JP6501413B2 - Composition for cell culture - Google Patents

Description

  The present invention relates to a composition for cell culture, in particular, a composition suitable for culturing pluripotent stem cells, a medium containing the composition, and a method for culturing pluripotent stem cells.

  Pluripotent stem cells are stem cells having the ability to differentiate into cells of any tissue (pluripotency) and the ability to proliferate almost infinitely, for example, embryonic stem cells (ES cells), artificial pluripotency These include sexual stem cells (iPS cells), embryonic germ cells (EG cells), pluripotent germ stem cells (mGS cells), and fusion cells of human ES cells and somatic cells. Since pluripotent stem cells can be induced to various tissue cells, their application to various fields such as regenerative medicine, materials thereof and drug screening is expected. In fact, administration of tissue cells obtained by differentiating pluripotent stem cells to humans has also been carried out (see Non-Patent Document 1).

  When pluripotent stem cells are used for producing research materials or regenerative medicine products, it is essential to culture and maintain the pluripotency of the cells. On the other hand, it is known that pluripotent stem cells such as ES cells and iPS cells may lose pluripotency, which is one of the properties of these cells, by passaging.

  Therefore, in order to culture and maintain pluripotent stem cells while maintaining pluripotency, culture is performed using serum or fibroblasts derived from a mouse as a feeder cell layer called feeder cells (non- Patent Documents 2 and 3). However, preparation of feeder cells is complicated, and preparation of such feeder cells has a problem that reproducibility is poor because the ability to maintain undifferentiated cells differs from lot to lot. In addition, in order to apply pluripotent stem cells to regenerative medicine, a culture environment that satisfies the Xeno-Free condition that excludes xenogeneic components from the culture system is also required, but using mouse-derived feeder cells There is an obstacle in clinical application because proteins of different origin may be mixed.

  For this reason, a feeder-free (feederless) culture method without using feeder cells has been proposed. For example, a feederless medium such as ReproFF (feederless medium for primate ES / iPS cells, manufactured by Reprocel) or mTeSR (registered trademark) (serum free medium for human ES / iPS cell maintenance, manufactured by Stemcell Technology) is commercially available. There is. Also, a feederless culture technique using a culture substrate coated with an E8 fragment of human laminin α5β1γ1 or an E8 fragment of human laminin α3β3γ2 is disclosed (see Patent Document 1).

  As described above, to maintain and culture pluripotent stem cells under feeder-free conditions, usually, basic fibroblast growth factor (bFGF) or transforming growth factor-β (also referred to as transforming growth factor-β) It is necessary to culture by adding (TGF-β) (see Patent Documents 2 and 3 and Non-patent Documents 4 and 5).

However, since the proteins bFGF and TGF-β, which are proteins, are expensive, there is a problem that the cost of the culture solution becomes high, and when a large amount of pluripotent stem cells are required for regenerative medicine etc. Has become an obstacle. Furthermore, bFGF and TGF-β may differ in quality between lots, and there is also a problem that it is difficult to culture and supply stable human pluripotent stem cells.
Therefore, for practical use of pluripotent stem cells, there is a need for a medium or method capable of culturing and maintaining the cells easily and stably while maintaining the undifferentiated state of pluripotent stem cells.

International Publication No. 2011/043405 International Publication No. 1999/020741 WO 2004/055155

Schwartz, S. D. et al, Lancet 379: 713-720, 2012 Takahashi, K. et al, Cell 131: 861-872, 2007. Thomson, J.A. A. et al, Science 282: 1145-1147, 1998 Chen G, et al, Nat Methods. 8: 424-429, 2011 Nakagawa M, et al. Sci Rep. 4: 3594, 2014

  An object of the present invention is to provide a composition that can be used for cell culture and the like, in particular, a new medium capable of maintaining and culturing pluripotent stem cells while maintaining an undifferentiated state.

  As a result of intensive studies, the present inventors have found that a compound represented by the following general formula (A) (hereinafter referred to as “compound A”) is a medium for maintaining and culturing pluripotent stem cells:

(In the formula,
R ¹ represents a hydrogen atom or an alkyl having 1 to 5 carbon atoms,
R ² represents a hydrogen atom or a hydroxyl group,
R ³ represents a hydrogen atom or a hydroxyl group,
R ⁴ and R ⁵ are the same or different and each represents a hydrogen atom or an alkyl having 1 to 5 carbon atoms,
R ⁶ represents a hydrogen atom or an alkyl having 1 to 15 carbon atoms,
R ⁷ represents a hydrogen atom or —O—CO—R ⁹ (wherein, R ⁹ represents a hydrogen atom, an alkyl having 1 to 5 carbon atoms, or an alkenyl having 2 to 5 carbon atoms),
R ⁸ represents a hydrogen atom or an alkyl having 1 to 5 carbon atoms,
-X-Y- ^is, 10 = CH- ^{(wherein, R 10} represents a hydrogen atom or a hydroxyalkyl of 1 to 5 carbon atoms) -CR, or ^-CR 11 ^R 12 -CHR ¹³ - (wherein, R ¹¹ represents a hydrogen atom or a hydroxyalkyl having 1 to 5 carbon atoms, and R ¹² and R ¹³ together represent an epoxy group with the adjacent carbon atom)),
A compound represented by the following structural formula (1) (hereinafter referred to as “compound 1”):

,
A compound represented by the following structural formula (2) (hereinafter referred to as “compound 2”):

And a compound represented by the following structural formula (3) (hereinafter referred to as “compound 3”):

The inventors have found that the above problems can be solved by adding at least one compound selected from the group consisting of or a salt thereof, and completed the present invention.

Examples of the present invention include the following.
[1] A composition for cell culture, which comprises at least one compound selected from the group consisting of Compound A, Compound 1, Compound 2 and Compound 3 or a salt thereof.
[2] A pluripotent stem cell maintenance culture medium comprising the composition for cell culture according to the above [1].
[3] A method for cultivating pluripotent stem cells, which is a method for culturing pluripotent stem cells while maintaining an undifferentiated state of pluripotent stem cells, said culture comprising compound A, compound 1, compound 2, And a culture method characterized in that it is carried out in the presence of at least one compound selected from the group consisting of compound 3 or a salt thereof.
[4] At least one member selected from the group consisting of Compound A, Compound 1, Compound 2 and Compound 3 for proliferating pluripotent stem cells while maintaining the undifferentiated state of pluripotent stem cells Use of a compound or a salt thereof.
[5] A method for producing iPS cells from somatic cells, wherein at least one compound selected from the group consisting of Compound A, Compound 1, Compound 2 and Compound 3 is a somatic cell into which a reprogramming factor has been introduced. Or culturing in the presence of a salt thereof.

  According to the present invention, pluripotent stem cells such as iPS cells are stably and efficiently cultured in an undifferentiated state under zeno-free conditions without using feeder cells and containing no animal-derived components. be able to. Therefore, according to the present invention, pluripotent stem cells such as iPS cells can be stably proliferated or established while maintaining their undifferentiated state under feeder-free and xeno-free conditions. Furthermore, according to the present invention, maintenance culture of pluripotent stem cells can be performed under feeder-free and xeno-free conditions using low molecular weight compounds instead of bFGF and TGF-β. Therefore, maintenance culture of pluripotent stem cells can be performed more simply and stably. Furthermore, the cost for maintenance culture of pluripotent stem cells can be reduced.

