WO2012128343A1

WO2012128343A1 - Agent for improving efficiency of generating induced pluripotent stem cells and efficient method for generating induced pluripotent stem cells by using same

Info

Publication number: WO2012128343A1
Application number: PCT/JP2012/057455
Authority: WO
Inventors: 直樹宮田; 孝禎鈴木; 誠人中川
Original assignee: 公立大学法人名古屋市立大学; 国立大学法人京都大学
Priority date: 2011-03-24
Filing date: 2012-03-23
Publication date: 2012-09-27
Also published as: JP2012200185A

Abstract

The present invention provides a method for improving the efficiency of generating iPS cells, the method involving bringing into contact, with somatic cells, one or more compounds selected from the group consisting of phenylcyclopropylamine derivatives, phenylalkyl hydrazine compounds such as the MAO inhibitor phenelzine, which is an antidepressant, and pharmaceutically acceptable salts, solvates and prodrugs thereof during the step of nucleus initialization.

Description

Establishing efficiency improvement agent for induced pluripotent stem cells and efficient method for establishing induced pluripotent stem cells using the same

The present invention relates to a method for improving the establishment efficiency of induced pluripotent stem cells (hereinafter referred to as iPS cells) and a drug therefor. More specifically, the present invention relates to an iPS cell establishment efficiency improving agent containing a phenylcyclopropylamine derivative or a phenylalkylhydrazine compound, and an iPS cell establishment efficiency improving method using the same.

In recent years, iPS cells derived from somatic cells without embryo destruction in mice and humans have been established one after another (Patent Literature 1, Non-Patent Literature 1-5), and serve as a transplant cell source in place of embryonic stem cells (ES cells). I have high expectations. Safer iPS cells without foreign gene integration have also been developed using protein transfer or adenoviruses or plasmids (Non-patent Documents 6-10), and application to regenerative medicine has become practical.
However, with the method that does not use retroviruses or lentiviruses, the efficiency of iPS cell establishment is still low. In particular, there are three factors except for the proto-oncogene c-Myc, which is likely to be tumorigenic in tissues and individuals differentiated from iPS cells. When human iPS cells are produced by introducing (Oct3 / 4, Sox2, Klf4) into somatic cells, there is a problem that the establishment efficiency is extremely low.

Various attempts have been made to improve iPS cell establishment efficiency. The Scripps Institute's Shen Ding et al. Group of signal transduction systems (eg TGFβ / ALK5 signal, MEK / ERK signal, Wnt signal) downstream of reprogramming factor 4 (Oct3 / 4, Sox2, Klf4, c-Myc) ) And epigenetic modifications (eg, histone deacetylation, DNA methylation), we are actively researching to complement or replace the function of the reprogramming gene using low molecular weight compounds. BIX01294 which is a transferase inhibitor, RG108 and 5aza-C which are DNA methyltransferase inhibitors, BayK which is an L-type calcium channel agonist, PD0325901 which is a MEK inhibitor, CHIR99021 which is a GSK3β inhibitor or a TGFβ receptor inhibitor SB431542 was used to improve iPS cell establishment efficiency (Patent Document 2), CHIR99021 is a lysine-specific demethylase 1 (LSD1) inhibitor, tranylcypromine (trans-2-pheny Reported PCPA) was able to establish iPS cells from human keratinocytes only two factors combine the Oct3 / 4 and Klf4 a (non-patent document 11); cyclopropylamine. Shen Ding et al. Further treated mouse epistem cells (EpiSC) with PCPA, SB431542, PD0325901, CHIR99021 and PD173074, a fibroblast growth factor receptor (FGFR) inhibitor, to give more undifferentiated ES cells. It has been reported that it can be transformed into like cells (Non-patent Document 12).
Patent Document 3 describes that when fibroblasts are cultured in the presence of a histone deacetylase (HDAC) inhibitor valproic acid (VPA) or PCPA, the expression level of Oct3 / 4 increases. Has been.

LSD1 is an enzyme that catalyzes the demethylation of monomethylated and dimethylated forms (H3K4me1 / 2) of the fourth lysine residue of histone H3 by flavin adenine dinucleotide (FAD) -dependent oxidation reaction, It is known that it is overexpressed in prostate cancer and interacts with the androgen receptor. When LSD1 is knocked down, the growth of cancer cells is suppressed.
As the LSD1 inhibitor, the above-mentioned PCPA and its derivatives are known (Non-patent Document 13), but PCPA has weak LSD1 inhibitory activity (IC ₅₀ = 21 μM), and LSD1 is a polyamine belonging to the same amine oxidase family. Because of its high homology with oxidase (PAO) and monoamine oxidase (MAO), conventionally known PCPA and its derivatives also inhibit MAO-A and MAO-B, and selectively inhibit only LSD1. There was a disadvantage that it was not possible.
Miyata and Suzuki et al. Synthesized a phenylcyclopropylamine derivative represented by the formula (1) or (2), and the compound or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof was synthesized. , LSD1 inhibitory activity is significantly higher than that of MAO-A and MAO-B, is an LSD1-selective inhibitor and has a cancer cell growth inhibitory effect, and is useful as an anticancer agent (Patent Literature 4, Non-Patent Literature 14).

