 ^7,10 ] -3-dodecene, 8-ethyltetracyclo [4.4.0.1 ^2,5 . 1 ^7,10 ] -3-dodecene, 8-ethylidenetetracyclo [4.4.0.1 ^2,5 . 1 ^7,10 ] -3-dodecene, 8,9-dimethyltetracyclo [4.4.0.1 ^2,5 . 1 ^7,10 ] -3-dodecene, 8-ethyl-9-methyltetracyclo [4.4.0.1 ^2,5 . 1 ^7,10 ] -3-dodecene, 8-ethylidene-9-methyltetracyclo [4.4.0.1 ^2,5 . 1 ^7,10 ] -3-dodecene, 8-methyl-8-carboxymethyltetracyclo [4.4.0.1 ^2,5 . 1 ^7,10 ] -3-dodecene,
7,8-benzotricyclo [4.3.0.1 ^2,5 ] dec-3-ene (common name methanotetrahydrofluorene: also called 1,4-methano-1,4,4a, 9a-tetrahydrofluorene) 1,4-methano-8-methyl-1,4,4a, 9a-tetrahydrofluorene, 1,4-methano-8-chloro-1,4,4a, 9a-tetrahydrofluorene, 1,4-methano-8 -Tetracyclic monomers such as bromo-1,4,4a, 9a-tetrahydrofluorene;

Other monomers capable of ring-opening copolymerization with norbornene monomers include cyclohexene, cycloheptene, cyclooctene, 1,4-cyclohexadiene, 1,5-cyclooctadiene, 1,5-cyclodecadiene, And monocyclic cycloolefin monomers such as 1,5,9-cyclododecatriene and 1,5,9,13-cyclohexadecatetraene.
These monomers may have one or more substituents. Examples of the substituent include an alkyl group, an alkylene group, an aryl group, a silyl group, an alkoxycarbonyl group, and an alkylidene group.

Other monomers capable of addition copolymerization with a norbornene monomer include α-olefin monomers having 2 to 20 carbon atoms such as ethylene, propylene, 1-butene, 1-pentene and 1-hexene; cyclobutene, cyclopentene, cyclohexene, cyclooctene, tetracyclo ^{[9.2.1.0 2,10.} Cycloolefin monomers such as 0 ^3,8 ] tetradeca-3,5,7,12-tetraene (also referred to as 3a, 5,6,7a-tetrahydro-4,7-methano-1H-indene); And non-conjugated diene monomers such as 4-hexadiene, 4-methyl-1,4-hexadiene, 5-methyl-1,4-hexadiene, and 1,7-octadiene.

Among these, as other monomers that can be addition copolymerized with norbornene monomers, α-olefin monomers are preferable, and ethylene is more preferable.
These monomers may have one or more substituents. Examples of the substituent include an alkyl group, an alkylene group, an aryl group, a silyl group, an alkoxycarbonyl group, and an alkylidene group.

A ring-opening polymer of a norbornene-based monomer, or a ring-opening polymer of a norbornene-based monomer and another monomer capable of ring-opening copolymerization with a monomer component is a known ring-opening polymerization. It can be obtained by polymerization in the presence of a catalyst. Examples of the ring-opening polymerization catalyst include a catalyst comprising a metal halide such as ruthenium or osmium, a nitrate or an acetylacetone compound, and a reducing agent, or a metal halide or acetylacetone such as titanium, zirconium, tungsten or molybdenum. A catalyst comprising a compound and an organoaluminum compound can be used.
The ring-opening polymer hydride of a norbornene-based monomer is usually obtained by adding a known hydrogenation catalyst containing a transition metal such as nickel or palladium to the polymerization solution of the above-described ring-opening polymer, and then adding a carbon-carbon unsaturated bond. Can be obtained by hydrogenation.

  An addition polymer of a norbornene-based monomer or an addition polymer of a norbornene-based monomer and another monomer copolymerizable therewith is a monomer component in the presence of a known addition polymerization catalyst. It can be obtained by polymerization. As the addition polymerization catalyst, for example, a catalyst composed of a titanium, zirconium or vanadium compound and an organoaluminum compound can be used.