IPS cells cultured in the presence of TGFβ1 and bFGF (positive control) or in the presence of TGFβ1 (control) were stained with Hoechst 33342 (upper row) and immunostained with an anti-Oct3 / 4 antibody. (Lower) is shown. In the figure, "bFGF +" is a positive control and "bFGF-" is a control. The fluorescence microscope image (upper stage) which stained the iPS cell of 1 week after culture | cultivating using each test compound with Hoechst33342 (upper stage) and the fluorescence microscope image (lower stage) which used the anti-Oct3 / 4 antibody are shown. The fluorescence microscope image (upper stage) which stained the iPS cell of 1 week after culture | cultivating using each test compound with Hoechst33342 (upper stage) and the fluorescence microscope image (lower stage) which used the anti-Oct3 / 4 antibody are shown. The fluorescence microscope image (upper stage) which stained the iPS cell of 1 week after culture | cultivating using each test compound with Hoechst33342 (upper stage) and the fluorescence microscope image (lower stage) which used the anti-Oct3 / 4 antibody are shown. A in FIG. 5 shows fluorescence microscopic images of 1 week after culture of 100 nM or 10 nM of Compound 1 with Hoechst 33342 stained with Hoechst 33342 (upper) and immunostained with anti-Oct 3/4 antibody Bottom row). In the figure, TGFβ1 + indicates a positive control, and TGFβ1- indicates a control. FIG. 5B shows fluorescence microscope images of 1 week after culture of Compound 2 with 1 μM, 100 nM or 10 nM, stained with Hoechst 33342 (upper) and immunostained with anti-Oct3 / 4 antibody. The image (bottom) is shown. The fluorescence microscope image (upper stage) which stained the iPS cell of 1 week after culture | cultivating using each test compound with Hoechst33342 (upper stage) and the fluorescence microscope image (lower stage) which used the anti-Oct3 / 4 antibody are shown. In the figure, "bFGF + TGFβ1 +" indicates a positive control, and "bFGF-TGFβ1-" indicates a control.

Hereinafter, the present invention will be described in detail.
(I) About the compound concerning this invention The meaning of each term in the compound A is as follows.
Examples of “alkyl” relating to R ¹ , R ⁴ , R ⁵ and R ⁸ include, for example, linear or branched alkyl having 1 to 5 carbon atoms, and specific examples thereof include Methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, 1-methylbutyl, 2-methylbutyl, n-pentyl, isopentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, There can be mentioned 2,2-dimethylpropyl and 1-ethylpropyl.
R ¹ , R ⁴ , R ⁵ , and R ⁸ are preferably alkyl having 1 to 5 carbon atoms, more preferably alkyl having 1 to 3 carbon atoms, and still more preferably methyl.

As the “alkyl” for R ⁶ , for example, linear or branched alkyl having 1 to 15 carbon atoms can be mentioned, and preferably linear or branched carbon having 1 to 12 carbon atoms Alkyl. Specifically, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1-methylbutyl, 2-methylbutyl, 1,1-dimethylbutyl, 1 , 2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, n-heptyl, n -Octyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, n-tridecyl, n-tetrade It can be exemplified Le, the n- pentadecyl.

Examples of the "alkyl" for R ⁹ include linear or branched alkyl having 1 to 5 carbon atoms. Specifically, for example, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, 1-methylbutyl, 2-methylbutyl, n-pentyl, isopentyl, 1,1-dimethylpropyl, 1 And 2-dimethylpropyl, 2,2-dimethylpropyl and 1-ethylpropyl can be mentioned. Among them, methyl, ethyl, propyl and isopropyl are preferable.

As the “alkenyl” for R ⁹ , for example, linear or branched alkenyl having 2 to 5 carbon atoms, for example, ethenyl, 1-methylethenyl, 1-propenyl, 2-propenyl, 1-butenyl, 2 -Butenyl, 3-butenyl, 1-methyl-1-propenyl, 2-methyl-1-propenyl, 1-methyl-2-propenyl, 2-methyl-2-propenyl, 1-pentenyl can be mentioned. Among them, alkenyl having 4 carbon atoms is preferable, and 1-methyl-1-propenyl is more preferable.

Examples of the “alkyl” of “hydroxyalkyl” relating to R ¹⁰ and R ¹¹ include, for example, the same as the alkyls 1 to 5 described above. Specific examples of "hydroxyalkyl" include, for example, hydroxymethyl and hydroxyethyl, preferably hydroxymethyl.
R ¹⁰ is preferably hydroxyalkyl having 1 to 3 carbon atoms, more preferably hydroxymethyl. R ¹¹ is preferably hydroxyalkyl having 1 to 3 carbon atoms, more preferably hydroxymethyl.

-CR ¹¹ R ¹² -CHR ¹³ - (In the ^{formula, R 11} represents a hydrogen atom or a hydroxyalkyl of 1 to 5 carbon ^{atoms, R 12} and ^{R 13} an epoxy group together with the carbon atom adjacent together Specific examples of the structure represented by (a) can include the structures shown below.

In the above formulae, R ¹¹ is as defined above.

  More specifically, examples of the compound A include compounds represented by the following structural formulas (4) to (10) (hereinafter referred to as “compound 4” to “compound 10”, respectively).

  Among the compounds according to the invention, compounds 1 to 10 are preferred, including their stereoisomers. More preferably, they are compounds 2, 5 and 10. As one example of the preferred embodiments of Compounds 2, 5 and 10, stereoisomers having a structure represented by the following Structural Formula (2-1), Structural Formula (5-1) and Structural Formula (10-1) are as follows: Although illustrated, it is not limited to these.

The compounds 1 to 10 are all known and commercially available. These compounds can be purchased, for example, from AnalytiCon Discovery GmbH.
Among the compounds A, compounds other than the compounds 4 to 10 can be appropriately produced according to the synthesis method of the compounds 1 to 10.

  As the “salt” of the compound according to the present invention, when the compound exhibits acidity, for example, a salt of an inorganic base such as sodium salt, potassium salt, calcium salt, magnesium salt, ammonium salt or methylamine, Mention may be made of salts of organic bases such as ethylamine, ethanolamine, lysine, arginine and the like. When the compound exhibits basicity, for example, salts of inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrofluoric acid and hydrobromic acid, or acetic acid, tartaric acid, lactic acid, citric acid, fumar Examples thereof include salts of organic acids such as acid, maleic acid, succinic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid and camphorsulfonic acid. Such salts can be obtained by methods known per se.

  The compound according to the present invention or a salt thereof may be a solvate (including a hydrate). Such a solvate can be usually obtained by recrystallizing a solvate from a corresponding solvent or a suitable mixed solvent containing the corresponding solvent. For example, the hydrate can be obtained by recrystallizing the compound according to the present invention from hydrous alcohol.

  The compounds according to the present invention and salts thereof are suitably used when culturing cells, and in particular, they are suitably used for culturing the pluripotent stem cells while maintaining the undifferentiated state of the cells. . Specifically, it can be suitably used, for example, for proliferating or establishing pluripotent stem cells while maintaining the undifferentiated state of pluripotent stem cells, and in particular, the undifferentiated state of pluripotent stem cells Can be suitably used to proliferate the pluripotent stem cells. Therefore, it is suitably used for maintenance culture of pluripotent stem cells and the like.

(II) Composition for cell culture according to the present invention and culture medium for pluripotent stem cell maintenance culture The composition for cell culture according to the present invention is selected from the group consisting of Compound A, Compound 1, Compound 2 and Compound 3. Or at least one compound or salt thereof. The composition can be used as it is as a medium for cell culture, but it can also be used as a medium supplement or a medium additive.