(Where
R ¹ represents any ^one of hydrogen, an alkyl group to which a substituent may be bonded, a phenyl group to which a substituent may be bonded, and a heterocyclic group to which a substituent may be bonded;
R ² represents an alkylene group which may have a branch and may have a substituent bonded thereto;
R ³ represents an alkyl group to which a substituent may be bonded, a phenyl group to which a substituent may be bonded, a heterocyclic group to which a substituent may be bonded, and a benzyl to which a substituent may be bonded. Any of the groups
R ⁴ represents an alkyl group to which a substituent may be bonded, a phenyl group to which a substituent may be bonded, a heterocyclic group to which a substituent may be bonded, or an alkyl to which a substituent may be bonded. An oxy group, a phenyloxy group to which a substituent may be bonded, an alkylamino group to which a substituent may be bonded, and a phenylamino group to which a substituent may be bonded;
X represents O, NH, NHCO, CONH, S or CH ₂ . )

However, the effect of improving the establishment efficiency of iPS cells by the phenylcyclopropylamine derivative has not been reported at all. Further, either the first and Shen Ding et al reported, PCPA from that used in one order lower concentration than an IC ₅₀ value of LSD1 inhibitors (2 [mu] M), iPS cell establishment efficiency improvement effect of PCPA is based on inhibition LSD1 Whether or not remains unknown.

International Publication No. 2007/069666 Pamphlet International Publication No. 2009/117439 Pamphlet International Publication No. 2010/056831 Pamphlet International Publication No. 2010/143582 Pamphlet

An object of the present invention is to provide a means for improving the establishment efficiency of iPS cells using a low molecular weight compound, and to provide an efficient method for producing iPS cells using the same.

As a result of intensive studies to achieve the above object, the present inventors introduced four genes of Oct3 / 4, Sox2, Klf4, and L-Myc into adult skin-derived fibroblasts, and then expressed the cells in the above formula. By culturing in the presence of a phenylalkylhydrazine compound such as the phenylcyclopropylamine derivative represented by (1) or (2) or the MAO inhibitor phenelzine (phenethylhydrazine) which is an antidepressant, The inventors have found that iPS cell establishment efficiency can be remarkably improved, and have completed the present invention.

That is, the present invention is as follows.
[1] In the nuclear initialization step, the formula (I):

(Where
R ¹ represents a hydrogen atom, an optionally substituted alkyl group, an optionally substituted aryl group, an optionally substituted heterocyclic group, or a formula —NHCO—R ⁴ (wherein R ⁴ is An optionally substituted alkyl group, an optionally substituted aryl group, an optionally substituted heterocyclic group, an optionally substituted alkoxy group, an optionally substituted aryloxy group, a substituted A mono- or di-alkylamino group which may be substituted, or a mono- or di-arylamino group which may be substituted;
R ² represents an optionally substituted alkylene group,
R ³ represents an alkyl group that may be substituted, an aryl group that may be substituted, a heterocyclic group that may be substituted, or an aralkyl group that may be substituted;
X represents O, NH, NHCO, CONH, S or CH ₂ . ) And formula (II):

(Where
R is a hydrogen atom or a C _1-4 alkyl group, a C _1-3 alkoxy group, an aryl group, an aralkyl group, a phenylalkoxy group, a phenoxy group, a hydroxy group, an alkylenedioxy group, and a halogen atom. Represents 5 substituents,
R ′ represents a hydrogen atom, a C _1-3 alkyl group, a C _3-6 cycloalkyl group or an aralkyl group,
R ″ represents a hydrogen atom, a C _1-6 alkyl group, a C _1-6 hydroxyalkyl group, a C _2-4 alkenyl group, an optionally substituted aryl group, an optionally substituted aralkyl group, C _{3- 6 represents a} cycloalkyl group, a C _2-4 alkynyl group, a thienylmethyl group or a pyridylmethyl group,
Y represents a linear or branched C _2-5 alkylene group. )
A method for improving iPS cell establishment efficiency, which comprises contacting a somatic cell with one or more compounds selected from the group consisting of a compound represented by the formula: and a pharmaceutically acceptable salt, solvate and prodrug thereof.
[2] The compound represented by the formula (I) has the formula

NCL-1, represented by the formula

NCL-2 represented by the formula

NCL-3 represented by

The method according to [1] above, which is NCL-4 represented by:
[3] The method according to [1] above, wherein the compound represented by the formula (II) is phenelzine (phenethylhydrazine).