(2) Monocyclic cyclic olefin polymer As the monocyclic olefin polymer, for example, an addition polymer of a monocyclic olefin monomer such as cyclohexene, cycloheptene, or cyclooctene can be used. it can.
(3) Cyclic conjugated diene polymer As the cyclic conjugated diene polymer, for example, a polymer obtained by subjecting a cyclic conjugated diene monomer such as cyclopentadiene or cyclohexadiene to 1,2- or 1,4-addition polymerization, and The hydride can be used.
(4) Vinyl alicyclic hydrocarbon polymer Examples of the vinyl alicyclic hydrocarbon polymer include polymers of vinyl alicyclic hydrocarbon monomers such as vinylcyclohexene and vinylcyclohexane, and hydrogenated products thereof; styrene; , Hydrides of aromatic ring portions of polymers of vinyl aromatic monomers such as α-methylstyrene; and the like. The vinyl alicyclic hydrocarbon polymer may be a copolymer with other monomers copolymerizable with these monomers.

  The molecular weight of the alicyclic structure-containing polymer is not particularly limited, but is usually a polyisoprene equivalent weight average molecular weight measured by gel permeation chromatography of a cyclohexane solution (or a toluene solution if the polymer does not dissolve). It is 5,000 or more, preferably 5,000 to 500,000, more preferably 8,000 to 200,000, and particularly preferably 10,000 to 100,000. When the weight average molecular weight is within this range, the mechanical strength and the moldability are highly balanced, which is preferable.

The glass transition temperature of the alicyclic structure-containing polymer may be appropriately selected depending on the purpose of use, but is usually 50 to 300 ° C, preferably 100 to 280 ° C, particularly preferably 100 to 250 ° C, and more preferably 100. ~ 200 ° C. When the glass transition temperature is within this range, heat resistance and molding processability are highly balanced and suitable.
In the present invention, the glass transition temperature of the alicyclic structure-containing polymer is measured based on JIS K7121.

The said alicyclic structure containing polymer can be used individually or in combination of 2 or more types, respectively.
In addition, for the alicyclic structure-containing polymer, a compounding agent usually used in thermoplastic resin materials, for example, a soft polymer, an antioxidant, an ultraviolet absorber, a light stabilizer, a near infrared absorber, a release agent. Additives such as colorants such as dyes and pigments, plasticizers, antistatic agents, fluorescent brighteners, and the like can be added in amounts that are usually employed.
In addition, the alicyclic structure-containing polymer may be mixed with another polymer other than the soft polymer (hereinafter simply referred to as “other polymer”). The amount of the other polymer mixed with the alicyclic structure-containing polymer is usually 200 parts by mass or less, preferably 150 parts by mass or less, more preferably 100 parts by mass with respect to 100 parts by mass of the alicyclic structure-containing polymer. It is as follows.
If the proportion of various compounding agents and other polymers to be blended with the alicyclic structure-containing polymer is too high, cells will not float easily, so all are blended within the range that does not impair the properties of the alicyclic structure-containing polymer. It is preferable.
The mixing method of the alicyclic structure-containing polymer and the compounding agent or other polymer is not particularly limited as long as the compounding agent is sufficiently dispersed in the polymer. Moreover, there is no special restriction | limiting in the order of a mixing | blending. As a blending method, for example, a method of kneading a resin in a molten state using a mixer, a uniaxial kneader, a biaxial kneader, a roll, a Brabender, an extruder, etc., after dissolving and dispersing in a suitable solvent, Examples thereof include a method of removing the solvent by a coagulation method, a casting method, or a direct drying method.
When a biaxial kneader is used, after kneading, it is usually extruded in a rod shape in a molten state, cut into an appropriate length with a strand cutter, and pelletized in many cases.