  The composition for cell culture of the present invention may consist of at least one compound selected from the group consisting of Compound A, Compound 1, Compound 2 and Compound 3 or a salt thereof, and the composition of the present invention. As long as the effect is exhibited, other components (for example, a medium basic component described later, an antioxidant, a serum substitute) may be included.

  The content of the compound according to the present invention and the salt thereof in the composition for cell culture according to the present invention is not particularly limited, but for example, the total concentration of the compound according to the present invention and the salt thereof is The content is in the range of 1 nM to 10 μM, and preferably in the range of 10 nM to 1 μM.

  The cells to be cultured are not particularly limited, but pluripotent stem cells are preferable. The pluripotent stem cells are not particularly limited as long as they have pluripotency capable of differentiating into all cells present in the living body and also have proliferation ability. As pluripotent stem cells, for example, embryonic stem cells (ES cells), embryonic stem cells derived from cloned embryos obtained by nuclear transplantation (nt ES cells), spermatogonial stem cells (GS cells), embryonic germ cells (EG cells), artificial Pluripotent stem cells (iPS cells), cultured fibroblasts or pluripotent cells derived from bone marrow stem cells (Muse cells), pluripotent germ stem cells (mGS cells), fusion cells of human ES cells and somatic cells, stimulation stimulation Pluripotent pluripotent cells (STAP cells) (WO 2013/163296) can be mentioned. Among them, ES cells or iPS cells are preferred.

Pluripotent stem cells are cells derived from mammals such as humans, monkeys, mice, rats, hamsters, rabbits, guinea pigs, cows, pigs, dogs, horses, cats, goats and sheep; cells derived from birds; cells derived from reptiles And so forth can be used. Preferably, those derived from mammals are used, and particularly preferably those derived from humans.
Particularly preferred pluripotent stem cells are ES cells or iPS cells of human origin.

  The composition for cell culture of the present invention is suitably used for pluripotent stem cell culture. For example, it can be used to proliferate or establish pluripotent stem cells in an undifferentiated state. In particular, it is suitably used to expand pluripotent stem cells while maintaining the undifferentiated state of pluripotent stem cells. In the present specification, a pluripotent stem cell in the “undifferentiated state” means that the pluripotent stem cell is in a pluripotent and proliferative ability state. Pluripotency means having differentiation ability as described above. The maintenance of the undifferentiated state of pluripotent stem cells can be confirmed using, for example, expression of at least Oct-3 / 4 as an index. In addition, the expression of one or more marker genes selected from the group consisting of Nanog, Sox2, SSEA-1, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81 is also indicated. Can be confirmed. In addition, the measurement of these marker genes can be suitably implemented using a commercially available kit (for example, ES / iPS Cell, Human, Characterization Kit (System Biosciences, LLC)).

  The method of using the composition for cell culture of the present invention is not particularly limited. For example, when used for culturing pluripotent stem cells, the composition for cell culture can be added to the culture medium. Moreover, the composition for cell culture can also be used as a pluripotent stem cell maintenance culture medium as it is.

The pluripotent stem cell maintenance culture medium containing the composition for cell culture is one of the preferred embodiments of the present invention. The pluripotent stem cell maintenance culture medium contains at least one compound selected from the group consisting of Compound A, Compound 1, Compound 2 and Compound 3 or a salt thereof.
The “pluripotent stem cell maintenance culture medium” refers to a culture medium suitable for culturing pluripotent stem cells or a composition thereof, which is capable of proliferating while maintaining the undifferentiated state of pluripotent stem cells. It is
By using the composition for cell culture of the present invention or the culture medium for pluripotent stem cell maintenance culture, pluripotent and proliferative in an undifferentiated state under feeder-free conditions without xeno-free components of animal origin. Maintenance stem cells can be maintained.

  In addition, the composition for cell culture and the pluripotent stem cell maintenance culture medium of the present invention can also be suitably used in culture for establishing pluripotent stem cells. For example, by using the composition for cell culture of the present invention or the culture medium for pluripotent stem cell maintenance culture, pluripotent and proliferative undifferentiated state under feeder-free and xeno-free conditions containing no animal-derived component Pluripotent stem cells can be established.

  The content of the composition for cell culture in the culture medium for pluripotent stem cell maintenance culture is not particularly limited. For example, the total concentration of Compound A or a salt thereof, Compound 1, Compound 2, and Compound 3 and their salts can be usually 1 nM to 10 μM, and preferably 10 nM to 1 μM. The total concentration is the concentration of the compound or the salt thereof when only one compound of the present invention or a salt thereof is used.

  The pluripotent stem cell maintenance culture medium usually contains a medium basic component. The medium basic component is not particularly limited as long as it can allow proliferation while maintaining the undifferentiated state of pluripotent stem cells when the above-described composition for cell culture is contained therein. Such medium basal components usually include carbon sources, nitrogen sources and inorganic salts that can be assimilated by cells. Specifically, sugars such as glucose, essential amino acids, inorganic salts (such as zinc, iron, magnesium, calcium, potassium), and vitamins are included. A buffer component can also be added to the medium basic component as needed.

As a medium basic component, a basic medium known to those skilled in the art can be used. Specific examples of the basal medium include, for example, Dulbecco's Modified Eagle's Medium (DMEM), Minimal essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, α Minimal essential Medium ( α MEM), Glasgow's Minimal essential Medium (GMEM), Iscove's Modified Dulbecco's Medium (IMDM) and the like can be used, and commercially available products can be used.
In addition, components such as nonessential amino acids and sodium pyruvate can also be added to the medium basic components.

In the pluripotent stem cell maintenance culture medium, in addition to the medium basic component, it is preferable to add an antioxidant and a serum replacement additive.
The antioxidant is not particularly limited. For example, 2-mercaptoethanol, dithiothreitol, ascorbic acid or an ester thereof can be used. Ascorbic acid or an ester thereof may form a salt. Among these, ascorbic acid or an ester thereof is preferred. The addition amount of the antioxidant is not particularly limited, and can be appropriately selected according to the type of the antioxidant. For example, in the medium, the antioxidant can be added in a concentration of usually 10 to 100 mg / L, preferably 50 to 75 mg / L.

Serum replacement additives are artificial liquid compositions that are usually configured to contain components similar to serum, and by adding them, it is possible to grow cells even in the absence of serum. It will be As serum replacement additives, for example, amino acids, inorganic salts, vitamins, albumin, insulin, transferrin, antioxidant components can be used. Examples of amino acids include glycine, L-alanine, L-asparagine, L-cysteine, L-aspartic acid, L-glutamic acid, L-phenylalanine, L-histidine, L-isoleucine, L-lysine, L-leucine, L- Examples include glutamine, L-arginine, L-methionine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine. As inorganic salts, for example, AgNO ₃ , AlCl ₃ .6H ₂ O, Ba (C ₂ H ₃ O ₂ ) ₂ , CdSO ₄ .8H ₂ O, CoCl ₂ .6H ₂ O, Cr ₂ (SO _{4) 3} 1H ₂ O, GeO ₂ , Na ₂ SeO ₃ , H ₂ SeO ₃ , KBr, KI, MnCl ₂ .4H ₂ O, NaF, Na ₂ SiO ₃ .9H ₂ O, NaVO ₃ , (NH ₄ ) 6Mo ₇ O _24. 4H ₂ O, NiSO ₄ · 6H ₂ O, RbCl, SnCl ₂ , ZrOCl ₂ · 8H ₂ O, sodium selenite. Examples of vitamins include thiamine and ascorbic acid. Examples of the antioxidant component include reduced glutathione. These may be used alone or in combination of two or more.
It is also possible to use commercially available serum replacement additives for ES cells, such as, for example, Knockout® Serum Replacement Additive (KSR) (Invitrogen).