[4] Formula (I):

(Where
R is a hydrogen atom or a C _1-4 alkyl group, a C _1-3 alkoxy group, an aryl group, an aralkyl group, a phenylalkoxy group, a phenoxy group, a hydroxy group, an alkylenedioxy group, and a halogen atom. Represents 5 substituents,
R ′ represents a hydrogen atom, a C _1-3 alkyl group, a C _3-6 cycloalkyl group or an aralkyl group,
R ″ represents a hydrogen atom, a C _1-6 alkyl group, a C _1-6 hydroxyalkyl group, a C _2-4 alkenyl group, an optionally substituted aryl group, an optionally substituted aralkyl group, C _{3- 6 represents a} cycloalkyl group, a C _2-4 alkynyl group, a thienylmethyl group or a pyridylmethyl group,
Y represents a linear or branched C _2-5 alkylene group. )
And an iPS cell establishment efficiency improving agent comprising at least one compound selected from the group consisting of a compound represented by formula (I) and a pharmaceutically acceptable salt, solvate and prodrug thereof.
[5] The compound represented by the formula (I) has the formula

NCL-1, represented by the formula

NCL-2 represented by the formula

NCL-3 represented by

The agent according to [4] above, which is NCL-4 represented by the formula:
[6] The agent according to [4] above, wherein the compound represented by the formula (II) is phenelzine (phenethylhydrazine).
[7] Formula (I):

(Where
R is a hydrogen atom or a C _1-4 alkyl group, a C _1-3 alkoxy group, an aryl group, an aralkyl group, a phenylalkoxy group, a phenoxy group, a hydroxy group, an alkylenedioxy group, and a halogen atom. Represents 5 substituents,
R ′ represents a hydrogen atom, a C _1-3 alkyl group, a C _3-6 cycloalkyl group or an aralkyl group,
R ″ represents a hydrogen atom, a C _1-6 alkyl group, a C _1-6 hydroxyalkyl group, a C _2-4 alkenyl group, an optionally substituted aryl group, an optionally substituted aralkyl group, C _{3- 6 represents a} cycloalkyl group, a C _2-4 alkynyl group, a thienylmethyl group or a pyridylmethyl group,
Y represents a linear or branched C _2-5 alkylene group. )
And a step of bringing a nuclear reprogramming substance into contact with one or more compounds selected from the group consisting of a compound represented by the formula (I) and pharmaceutically acceptable salts, solvates and prodrugs thereof: iPS cell production method.
[8] The compound represented by the formula (I) is represented by the formula:

NCL-1, represented by the formula

NCL-2 represented by the formula

NCL-3 represented by

The method according to [7] above, which is NCL-4 represented by the formula:
[9] The method according to [7] above, wherein the compound represented by the formula (II) is phenelzine (phenethylhydrazine).
[10] The nuclear reprogramming substance is selected from the group consisting of Oct family members, Sox family members, Klf4 family members, Myc family members, Lin family members and Nanog, and nucleic acids encoding them. The method according to any one of [7] to [9] above.
[11] The method according to any one of [7] to [9] above, wherein the nuclear reprogramming substance is Oct3 / 4, Klf4 and Sox2, or a nucleic acid encoding them.
[12] The above [7] to [9], wherein the nuclear reprogramming substance is Oct3 / 4, Klf4, Sox2, and c-Myc or L-Myc and / or Nanog and / or Lin28 or Lin28B, or a nucleic acid encoding them. ] The method in any one of.
[13] Formula (I):

(Where
R is a hydrogen atom or a C _1-4 alkyl group, a C _1-3 alkoxy group, an aryl group, an aralkyl group, a phenylalkoxy group, a phenoxy group, a hydroxy group, an alkylenedioxy group, and a halogen atom. Represents 5 substituents,
R ′ represents a hydrogen atom, a C _1-3 alkyl group, a C _3-6 cycloalkyl group or an aralkyl group,
R ″ represents a hydrogen atom, a C _1-6 alkyl group, a C _1-6 hydroxyalkyl group, a C _2-4 alkenyl group, an optionally substituted aryl group, an optionally substituted aralkyl group, C _{3- 6 represents a} cycloalkyl group, a C _2-4 alkynyl group, a thienylmethyl group or a pyridylmethyl group,
Y represents a linear or branched C _2-5 alkylene group. )
IPS from a somatic cell, comprising one or more compounds selected from the group consisting of compounds represented by the formula (I) and pharmaceutically acceptable salts, solvates and prodrugs thereof, and a nuclear reprogramming substance. Cell inducer.
[14] The compound represented by the formula (I) is represented by the formula:

NCL-1, represented by the formula

NCL-2 represented by the formula

NCL-3 represented by

The agent according to [13] above, which is NCL-4 represented by
[15] The agent according to [13] above, wherein the compound represented by the formula (II) is phenelzine (phenethylhydrazine).
[16] The nuclear reprogramming substance is selected from the group consisting of Oct family members, Sox family members, Klf4 family members, Myc family members, Lin family members and Nanog, and nucleic acids encoding them. The agent according to any one of [13] to [15] above.
[17] The agent according to any one of [13] to [15] above, wherein the nuclear reprogramming substance is Oct3 / 4, Klf4 and Sox2, or a nucleic acid encoding them.
[18] The above [13] to [15], wherein the nuclear reprogramming substance is Oct3 / 4, Klf4, Sox2, and c-Myc or L-Myc and / or Nanog and / or Lin28 or Lin28B, or a nucleic acid encoding them. ] The agent in any one of.
[19] The following steps:
(1) a step of producing iPS cells by the method according to any one of [7] to [12], and (2) subjecting the iPS cells obtained in step (1) to differentiation induction treatment, somatic cells. The process of differentiating into
A method for producing somatic cells, comprising:
[20] Formula (I) for improving iPS cell establishment efficiency:

(Where
R is a hydrogen atom or a C _1-4 alkyl group, a C _1-3 alkoxy group, an aryl group, an aralkyl group, a phenylalkoxy group, a phenoxy group, a hydroxy group, an alkylenedioxy group, and a halogen atom. Represents 5 substituents,
R ′ represents a hydrogen atom, a C _1-3 alkyl group, a C _3-6 cycloalkyl group or an aralkyl group,
R ″ represents a hydrogen atom, a C _1-6 alkyl group, a C _1-6 hydroxyalkyl group, a C _2-4 alkenyl group, an optionally substituted aryl group, an optionally substituted aralkyl group, C _{3- 6 represents a} cycloalkyl group, C _2-4 alkynyl group, thienylmethyl group or pyridylmethyl group,
Y represents a linear or branched C _2-5 alkylene group. )
And one or more compounds selected from the group consisting of pharmaceutically acceptable salts, solvates and prodrugs thereof.
[21] The compound represented by the formula (I) is represented by the formula:

NCL-1, represented by the formula

NCL-2 represented by the formula

NCL-3 represented by

The use according to [20] above, which is NCL-4 represented by:
[22] The use according to [20] above, wherein the compound represented by the formula (II) is phenelzine (phenethylhydrazine).
[23] Formula (I) for improving iPS cell establishment efficiency:

(Where
R is a hydrogen atom or a C _1-4 alkyl group, a C _1-3 alkoxy group, an aryl group, an aralkyl group, a phenylalkoxy group, a phenoxy group, a hydroxy group, an alkylenedioxy group, and a halogen atom. Represents 5 substituents,
R ′ represents a hydrogen atom, a C _1-3 alkyl group, a C _3-6 cycloalkyl group or an aralkyl group,
R ″ represents a hydrogen atom, a C _1-6 alkyl group, a C _1-6 hydroxyalkyl group, a C _2-4 alkenyl group, an optionally substituted aryl group, an optionally substituted aralkyl group, C _{3- 6 represents a} cycloalkyl group, a C _2-4 alkynyl group, a thienylmethyl group or a pyridylmethyl group,
Y represents a linear or branched C _2-5 alkylene group. )
And one or more compounds selected from the group consisting of pharmaceutically acceptable salts, solvates and prodrugs thereof, wherein the compound is contacted with a somatic cell together with a nuclear reprogramming substance. Use, characterized by letting
[24] The compound represented by the formula (I) is represented by the formula:

NCL-1, represented by the formula

NCL-2 represented by the formula

NCL-3 represented by

The use according to [23] above, which is NCL-4 represented by:
[25] Use according to the above [23], wherein the compound represented by the formula (II) is phenelzine (phenethylhydrazine).
[26] The nuclear reprogramming substance is selected from the group consisting of Oct family members, Sox family members, Klf4 family members, Myc family members, Lin family members and Nanog, and nucleic acids encoding them. The use according to any one of [23] to [25] above.
[27] The use according to any of [23] to [25] above, wherein the nuclear reprogramming substance is Oct3 / 4, Klf4 and Sox2, or a nucleic acid encoding them.
[28] The above [23] to [25], wherein the nuclear reprogramming substance is Oct3 / 4, Klf4, Sox2, and c-Myc or L-Myc and / or Nanog and / or Lin28 or Lin28B, or a nucleic acid encoding them. ] Use in any one of.