The molding method of the container composed of the alicyclic structure-containing polymer can be arbitrarily selected according to the desired shape of the culture container. Examples of molding methods include injection molding, extrusion molding, cast molding, inflation molding, blow molding, vacuum molding, press molding, compression molding, rotational molding, calendar molding, and rolling molding. , Cutting molding method, spinning and the like, and these molding methods can be combined, or post-treatment such as stretching can be carried out as necessary after molding.
Examples of the shape of the culture container include a dish, a plate, a microchannel chip, a bag, a tube, a scaffold, a cup, and a jar fermenter.

In the present invention, the culture container is preferably sterilized.
There are no particular restrictions on the method of sterilization, heating methods such as the high-pressure steam method and dry heat method; radiation methods that irradiate radiation such as γ rays and electron beams; irradiation methods that irradiate high frequencies; ethylene oxide gas (EOG) The method can be selected from methods generally employed in the medical field, such as a gas method in which a gas is brought into contact; a filtration method using a sterilizing filter; Among these, the gas method is preferable because the change in the polar state of the surface is small.

In addition, the inner surface of the culture vessel including the culture surface can be subjected to treatments other than the sterilization purpose generally applied to the culture vessel such as plasma treatment, corona discharge treatment, ozone treatment, and ultraviolet irradiation treatment. However, these inner surface treatments are possible because the costs generated by performing these surface treatment operations can be reduced, and the cleanliness may be impaired by partial decomposition of the inner surface of the culture vessel accompanying the surface treatment. Weak surface with water contact angle of ± 20%, preferably ± 10% of the bottom surface of the container when not used or with respect to the water contact angle of the bottom surface of the container (the side in contact with the culture solution) before the inner surface treatment It is preferred that it has only been treated. Here, the water contact angle is determined by using a fully automatic contact angle meter (“LCD-400S” manufactured by Kyowa Interface Science Co., Ltd.), cutting the bottom of the dish with a Φ30 mm circle cutter, and the center of the sample. Using a total of 5 points of 4 apexes of a 20 mm square as the measurement points, the radius r and height h of the droplet are obtained, and tan θ1 = h / r, θ = 2θ ₁ → θ = 2 arctan (h / r) The required θ (θ / 2 method).

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated concretely, this invention is not limited to these Examples.

Example I
As an alicyclic structure-containing polymer, using ZEONOR (registered trademark) 1060R (manufactured by Nippon Zeon Co., Ltd., a ring-opening polymer hydride of a norbornene monomer; hereinafter simply referred to as “1060R”), an injection molding method is used. A 6-well plate having 6 wells with a diameter of 35 mm was obtained, and then sterilized with ethylene oxide. Hereinafter, this culture container is referred to as “1060R plate”.
As a culture vessel, 2 ml of a coating agent obtained by dissolving vitronectin (Life Technologies A147000) dissolved in a phosphate buffer solution (PBS) so as to have the concentration shown in Table 1 was added to a 1060R plate as a culture container. After incubation for a period of time, the coating agent was removed from the container to obtain a vitronectin-coated plate.
Usually, when vitronectin is used as a coating agent, it is recommended to use 5.0 μg / ml. Therefore, a dilution column was prepared with the concentration as a reference value (1 time), and each was used as a coating agent.
In each well of the obtained plate, 3% serum replacement KnockOut (registered trademark) Serum Replacement (hereinafter referred to as “KSR”; Thermofisher Scientific, Inc.), ACTIVIN A (R & D, final concentration: 15 ug / mL) and BMP4 (manufactured by R & D, final concentration: 3.75 ng / mL) are added to a basal medium (manufactured by Kojin Bio) to produce a synthetic medium (hereinafter referred to as “iPS differentiation-inducing medium”), manufactured by 1060R Placed in each well of the plate. Next, after seeding the artificial pluripotent stem cells at 3.3 × 10 ⁵ cells / well in each well of the 1060R plate, a ROCK inhibitor (manufactured by Wako, final concentration: 10 μM) was added, and 5% CO 2 was added. _The cells were cultured at 37 ° C. for 24 hours under _two atmospheres. Thereafter, the medium was removed, and the iPS differentiation-inducing culture medium was added again, and the culture was continued for 2 days under the same conditions. Medium change was performed every 24 hours. The microscope observation image of the cell in culture is shown in FIG.
When the vitronectin concentration of the coating agent was 0.60 μg / ml, a slight decrease in the number of adherent cells was confirmed, but when the vitronectin concentration was 0.60 μg / ml or more, there was no problem in providing a differentiation induction test. Sufficient adhesion was maintained. Even when the vitronectin concentration is lowered to 0.50 μg / ml, the cell adhesion state is maintained. On the other hand, at vitronectin concentrations: 0.30 μg / ml and 0.15 μg / ml, cells did not maintain sufficient adhesion, and some floating cells (rounded portions) were also observed. It was difficult to do.
Further, from these cell samples, the amount of transcription of the CER1 gene, which is a mesendoderm marker, was quantified by a real-time PCR method (internal standard gene is GAPDH gene), and a plate coated with a vitronectin concentration of 0.60 μg / ml or more. In all the cell samples cultured in the above, the above-mentioned mesendoderm marker was expressed.
In addition, when the same cell culture was performed using a 6-well plate made of polystyrene, there was no difference in the number of adherent cells compared to the 1060R-made plate, and the cell adhesion was sufficiently maintained in the 1060R-made plate. It was confirmed.