  The addition amount of the serum replacement additive is not particularly limited, and can be appropriately selected according to the type of serum replacement additive. For example, in the medium, the concentration of serum replacement additive can be added so that the concentration is usually 5 to 30 v / v%, preferably 10 to 20 v / v%.

  The composition for cell culture and the culture medium for pluripotent stem cell maintenance culture according to the present invention can also contain animal-derived components such as feeder cells used for maintenance culture of pluripotent stem cells, serum, etc. Preferably, it does not contain feeder cells and / or serum, and more preferably does not contain feeder cells and serum. Also, particularly preferably, it is a xeno-free one containing no animal-derived component. The composition for cell culture and the culture medium for pluripotent stem cell maintenance culture according to the present invention can also contain bFGF and / or TGFβ which are used for maintenance culture of pluripotent stem cells in a feeder-free manner. However, preferably it does not contain bFGF and / or TGFβ, more preferably it does not contain bFGF and TGFβ. Particularly preferably, they do not contain feeder cells, animal-derived components such as serum, bFGF and TGFβ.

  In the composition for cell culture according to the present invention and the culture medium for pluripotent stem cell maintenance culture, optional components such as pH adjusters, moisturizers, preservatives, viscosity adjusters and the like can also be used, as needed. The addition amount of such optional components is not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected.

  The composition for cell culture according to the present invention and each of the above-mentioned components may be added to the medium in an amount such that the desired concentration is obtained from the beginning, and may be added in two or more divided doses. It may be added in such an amount that the final concentration will be achieved. Preferably, it is added in such an amount as to obtain the desired concentration from the beginning. The pH of the pluripotent stem cell maintenance culture medium is usually adjusted to 6.5 to 7.5, preferably to 6.8 to 7.2.

  The composition for cell culture and the culture medium for pluripotent stem cell maintenance culture according to the present invention may be prepared in a solution form or a dry form, respectively. When in solution form, it may be provided as a concentrated composition (e.g. 1 to 1000 times) and may be suitably diluted on use. The liquid used to dilute or dissolve the composition or medium in solution form or dry form is not particularly limited, but generally, water, buffer, saline can be used, and it is appropriately selected as necessary. be able to.

  In a preferred embodiment of the present invention, the composition for cell culture and the culture medium for pluripotent stem cell maintenance culture are sterilized to prevent contamination. The sterilization method is not particularly limited, and a usual method can be adopted, and for example, it can be sterilized by ultraviolet irradiation, heat sterilization, radiation, or filtration.

(III) Culture Method The present invention also encompasses a method of culturing pluripotent stem cells for proliferating or establishing the pluripotent stem cells while maintaining the undifferentiated state of the pluripotent stem cells. In the culture method of the present invention, the pluripotent stem cells are cultured in the presence of at least one compound selected from the group consisting of Compound A, Compound 1, Compound 2 and Compound 3 or a salt thereof.
The culture method of the present invention is preferably a culture method of pluripotent stem cells for proliferating pluripotent stem cells while maintaining an undifferentiated state of pluripotent stem cells.
Undifferentiating pluripotent stem cells by culturing pluripotent stem cells in the presence of at least one compound selected from the group consisting of compound A, compound 1, compound 2 and compound 3 or a salt thereof The pluripotent stem cells can be proliferated or established while maintaining the state.

Hereinafter, a culture method of pluripotent stem cells for proliferating pluripotent stem cells while maintaining the undifferentiated state of pluripotent stem cells will be described.
The method of the present invention is preferably used when maintaining and culturing pluripotent stem cells (preferably human iPS cells or ES cells described above) under feeder-free and xeno-free culture conditions.

Culturing of pluripotent stem cells in the method of the present invention is preferably performed using the pluripotent stem cell maintenance culture medium described above. The components of the pluripotent stem cell maintenance culture medium and the preferable embodiments thereof are the same as those described above. Moreover, it is preferable to use the culture medium for pluripotent stem cell maintenance culture in the form of a solution (culture solution).
For example, using the above pluripotent stem cell maintenance culture medium as a culture medium, the undifferentiated state of pluripotent stem cells is maintained by culturing under the conditions usually used for maintenance culture of pluripotent stem cells. While, pluripotent stem cells can be proliferated.

  Each component of the pluripotent stem cell maintenance culture medium may be added to the medium in an amount such that the desired concentration is obtained from the beginning, and may be added twice or more times during the culture, It may be added in such an amount that the final concentration will be achieved. Preferably, it is added in such an amount as to obtain the desired concentration from the beginning. The pH of the pluripotent stem cell maintenance culture medium is usually adjusted to 6.5 to 7.5, preferably to 6.8 to 7.2.

  In the method of the present invention, for example, the total concentration of Compound A, Compound 1, Compound 2 and Compound 3 and their salts in the medium can be preferably 1 nM to 10 μM, more preferably Culture is performed under conditions of 10 nM to 1 μM. When the concentration of the compound according to the present invention and the salt thereof is in such a range, pluripotent stem cells can be efficiently proliferated while maintaining the undifferentiated state of pluripotent stem cells. The compound according to the present invention or a salt thereof may be added to the culture medium in an amount such that the desired concentration is obtained from the beginning, and may be added twice or more times during the culture, and finally added. It may be added in an amount to achieve the desired concentration.

  In the culture method of the present invention, pluripotent stem cells are preferably cultured under conditions that do not contain feeder cells. It is also preferred to culture pluripotent stem cells in the absence of serum. More preferably, the culture of multifunctional cells is performed in the absence of feeder cells and serum. Furthermore, it is preferable to culture pluripotent stem cells under feeder-free and xeno-free conditions. Moreover, in the culture method of the present invention, it is preferable to culture a multifunctional cell in the absence of bFGF and / or TGFβ, and more preferably, culture is performed in the absence of bFGF and TGFβ.

  In the present invention, as a preferred embodiment of the culture method under feeder-free conditions, for example, a method of culturing using a culture vessel coated with an extracellular matrix is exemplified. The coating treatment may be carried out by appropriately removing the solution after the solution containing the extracellular matrix is put in the culture vessel. In the present invention, it is preferable to culture pluripotent stem cells in a culture vessel coated with an extracellular matrix as described above.

  In the present invention, the extracellular matrix is a supramolecular structure existing outside the cell, and may be naturally occurring or artificial (recombinant). For example, substances such as collagen, proteoglycan, fibronectin, hyaluronic acid, tenascin, entactin, elastin, fibrillin, laminin or fragments thereof can be mentioned. These extracellular matrices may be used in combination, for example, preparations from cells such as BD Matrigel®. As an artifact, a fragment of laminin is exemplified. In the present invention, laminin is a protein having a heterotrimeric structure having one each of an α chain, a β chain, and a γ chain, and is not particularly limited. For example, the α chain is α1, α2, α3,. α4 or α5, β chain is β1, β2 or β3, and γ chain is exemplified by γ1, γ2 or γ3. In the present invention, the fragment of laminin is not particularly limited as long as it is a fragment of laminin having integrin binding activity, and examples thereof include E8 fragment which is a fragment obtained by digestion with elastase.

In the present invention, the number of pluripotent stem cells to be cultured is not particularly limited, but, for example, usually 50 to 50000 cells / cm ^{2 of} pluripotent stem cells, preferably 100 to 5000 The culture can be started as individual cells / cm ² .
In addition, since the culture method of the present invention can culture and proliferate, for example, one pluripotent stem cell, it is possible to form a clonal cell population.