When a compound represented by the formula (I) or (II) is brought into contact with a somatic cell at the time of nuclear reprogramming, the establishment efficiency of iPS cells can be remarkably improved. It is particularly useful for induction of human iPS cells by 3 factors except Myc.

Effects of NCL-1 to 4 and phenelzine on the efficiency of iPS cell establishment from adult skin-derived fibroblasts (HDF) by introduction of 4 genes (Oct3 / 4, Sox2, Klf4, L-Myc) (d1-d5: gene 1 to 5 days after introduction; d4-d9: days 4 to 9 after gene introduction; d7-d14: days 7 to 14 after gene introduction). The vertical axis represents the number of human iPS cell colonies. DMSO indicates the case where only the solvent is added. Effects of addition of NCL-3 and phenelzine on the efficiency of iPS cell establishment from adult skin-derived fibroblasts (HDF) by introduction of 4 genes (Oct3 / 4, Sox2, Klf4, L-Myc) (1, 5, 10, 50 , 100 and 500 μM). The vertical axis represents the number of human iPS cell colonies. DMSO indicates the case where only the solvent is added.

The present invention relates to a group consisting of LSD1 inhibitors represented by the above formulas (I) and (II) and their pharmaceutically acceptable salts, solvates and prodrugs in the nuclear reprogramming step of somatic cells. Provided is a method for improving iPS cell establishment efficiency by contacting one or more selected compounds (hereinafter collectively referred to as factors for establishing establishment efficiency of the present invention) with the somatic cell. Here, since nuclear reprogramming of somatic cells is performed by introducing a nuclear reprogramming substance into somatic cells, the present invention also provides iPS cells by contacting somatic cells with the above factors and nuclear reprogramming substances. A manufacturing method is provided. In the present specification, iPS cells cannot be established only with a nuclear reprogramming substance, and even when iPS cells are established by contacting somatic cells together with the establishment efficiency improving factor of the present invention, “improvement of establishment efficiency” Treat as applicable.

(A) Somatic cell source Somatic cells that can be used as starting materials for preparing iPS cells in the present invention are germ cells derived from mammals (eg, humans, mice, monkeys, cows, pigs, rats, dogs, etc.). May be any cell other than, for example, keratinized epithelial cells (eg, keratinized epidermal cells), mucosal epithelial cells (eg, epithelial cells of the tongue surface layer), exocrine glandular epithelial cells (eg, mammary cells), Hormone-secreting cells (eg, adrenal medullary cells), cells for metabolism and storage (eg, hepatocytes), luminal epithelial cells that make up the interface (eg, type I alveolar cells), luminal epithelium of inner chain vessels Cells (eg, vascular endothelial cells), ciliated cells with transport ability (eg, airway epithelial cells), cells for extracellular matrix secretion (eg, fibroblasts), contractile cells (eg, smooth muscle cells), Blood and immune system cells (eg, T lymphocytes), sensory Vesicles (eg, sputum cells), autonomic nervous system neurons (eg, cholinergic neurons), sensory organ and peripheral neuron support cells (eg, associated cells), central nervous system neurons and glial cells (eg, astrocytes) Glial cells), pigment cells (eg, retinal pigment epithelial cells), and their precursor cells (tissue precursor cells). There is no particular limitation on the degree of differentiation of the cells and the age of the animal from which the cells are collected, and this is the same for both undifferentiated progenitor cells (including somatic stem cells) and final differentiated mature cells. It can be used as the source of somatic cells in the invention. Examples of undifferentiated progenitor cells include tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells.

There are no particular limitations on the mammalian individual from which somatic cells are collected, but when the resulting iPS cells are used for human regenerative medicine, the patient or the type of HLA is used from the viewpoint that rejection does not occur. It is particularly preferred to collect somatic cells from others who are identical or substantially identical. Here, the type of HLA is “substantially the same” means that when the cells obtained by inducing differentiation from iPS cells derived from the somatic cells are transplanted into a patient by using an immunosuppressant or the like, the transplanted cells are This means that the HLA types match to the extent that they can be engrafted. For example, the case where the main HLA (for example, 3 loci of HLA-A, HLA-B, and HLA-DR) is the same is mentioned (the same applies hereinafter). Also, when not being administered (transplanted) to humans, for example, when iPS cells are used as a source of screening cells for evaluating the patient's drug sensitivity and the presence or absence of side effects, It is desirable to collect somatic cells from others who have the same genetic polymorphism that correlates with side effects.

Somatic cells isolated from mammals can be pre-cultured in a medium known per se suitable for culturing according to the type of cells prior to being subjected to the nuclear reprogramming step. Examples of such a medium include a minimum essential medium (MEM), Dulbecco's modified Eagle medium (DMEM), RPMI1640 medium, 199 medium, and F12 medium containing about 5 to 20% fetal calf serum. It is not limited to. When contacting with the establishment efficiency improving factor and nuclear reprogramming substance of the present invention (and, if necessary, other iPS cell establishment efficiency improving substances described later), for example, when using an introduction reagent such as a cationic liposome In some cases, it may be preferable to replace the medium with a serum-free medium in order to prevent a reduction in introduction efficiency.