Example II
The induced pluripotent stem cells were cultured in the same manner as in Example I except that the concentration of the coating agent used in the 1060R plate was changed to the amount shown in Table 2.
Thereafter, the medium was replaced with an endoderm cell differentiation medium from which only BMP4 was removed from the above-mentioned iPS differentiation induction medium, and further cultured at 37 ° C. in a 5% CO ₂ atmosphere to induce differentiation into endoderm cells.
Next, the amount of transcription of the SOX17 gene, which is an endoderm marker, was quantified by a real-time PCR method (internal standard gene is GAPDH gene).
As a comparative control, a vessel surface-treated with a coating agent was prepared in the same manner as described above except that the culture vessel was changed to a commercially available polystyrene 6-well plate (manufactured by FALCON). After performing differentiation induction operation and confirming the expression of the mesendoderm marker of cultured cells, the expression of the endoderm marker was quantified.
Each plate when a coating agent having a vitronectin concentration of 5.00 μg / ml was applied to a polystyrene 6-well plate in the same manner as in Example I and the amount of endoderm marker quantified in the same manner as in Example I was 1. FIG. 2 shows the relative amount of the endoderm marker amount. In FIG. 2, “TCPS” means a polystyrene 6-well plate, and “COP” means a 1060R plate.
As shown in FIG. 2, the amount of endoderm marker per cell increased at a vitronectin concentration of 1.20 μg / ml, a vitronectin concentration of 0.80 μg / ml, and a vitronectin concentration of 0.50 μg / ml.

  From the results of FIGS. 1 and 2, it is shown that, when a coating agent having a vitronectin concentration in the range of 0.50 to 1.20 μg / ml is used, the induction of endoderm differentiation is promoted while maintaining the cell adhesion ability. It was done.

  According to the induced differentiation method of induced pluripotent stem cells of the present invention, induced induced pluripotent stem cells can be efficiently induced to differentiate into endoderm cells.

Claims (2)

At least the culture surface is coated using a coating agent having a vitronectin concentration of 0.50 to 1.20 μg / ml in a culture vessel in which at least the culture surface is composed of an alicyclic structure-containing polymer,
Inducing differentiation of induced pluripotent stem cells by culturing induced pluripotent stem cells in the culture vessel coated with the coating, and inducing differentiation into endoderm cells by attaching the induced pluripotent stem cells to the culture surface Method.   The method for inducing differentiation of induced pluripotent stem cells according to claim 1, wherein the alicyclic structure-containing polymer does not have a polar group.