Pluripotent stem cells to be cultured are similar to those described above.
Pluripotent stem cells to be subjected to culture are in an undifferentiated state. The method for preparing pluripotent stem cells to be cultured is not particularly limited. For example, pluripotent stem cells established under feederless conditions or pluripotent stem cells established in a co-culture system with feeder cells may be used. Can. Specifically, for example, pluripotent stem cells established or subcultured by a known method can be used. In addition, pluripotent stem cells established by culturing according to the method of the present invention described later can be used. In addition, pluripotent stem cells can be recovered from a co-culture system with feeder cells, and the recovered pluripotent stem cells can be used as they are for culture, but the recovered pluripotent stem cells can be passaged over several stages. It can also be used for culture. The subculture in this case is preferably performed under feeder-free conditions.

  In the method of the present invention, pluripotent stem cells to be subjected to culture may be in the state of a single cell by being substantially separated (or dissociated) by any method, or cells may adhere to each other. It may be in the form of isolated cell mass. Specifically, for example, before being subjected to culture, treatment may be performed to separate pluripotent stem cells as necessary, and the cells may be transferred to a new culture vessel and cultured in a single cell state or a cell mass state. Here, as a method of separation, for example, a separation solution having mechanical separation, protease activity and collagenase activity (for example, a solution containing trypsin and collagenase, Accutase (registered trademark) and Accumax (Innovative Cell Technologies) , Inc.) or separation using only a separate solution having collagenase activity.

  In the case of dispersing pluripotent stem cells into single cells, the medium preferably contains a ROCK inhibitor.

  In the present invention, the ROCK inhibitor is not particularly limited as long as it can suppress the function of Rho-kinase (ROCK), and, for example, Y-27632 (Cas No .: 146986-50-7) (eg, Ishizaki) et al., Mol. Pharmacol. 57, 976-983 (2000); Narumiya et al., Methods Enzymol. 325, 273-284 (2000), Fasudil / HA 1077 (e.g., Uenata et al., Nature 389: 990-994 (1997), H-1152 (e.g. Sasaki et al., Pharmacol. Ther. 93: 225-232 (2002)), Wf-536 (e.g. Nakajima). 52 (4): 319-324 (2003) and derivatives thereof, and antisense nucleic acids against ROCK, RNA interference-inducing nucleic acids (eg, siRNA), dominant negative mutants, and These include expression vectors. In addition, other known low molecular weight compounds can also be used as ROCK inhibitors (for example, US Patent Application Publication Nos. 2005/0209261, 2005/0192304, 2004/0014755, and 2004/0002508). 2004/0002507, 2003/0125344, 2003/0087919, and WO 2003/062227, 2003/059913, 2003/062225, 2002/076976. No. 2004/039796)). In the present invention, one or more ROCK inhibitors may be used. Preferred ROCK inhibitors for use in the present invention include Y-27632. In the present invention, the concentration of the ROCK inhibitor in the pluripotent stem cell maintenance culture medium can be appropriately selected by those skilled in the art according to the ROCK inhibitor to be used, but for example, Y-27632 is used as the ROCK inhibitor In the case, it is generally 0.1 μM to 100 μM, preferably 1 μM to 50 μM, and more preferably 5 μM to 20 μM.

In the culture method of pluripotent stem cells of the present invention, the culture temperature is not particularly limited, but is usually about 30 to 40 ° C, preferably about 37 ° C. In general, the culture is performed under an atmosphere of CO ₂ -containing air, and the CO ₂ concentration is preferably about 2 to 5%.
It is preferable to change the medium usually every one to two days, preferably daily.
In addition, pluripotent stem cells can be passaged as appropriate. For example, in the case of ES cells, it is usually preferable to pass every 3 to 4 days. Passaging can be performed by a known method.

The culture method of the present invention can be used for culture to establish pluripotent stem cells in an undifferentiated state. For example, in the culture for establishing pluripotent stem cells in an undifferentiated state, the presence of at least one compound selected from the group consisting of compound A, compound 1, compound 2 and compound 3 or a salt thereof By performing under the cells, pluripotent stem cells can be established. The method of the present invention is suitably used, for example, in establishing pluripotent stem cells under feeder-free and xeno-free culture conditions.
The pluripotent stem cells are similar to those described above, and are preferably iPS cells or ES cells. The pluripotent stem cells are preferably derived from mammals, and the pluripotent stem cells are preferably derived from human. Among them, human-derived iPS cells are particularly preferable.

  For example, when establishing pluripotent stem cells, using the above-described composition for cell culture or pluripotent stem cell maintenance culture medium as a culture medium, pluripotent under conditions usually used for establishment of pluripotent stem cells By culturing adult stem cells, undifferentiated pluripotent stem cells can be established. Preferred embodiments of the culture medium are as described above. Culture conditions and the like for establishing pluripotent stem cells in an undifferentiated state differ depending on the type of pluripotent stem cells, and can be appropriately selected.

The concentration of the compound of the present invention or a salt thereof in the medium can be the same as in the above-described maintenance culture, and the medium of compound A, compound 1, compound 2, and compound 3 and their salts is used. The culture is carried out under conditions of a total concentration of preferably 1 nM to 10 μM, more preferably 10 nM to 1 μM. The culture temperature is not particularly limited, but is usually about 30 to 40 ° C, preferably about 37 ° C. In general, the culture is performed under an atmosphere of CO ₂ -containing air, and the CO ₂ concentration is preferably about 2 to 5%.
Medium exchange is usually performed every one to two days, preferably daily.

  For example, in the case of ES cells, human ES cells can be established by culturing cell masses isolated from human blastocysts in the presence of the compound of the present invention or a salt thereof. Preferably, the above-mentioned composition for cell culture or a culture medium for pluripotent stem cell maintenance culture is used as a medium (culture solution) for producing ES cells. Maintenance of ES cells by subculture can be performed, for example, by the culture method of the present invention described above.

  Generally, ES cells can be selected by Real-Time PCR using expression of gene markers such as alkaline phosphatase, Oct-3 / 4 and Nanog as indicators. In particular, in selection of human ES cells, expression of gene markers such as OCT-3 / 4, NANOG, ECAD can be used as an index (E. Kroon et al. (2008), Nat. Biotechnol., 26: 443). -452).

For example, in culture for establishing iPS cells, iPS cells can be established by contacting somatic cells with a reprogramming factor in the presence of the compound according to the present invention or a salt thereof, and culturing. . In addition, iPS cells can be established by culturing somatic cells into which a reprogramming factor has been introduced in the presence of the compound of the present invention or a salt thereof.
Preferably, the above-mentioned composition for cell culture or a culture medium for pluripotent stem cell maintenance culture is used as a medium (culture solution) for iPS cell induction.

Examples of culture methods for establishing iPS cells include, for example, a culture temperature of about 30 to 40 ° C., a CO ₂ concentration of about 2 to 5%, and preferably a culture temperature of about 37 ° C., about 5% The somatic cells are contacted with a reprogramming factor in the presence of CO ₂ , transferred to a dish coated with extracellular matrix, and then cultured in a pluripotent stem cell maintenance culture medium, and the contact is carried out for about 20 to about 30 After days or more iPS-like colonies can be generated. Immediately after contacting with the reprogramming factor, the cells may be cultured in a medium suitable for somatic cells.