(B) Establishment Efficiency Improvement Factor of the Present Invention The first establishment efficiency improvement factor of the present invention is a phenylcyclopropylamine derivative represented by formula (I) and having LSD1-selective inhibitory activity (hereinafter referred to as phenylcyclopropylamine). Derivative (I)), or a pharmaceutically acceptable salt, solvate or prodrug thereof.

(Where
R ¹ represents a hydrogen atom, an optionally substituted alkyl group, an optionally substituted aryl group, an optionally substituted heterocyclic group, or a formula —NHCO—R ⁴ (wherein R ⁴ is An optionally substituted alkyl group, an optionally substituted aryl group, an optionally substituted heterocyclic group, an optionally substituted alkoxy group, an optionally substituted aryloxy group, a substituted A mono- or di-alkylamino group which may be substituted, or a mono- or di-arylamino group which may be substituted;
R ² represents an optionally substituted alkylene group,
R ³ represents an alkyl group that may be substituted, an aryl group that may be substituted, a heterocyclic group that may be substituted, or an aralkyl group that may be substituted;
X represents O, NH, NHCO, CONH, S or CH ₂ . )

In the present specification, “alkyl (group)” means linear or branched alkyl (group), for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl. C _1-10 alkyl (group) such as pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 2-ethylbutyl, heptyl, octyl, nonyl, decyl, etc. Of these, C _1-6 alkyl (group) is preferred.

In the present specification, “alkoxy (group)” means a linear or branched alkoxy (group), for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert- Butoxy, pentyloxy, isopentyloxy, neopentyloxy, tert-pentyloxy, hexyloxy, 2,2-dimethylbutoxy, 3,3-dimethylbutoxy, 2-ethylbutoxy, heptyloxy, octyloxy, nonyloxy, decyloxy, etc. C _1-10 alkoxy (group) is preferable, and C _1-6 alkoxy (group) is particularly preferable.

In the present specification, “alkylene (group)” means a linear or branched alkylene (group), for example, methylene, ethylene, trimethylene, tetramethylene, pentamethylene, hexamethylene, 1-methylethylene. C ₁ such as 1-methyltrimethylene, 2-methyltrimethylene, 1,1-dimethylethylene, 1-ethylethylene, 1-methyltetramethylene, 2-ethyltrimethylene, heptamethylene, octamethylene, nonamethylene, decamethylene, etc. _-10 alkylene (group) is mentioned, among them, C _1-6 alkylene (group) is preferable, and ethylene is more preferable.

In the present specification, “aryl (group)” means an aromatic hydrocarbon group, for example, C _6-12 aryl (group) such as phenyl, 1-naphthyl, 2-naphthyl, anthryl, phenanthryl, biphenyl and the like. Among them, C _6-10 aryl (group) is preferable, and phenyl is more preferable.

In the present specification, regarding the “aralkyl (group)”, the aryl moiety includes the same group as the above “aryl (group)”, and the alkyl moiety thereof is the same as the above “alkyl (group)”. Groups. Preferable specific examples of “aralkyl (group)” include, for example, benzyl, 1-phenylethyl, 2-phenylethyl, 1-phenylpropyl, 1-phenylbutyl, 1-phenylpentyl, (1-naphthyl) methyl, C _6-10 aryl- such as (2-naphthyl) methyl, 1- (1-naphthyl) ethyl, 1- (2-naphthyl) ethyl, 2- (1-naphthyl) ethyl, 2- (2-naphthyl) ethyl, etc. _Examples thereof include C _1-6 alkyl (group), among which phenyl-C _1-3 alkyl (group) is preferable, and benzyl is more preferable.

In the present specification, examples of the “heterocyclic group (including“ heterocycle- ”)” include an aromatic heterocyclic group and a non-aromatic heterocyclic group.
Examples of the aromatic heterocyclic group include 4 to 7 members (preferably 5 or 6 members) containing 1 to 4 heteroatoms selected from oxygen atoms, sulfur atoms, and nitrogen atoms in addition to carbon atoms as ring constituent atoms. And monocyclic aromatic heterocyclic groups and condensed aromatic heterocyclic groups. Examples of the condensed aromatic heterocyclic group include a ring corresponding to the 4- to 7-membered monocyclic aromatic heterocyclic group and a 5- or 6-membered aromatic heterocyclic ring containing 1 or 2 nitrogen atoms. (Eg, pyrrole, imidazole, pyrazole, pyrazine, pyridine, pyrimidine) From a ring in which 1 or 2 selected from a 5-membered aromatic heterocycle containing one sulfur atom (eg, thiophene) and a benzene ring are condensed Examples include groups to be derived.
Specific examples of the aromatic heterocyclic group include, for example,
Furyl, thienyl, pyrrolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, imidazolyl, pyrazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, furazanyl, 1,2,3- Monocyclic such as thiadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, tetrazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl An aromatic heterocyclic group; and benzofuranyl, isobenzofuranyl, benzo [b] thienyl, indolyl, isoindolyl, 1H-indazolyl, benzimidazolyl, benzoxazolyl, benzo [d] isoxazolyl, benzothiazolyl, benzo [d] i Thiazolyl, 1H-benzotriazolyl, quinolyl, isoquinolyl, cinnolinyl, quinazolinyl, quinoxalinyl, phthalazinyl, naphthyridinyl, purinyl, pteridinyl, carbazolyl, α-carbolinyl, β-carbolinyl, γ-carbolinyl, acridinyl, phenoxazinyl, phenothazinyl, phenothiazinyl Phenoxathiinyl, thiantenyl, phenathidinyl, phenatridinyl, phenanthrolinyl, indolizinyl, pyrrolo [1,2-b] pyridazinyl, pyrazolo [1,5-a] pyridyl, imidazo [1,2-a] pyridyl, imidazo [ 1,5-a] pyridyl, imidazo [1,2-a] pyridazinyl, imidazo [1,2-a] pyrimidinyl, 1,2,4-triazolo [4,3-a] pyridyl, 1,2,4- Thoria B [4,3-b] fused aromatic heterocyclic group pyridazinyl and the like;
Is mentioned.
Examples of the non-aromatic heterocyclic group include 4 to 7 members (preferably 5 or 6 members) containing 1 to 4 heteroatoms selected from oxygen atoms, sulfur atoms and nitrogen atoms in addition to carbon atoms as ring constituent atoms. ) Monocyclic non-aromatic heterocyclic group and condensed non-aromatic heterocyclic group. Examples of the condensed non-aromatic heterocyclic group include a ring corresponding to the 4- to 7-membered monocyclic non-aromatic heterocyclic group, and a 5- or 6-membered aromatic containing 1 or 2 nitrogen atoms. 1 or 2 rings selected from a heterocyclic ring (eg, pyrrole, imidazole, pyrazole, pyrazine, pyridine, pyrimidine), a 5-membered aromatic heterocyclic ring containing 1 sulfur atom (eg, thiophene) and a benzene ring Examples thereof include a group derived from a condensed ring and a group obtained by partial saturation of the group.
Specific examples of the non-aromatic heterocyclic group include, for example,
Monocyclic non-aromatic heterocyclic groups such as azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, tetrahydrofuryl, thiolanyl, imidazolidinyl, pyrazolidinyl, oxazolidinyl, thiazolidinyl, piperidyl, tetrahydropyranyl, morpholinyl, thiomorpholinyl, piperazinyl, etc .; , Dihydroquinolyl, isochromenyl, chromenyl (2H-chromenyl, 4H-chromenyl), 1,2,3,4-tetrahydroisoquinolyl, 1,2,3,4-tetrahydroquinolyl, 2,3-dihydrobenzo Condensed non-aromatic heterocyclic groups such as furanyl, benzo [1,3] dioxolyl;
Is mentioned.

In the present specification, “halogen atom” means a fluorine atom, a chlorine atom, a bromine atom or an iodine atom.

In the present specification, “acyl (group)” means a carbonyl group to which a formyl group; or an “alkyl group (as defined above)” or “alkoxy group (as defined above)” is bonded. Preferred examples of “acyl (group)” include formyl; C _1-6 alkyl-carbonyl group such as acetyl, propionyl, butyryl; formyloxy; C _1-6 alkoxy- such as acetyloxy, propionyloxy, butyryloxy, etc. A carbonyl group etc. are mentioned.

In the present specification, “cycloalkyl (group)” means a cyclic non-aromatic hydrocarbon group, for example, C ₃ such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl and the like. _-10 cycloalkyl (group) can be mentioned, and C _3-6 cycloalkyl (group) is particularly preferable.