Alternatively, feeder cells (eg, mitomycin C-treated STO cells under a culture temperature of about 30 to 40 ° C., a CO ₂ concentration of about 2 to 5%, preferably at about 37 ° C., in the presence of about 5% CO ₂₎ (SNL cells, etc.) in pluripotent stem cell maintenance culture medium, and can give rise to ES-like colonies after about 25 to about 30 days or more. Preferably, a method using (Takahashi K, et al. (2009), PLoS One. 4: e8067 or WO 2010/137746) in which somatic cells to be initialized are used instead of feeder cells is exemplified.
Furthermore, to increase establishment efficiency, iPS cells may be established under hypoxic conditions (oxygen concentration of 0.1% or more and 15% or less) (Yoshida Y, et al. (2009), Cell Stem Cell. 5 : 237-241 or WO 2010/013845).

During the above culture, it is preferable to perform culture medium exchange with fresh culture medium once every day from the second day after the start of culture. Further, the number of somatic cells used for nuclear reprogramming is not limited, but is preferably in the range of about 5 × 10 ³ to about 5 × 10 ⁶ cells per 100 cm ^{2 of} culture dish.

  iPS cells can be selected by the shape of the formed colony. On the other hand, when a drug resistance gene that is expressed in conjunction with a gene (for example, Oct3 / 4, Nanog) that is expressed when somatic cells are reprogrammed is introduced as a marker gene, a culture solution containing the corresponding drug (selection The established iPS cells can be selected by culturing in a culture solution). In addition, if the marker gene is a fluorescent protein gene, iPS cells are selected by observing with a fluorescent microscope, by adding a luminescent substrate in the case of a luminescent enzyme gene, or by adding a chromogenic substrate in the case of a chromogenic enzyme gene. can do.

  The somatic cells used for producing iPS cells include all animal cells (preferably mammalian cells including human) except germline cells such as oocytes, oocytes and ES cells or totipotent cells. It can be used. Somatic cells include, but are not limited to, fetal (child) somatic cells, neonatal (child) somatic cells, and any mature healthy or diseased somatic cells, and also primary culture cells. Also included are passage cells and cell lines. Specifically, somatic cells include, for example, (1) tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, dental pulp stem cells, (2) tissue precursor cells, (3) lymphocytes, epithelium Cells, endothelial cells, muscle cells, fibroblasts (skin cells etc.), hair cells, hepatocytes, gastric mucous cells, enterocytes, spleen cells, pancreatic cells (pancreatic exocrine cells etc.), brain cells, lung cells, kidney cells And differentiated cells such as adipocytes.

  As the reprogramming factor, a known gene can be used, and a gene specifically expressed in ES cells, a gene product of the gene or a gene that plays an important role in maintaining non-differentiation of non-coding RNA or ES cells, The gene product or non-coding RNA, or a low molecular weight compound may be used. As genes included in the reprogramming factor, for example, Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15 -2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3 or Glis1 etc. are exemplified, and these reprogramming factors may be used alone or in combination. As a combination of initialization factors, WO2007 / 069666, WO2008 / 118820, WO2009 / 007852, WO2009 / 032194, WO2009 / 058413, WO2009 / 057831, WO2009 / 075119, WO2009 / 079007, WO2009 / 091659, WO2009 / 101084, WO2009 / 01084 101407, WO2009 / 102983, WO2009 / 114949, WO2009 / 117439, WO2009 / 126250, WO2009 / 126251, WO2009 / 126655, WO2009 / 157593, WO2010 / 009015, WO2010 / 033906, WO2010 / 033920, WO2010 / 042800, WO2010 / 05 626, WO2010 / 056831, WO2010 / 068955, WO2010 / 098419, WO2010 / 102267, WO2010 / 111409, WO2010 / 111422, WO2010 / 115050, WO2010 / 124290, WO2010 / 147395, WO2010 / 147612, Huangfu D, et al. (2008), Nat. Biotechnol. , 26: 795-797, Shi Y, et al. (2008), Cell Stem Cell, 2: 525-528, Eminli S, et al. (2008), Stem Cells. 26: 2467-2474, Huangfu D, et al. (2008), Nat Biotechnol. 26: 1269-1275, Shi Y, et al. (2008), Cell Stem Cell, 3, 568-574, Zhao Y, et al. (2008), Cell Stem Cell, 3: 475-479, Marson A, (2008), Cell Stem Cell, 3, 132-135, Feng B, et al. (2009), Nat Cell Biol. 11: 197-203, R.S. L. Judson et al. , (2009), Nat. Biotech. , 27: 459-461, Lyssiotis CA, et al. (2009), Proc Natl Acad Sci US A. 106: 8912-8917, Kim JB, et al. (2009), Nature. 461: 649-643, Ichida JK, et al. (2009), Cell Stem Cell. 5: 491-503, Heng JC, et al. (2010), Cell Stem Cell. 6: 167-74, Han J, et al. (2010), Nature. 463: 1096-100, Mali P, et al. (2010), Stem Cells. 28: 713-720, Maekawa M, et al. (2011), Nature. 474: 225-9. The combination as described in is illustrated.

  Examples of the reprogramming factor include histone deacetylase (HDAC) inhibitors [eg, valproic acid (VPA), trichostatin A, sodium butyrate, small molecule inhibitors such as MC 1293, M344, siRNA against HDAC (eg Nucleic acid expression inhibitors such as HDAC1 siRNA Smartpool (registered trademark) (Millipore), HuSH 29mer shRNA Constructs against HDAC1 (OriGene), etc.], MEK inhibitors (eg PD184352, PD98059, U0126, SL327 and PD0325901), Glycogen synthase kinase-3 inhibitor (eg, Bio and CHIR99021), DNA methyltransferase inhibitor (eg, , 5-azacytidine), histone methyltransferase inhibitors (for example, small molecule inhibitors such as BIX-01294, nucleic acid expression inhibitors such as Suv39hl, Suv39h2, SetDBl and G9a, and nucleic acid expression inhibitors such as shRNA), L-channel calcium agonist (Eg, Baik 8644), butyric acid, TGFβ inhibitors or ALK5 inhibitors (eg, LY364947, SB431542, 616453 and A-83-01), p53 inhibitors (eg, siRNA and shRNA against p53), ARID3A inhibitors (eg, ARID3A) siRNA and shRNA), miRNA such as miR-291-3p, miR-294, miR-295 and mir-302, Wnt Sig to increase the establishment efficiency of naling (eg soluble Wnt3a), neuropeptide Y, prostaglandins (eg prostaglandin E2 and prostaglandin J2), hTERT, SV40LT, UTF1, IRX6, GLIS1, PITX2, DMRTB1 etc. The factors used for the purpose are also included, and in the present specification, the factors used for the purpose of improving the establishment efficiency are also not distinguished from the reprogramming factors.

  The reprogramming factor may be introduced into somatic cells in the form of a protein, for example, by lipofection, fusion with a cell membrane permeable peptide (eg, TAT and polyarginine derived from HIV), microinjection and the like.

  On the other hand, in the case of the form of DNA, it can be introduced into somatic cells by techniques such as viruses, plasmids, vectors such as artificial chromosomes, lipofection, liposomes, microinjection and the like. Examples of viral vectors include retrovirus vectors, lentivirus vectors (above, Cell, 126, pp. 663-676, 2006; Cell, 131, pp. 861-872, 2007; Science, 318, pp. 1917-1920, 2007) And adenovirus vectors (Science, 322, 945-949, 2008), adeno-associated virus vectors, Sendai virus vectors (WO 2010/008054) and the like. Moreover, examples of artificial chromosome vectors include human artificial chromosome (HAC), yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC, PAC) and the like. As a plasmid, a plasmid for mammalian cells can be used (Science, 322: 949-953, 2008). The vector can contain regulatory sequences such as a promoter, enhancer, ribosome binding sequence, terminator, polyadenylation site, etc. so that the nuclear reprogramming substance can be expressed, and further, if necessary, a drug resistance gene ( For example, kanamycin resistance gene, ampicillin resistance gene, puromycin resistance gene, etc.), thymidine kinase gene, selective marker sequences such as diphtheria toxin gene etc., reporter gene sequences such as green fluorescent protein (GFP), β-glucuronidase (GUS), FLAG etc. Can be included. In addition, the above vector has a LoxP sequence before and after the gene for introducing into the somatic cell, in order to excise the gene encoding the reprogramming factor or promoter together with the gene encoding the reprogramming factor linked thereto. May be

  Also, in the form of RNA, it may be introduced into somatic cells by, for example, lipofection, microinjection, etc., and RNA incorporated with 5-methylcytidine and pseudouridine (TriLink Biotechnologies) is used to inhibit degradation. (Warren L, (2010) Cell Stem Cell. 7: 618-630).

In addition, embryonic germ cells are established by culturing embryonic primordial germ cells in the presence of the compound of the present invention or a salt thereof, preferably using a pluripotent stem cell maintenance culture medium.
Also, for example, for the production of nt ES cells, a combination of nuclear transfer technology (JB Cibelli et al. (1998), Nature Biotechnol., 16: 642-646) and ES cell production technology usually It is utilized (Kiyoka Wakayama et al. (2008), Experimental Medicine, Vol. 26, No. 5 (extra edition), 47-52). In nuclear transfer, the nucleus of a somatic cell is injected into an enucleated unfertilized egg of a mammal, and preferably in the presence of the compound according to the present invention or a salt thereof, preferably using a pluripotent stem cell maintenance culture medium. It can be initialized by culturing for time.

  When the compound according to the present invention or a salt thereof is used, pluripotent stem cells can be established even in the culture in the absence of feeder cells, so culture conditions in the absence of feeder cells should be used. Can.

  Pluripotent stem cells cultured by the culture method of the present invention are usually pluripotent stem cells in an undifferentiated state having pluripotency. Such pluripotent stem cells can be used as they are or differentiated into desired cells, and used for producing research materials or regenerative medicine products. The composition for cell culture, pluripotent stem cell maintenance culture medium of the present invention, and culture method are compositions for proliferating or establishing pluripotent stem cells while maintaining the undifferentiated state of pluripotent stem cells. , Media and methods are also suitably used.

  Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited to the following examples.

1. Cell human iPS cells can be used after culturing the 201B7 strain prepared by introducing four factors (OCT3 / 4, SOX2, KLF4 and c-MYC) into human fibroblasts using retrovirus under the following conditions: (Takahashi K, et al, Cell. 131: 861-872, 2007.). The 201B7 strain is seeded on a dish coated with 0.5 μg / cm ² of a laminin 511 E8 fragment (trade name “iMatrix-511”, Nippi), and 64 mg / L L-ascorbic acid magnesium 2-phosphate, 14 μg / L L cultured in DMEM / F12 supplemented with sodium selenite (sodium selenium), 19.4 mg / L insulin, 543 mg / L NaHCO ₃ , 10.7 mg / L transferrin, 100 μg / L bFGF and 2 μg / L TGFβ 1, Passaging was continued by the culture method without using feeder cells.

2. Compounds The chemical structures of Compounds 1 to 10 used in this Example are shown in Tables 1 to 3. Compounds 1 to 10 were purchased from AnalytiCon Discovery GmbH.

3. Evaluation The confirmation that the iPS cells are in an undifferentiated state (having pluripotency) was performed by confirming the expression of Oct-3 / 4.
In the following examples, immunostaining with an anti-Oct3 / 4 antibody and nuclear staining with Hoechst 33342 are described by Nakagawa M, et al. Sci Rep. 4: 3594, it carried out by the method as described in 2014.

Example 1
Examination of Alternative Compound for bFGF The human iPS cells were seeded at 1,300 cells / well on a 96-well plate coated with 0.5 μg / cm ² of a laminin 511 E8 fragment (trade name "iMatrix-511", Nippi) . 64 mg / L L-ascorbic acid-2-phosphate magnesium, 14 μg / L sodium selenite, 19.4 mg / L insulin, 543 mg / L NaHCO ₃ , 10.7 mg / L transferrin, and 2 μg / L TGFβ1 were added DMEM / F12 was the control medium for bFGF. 1 μM of each test compound (Compounds 1, 2, 4, 5, 6, 7, 8, 9 and 10) shown in Tables 1 to 3 was added to this control medium to prepare a medium. Each obtained medium was added to the seeded human iPS cells and cultured at 37 ° C. As a positive control, a control medium containing 100 μg / L bFGF was used. One week later, cells were fixed and subjected to immunostaining with anti-Oct3 / 4 antibody and nuclear staining.

The results are shown in FIGS. FIG. 1 is a fluorescence microscope image of human iPS cells cultured in a positive control (indicated by “bFGF +” in the figure) and a control medium (indicated by “bFGF-” in the figure).
FIGS. 2 and 3 are fluorescence microscopic images of human iPS cells cultured using a medium to which each test compound was added.

  As a result, in any of the compounds, nuclear-stained cells expressed Oct3 / 4. From the above, the test compounds (Compounds 1, 2, 4, 5, 6, 7, 8, 9 and 10) are not pluripotent stem cells as in the case where bFGF is used (“bFGF +” in FIG. 1). It has been confirmed that differentiation maintenance culture is possible.

Example 2
Examination of Alternative Compound for TGFβ1 The human iPS cells were seeded at 1,300 cells / well on a 96 well plate coated with 0.5 μg / cm ² of a laminin 511 E8 fragment (trade name "iMatrix-511", Nippi) . 64 mg / L L-ascorbic acid-2-phosphate magnesium, 14 μg / L sodium selenite, 19.4 mg / L insulin, 543 mg / L NaHCO ₃ , 10.7 mg / L transferrin, and 100 μg / L bFGF were added DMEM / F12 was a control medium of TGFβ1. 1 μM of each test compound (1, 2, 4 and 5) shown in Tables 1 to 3 was added to this control medium to prepare a medium. Each obtained medium was added to the seeded human iPS cells and cultured at 37 ° C. As a positive control, a medium in which 2 μg / L TGFβ1 was added to a control medium (the same as the medium used as a positive control in Example 1) was used. One week later, cells were fixed and subjected to immunostaining with anti-Oct3 / 4 antibody and nuclear staining.

  FIG. 4 shows a fluorescence microscopic image of human iPS cells cultured using a medium to which each test compound was added. In any of the compounds, nuclear stained cells expressed Oct3 / 4. From the above, it is confirmed that each compound (Compounds 1, 2, 4 and 5) enables undifferentiated maintenance culture of pluripotent stem cells as in the case of using TGFβ1 (“bFGF +” in FIG. 1). It was done.

Example 3
Subsequently, using the control medium (control medium of TGFβ1) used in Example 2, effective concentrations of Compounds 1 and 2 were examined. As a positive control, a medium to which 2 μg / L of TGFβ1 was added was used as the control medium used in Example 2 (“TGFβ1 +” in A of FIG. 5), and the control medium used in Example 2 was used as a control. (“TGFβ1-” in FIG. 5A).
When the compound 1 was cultured at a concentration of 100 nM or 10 nM under the same conditions as in Example 2, it was confirmed that an effect equivalent to that obtained when TGFβ1 was added (positive control) was obtained. These results are shown in A of FIG.

Similarly, when culture was conducted at a concentration of 100 nM or 10 nM in Example 2, it was confirmed that the same effect as when TGFβ1 was added (positive control) was obtained. These results are shown in B of FIG.
From the above, it was confirmed that pluripotent stem cells can be cultured in an undifferentiated state by adding Compound 1 and Compound 2 at a concentration of 10 nM in the medium.

Example 4
Examination of alternative compounds for bFGF and TGFβ1 The human iPS cells were applied at 1,300 cells / well to a 96-well plate coated with 0.5 μg / cm ² of a laminin 511E8 fragment (trade name “iMatrix-511”, Nippi) Sowed. 64 mg / L L-ascorbic acid-2-phosphate magnesium, 14 μg / L sodium selenite, 19.4 mg / L insulin, 543 mg / L NaHCO ₃ and 10.7 mg / L transferrin added DMEM / F12, bFGF And a control medium of TGFβ1. 1 μM of each test compound (compounds 3, 6, 7, 8, 9 and 10) was added to this control medium to prepare a medium. Each obtained medium was added to the seeded human iPS cells and cultured at 37 ° C. One week later, cells were fixed and subjected to immunostaining with anti-Oct3 / 4 antibody and nuclear staining. As a positive control, a medium was used in which 100 μg / L of bFGF and 2 μg / L of TGFβ1 were added to the control medium (“bFGF + TGFβ1 +” in FIG. 6). As a control, the above-described control medium of bFGF and TGFβ1 (medium without addition of bFGF, TGFβ1 and each compound) was used (“bFGF-TGFβ1-” in FIG. 6).

  The results are shown in FIG. From FIG. 6, the nuclear-stained cells expressed Oct3 / 4 even when any of the compounds was used. From the above, each compound (compounds 3, 6, 7, 8, 9 and 10) enables undifferentiated maintenance culture of pluripotent stem cells as in the case of using two components of bFGF and TGFβ1. confirmed.

Claims (16)

The compound represented by the following general formula (A):
R ¹ represents methyl ,
R ² represents a hydrogen atom or a hydroxyl group,
R ³ represents a hydrogen atom or a hydroxyl group,
R ⁴ and R ⁵ both represent methyl ,
R ⁶ represents alkyl having 1 to 12 carbon atoms ,
R ⁷ represents a hydrogen atom or —O—CO—R ⁹ (wherein, R ⁹ represents an alkyl having 1 to 3 carbon atoms, or 1-methyl-1-propenyl ),
R ⁸ represents methyl ,
-X-Y- ^is -CR 10 = CH- ^{(wherein, R 10} represents a hydroxymethyl), or ^-CR 11 ^R 12 -CHR ¹³ - ^{(wherein, R 11} represents hydroxymethyl, R ¹² and R ¹³ are taken together to form an epoxy group with the adjacent carbon atom)),
The compound represented by the following structural formula (1):
,
The compound represented by the following structural formula (2):
And a compound represented by the following structural formula (3):
A composition for pluripotent stem cell culture, comprising at least one compound selected from the group consisting of: or a salt thereof. The composition for pluripotent stem cell culture according to claim 1, wherein the compound represented by the general formula (A) is a compound represented by any one of the following structural formulas (4) to (10).
The composition for pluripotent stem cell culture according to claim 1 or 2 , which is used to expand the pluripotent stem cells while maintaining the undifferentiated state of pluripotent stem cells. The composition for pluripotent stem cell culture according to any one of claims 1 to 3 , wherein the pluripotent stem cells are cells of human origin. The composition for pluripotent stem cell culture according to any one of claims 1 to 4 , wherein the pluripotent stem cells are ES cells or iPS cells. A pluripotent stem cell maintenance culture medium comprising the composition for pluripotent stem cell culture according to any one of claims 1 to 5 . The pluripotent stem cell maintenance culture medium according to claim 6 , which does not contain serum. The compound represented by the general formula (A), the compound represented by the structural formula (1), the compound represented by the structural formula (2), the compound represented by the structural formula (3), and a salt thereof The pluripotent stem cell maintenance culture medium according to claim 6 or 7 , wherein the total concentration is 10 nM to 1 μM. A method for culturing pluripotent stem cells for propagating the pluripotent stem cells while maintaining the undifferentiated state of pluripotent stem cells, the culture comprising a compound represented by the following general formula (A): :
(In the formula,
R ¹ represents methyl ,
R ² represents a hydrogen atom or a hydroxyl group,
R ³ represents a hydrogen atom or a hydroxyl group,
R ⁴ and R ⁵ both represent methyl ,
R ⁶ represents alkyl having 1 to 12 carbon atoms ,
R ⁷ represents a hydrogen atom or —O—CO—R ⁹ (wherein, R ⁹ represents an alkyl having 1 to 3 carbon atoms, or 1-methyl-1-propenyl ),
R ⁸ represents methyl ,
-X-Y- ^is -CR 10 = CH- ^{(wherein, R 10} represents a hydroxymethyl), or ^-CR 11 ^R 12 -CHR ¹³ - ^{(wherein, R 11} represents hydroxymethyl, R ¹² and R ¹³ are taken together to form an epoxy group with the adjacent carbon atom)),
The compound represented by the following structural formula (1):
,
The compound represented by the following structural formula (2):
And a compound represented by the following structural formula (3):
A culture method characterized in that it is carried out in the presence of at least one compound selected from the group consisting of or a salt thereof. The culture method according to claim 9 , wherein the compound represented by the general formula (A) is a compound represented by any one of the following structural formulas (4) to (10).
The culture method according to claim 9 or 10 , wherein the pluripotent stem cells are cells of human origin. The culture method according to any one of claims 9 to 11 , wherein the pluripotent stem cells are ES cells or iPS cells. The culture method according to any one of claims 9 to 12 , wherein pluripotent stem cells are cultured under conditions not containing feeder cells. The culture method according to any one of claims 9 to 13 , wherein pluripotent stem cells are cultured in the absence of serum. The compound represented by the general formula (A), the compound represented by the structural formula (1), the compound represented by the structural formula (2), the compound represented by the structural formula (3), and a salt thereof The culture method according to any one of claims 9 to 14 , wherein the culture is carried out under conditions of a total concentration of 10 nM to 1 μM. A compound represented by the following general formula (A) for proliferating pluripotent stem cells while maintaining the undifferentiated state of pluripotent stem cells:
(In the formula,
R ¹ represents methyl ,
R ² represents a hydrogen atom or a hydroxyl group,
R ³ represents a hydrogen atom or a hydroxyl group,
R ⁴ and R ⁵ both represent methyl ,
R ⁶ represents alkyl having 1 to 12 carbon atoms ,
R ⁷ represents a hydrogen atom or —O—CO—R ⁹ (wherein, R ⁹ represents an alkyl having 1 to 3 carbon atoms, or 1-methyl-1-propenyl ),
R ⁸ represents methyl ,
-X-Y- ^is -CR 10 = CH- ^{(wherein, R 10} represents a hydroxymethyl), or ^-CR 11 ^R 12 -CHR ¹³ - ^{(wherein, R 11} represents hydroxymethyl, R ¹² and R ¹³ are taken together to form an epoxy group with the adjacent carbon atom)),
The compound represented by the following structural formula (1):
,
The compound represented by the following structural formula (2):
And a compound represented by the following structural formula (3):
Use of at least one compound selected from the group consisting of or a salt thereof.