In “optionally substituted alkyl group”, “optionally substituted alkoxy group”, “optionally substituted mono- or di-alkylamino group” and “optionally substituted alkylene group” The “substituent” is not particularly limited.
(1) a halogen atom,
(2) hydroxyl group,
(3) an alkoxy group,
(4) aryloxy group,
(5) Aralkyloxy group,
(6) Heterocycle-oxy group,
(7) an acyloxy group,
(8) sulfanyl group,
(9) an alkylsulfanyl group,
(10) arylsulfanyl group,
(11) Aralkylsulfanyl group,
(12) heterocyclic-sulfanyl group,
(13) an alkylsulfonyl group,
(14) arylsulfonyl group,
(15) Aralkylsulfonyl group,
(16) Heterocycle-sulfonyl group,
(17) mono- or di-alkylamino groups,
(18) mono- or di-arylamino groups,
(19) mono- or di-aralkylamino groups,
(20) mono- or di-heterocyclic-amino groups,
(21) mono- or di-acylamino groups,
(22) carboxyl group,
(23) an alkoxycarbonyl group,
(24) alkylcarbonyl group,
(25) an arylcarbonyl group,
(26) aryloxycarbonyl group,
(27) aralkylcarbonyl group,
(28) Aralkyloxycarbonyl group,
(29) Heterocycle-carbonyl group,
(30) mono- or di-alkylcarbamoyl groups,
(31) mono- or di-arylcarbamoyl groups,
(32) mono- or di-aralkylcarbamoyl groups,
(33) mono- or di-heterocyclic-carbamoyl groups,
(34) a cycloalkyl group,
(35) an aryl group,
(36) Heterocyclic groups and the like can be mentioned.
These substituents are present at substitutable positions, and the number thereof is 1 to several, preferably 1 to 2, more preferably 1. When the number of substituents is 2 or more, they may be the same or different.
The above substituents may be further substituted with a substituent such as an alkyl group, an amino group, a hydroxyl group, an alkoxy group, a halogen atom, or a guanidino group.

“Optionally substituted aryl group”, “optionally substituted heterocyclic group”, “optionally substituted aryloxy group”, “optionally substituted mono- or di-arylamino group” The “substituent” in the “optionally substituted aralkyl group” is not particularly limited.
(1) groups exemplified as substituents such as the above-mentioned “optionally substituted alkyl group”, (2) alkyl groups (the alkyl groups are amino groups, hydroxyl groups, alkoxy groups, halogen atoms, guanidino groups, etc. (It may be substituted with a substituent.)
Etc.
These substituents are present at substitutable positions, and the number thereof is 1 to several, preferably 1 to 2, more preferably 1. When the number of substituents is 2 or more, they may be the same or different.

In formula (I), R ¹ is
Preferably, it is a hydrogen atom or the formula —NHCO—R ⁴ (wherein R ⁴ has the same meaning as described above),
More preferably, it is a hydrogen atom or a formula —NHCO—R ⁴ (wherein R ⁴ is an optionally substituted C _6-10 aryl group),
More preferably, a hydrogen atom or a formula —NHCO—R ⁴ (wherein R ⁴ is an optionally substituted phenyl (suitable substituents are a halogen atom, a C _1-6 alkyl group (substituted with a guanidino group) A C _6-10 aryl group, a non-aromatic heterocyclic-carbonyl group (preferably piperazinylcarbonyl), a C _1-6 alkyl-carbamoyl group (which may be substituted with an amino group) )
Particularly preferred is a hydrogen atom or the formula —NHCO—R ⁴ (wherein R ⁴ is phenyl).

In the formula (I), R ² is preferably an optionally substituted C _1-6 alkylene group, more preferably an optionally substituted ethylene, and particularly preferably ethylene.

In the formula (I), R ³ is
Preferably, it is an aryl group which may be substituted, or an aralkyl group which may be substituted,
More preferably, it is an optionally substituted C _6-10 aryl group, or an _optionally substituted C _6-10 aryl-C _1-3 alkyl group,
More preferably, it is an optionally substituted C _6-10 aryl-C _1-3 alkyl group,
Even more preferably, it is an optionally substituted benzyl (suitable substituents are a halogen atom, a C _1-6 alkyl group, a C _3-6 cycloalkyl group),
Particularly preferred is benzyl.

In the formula (I), X is preferably O.

In formula (I), the amino-substituted cyclopropyl group is preferably bonded to the meta position or the para position with respect to the group X. That is, formula (I) is preferably

Or

It is.

Suitable phenylcyclopropylamine derivatives (I) are:
R ¹ is a hydrogen atom or a group represented by the formula —NHCO—R ⁴ (wherein R ⁴ is as defined above),
R ² is an optionally substituted C _1-6 alkylene group,
R ³ is an aryl group which may be substituted, or an aralkyl group which may be substituted;
A compound in which X is O.

More preferred phenylcyclopropylamine derivatives (I) are:
R ¹ is a hydrogen atom or a group represented by the formula —NHCO—R ⁴ (wherein R ⁴ is an optionally substituted C _6-10 aryl group);
R ² is an optionally substituted C _1-6 alkylene group,
R ³ is an optionally substituted C _6-10 aryl group, or an _optionally substituted C _6-10 aryl-C _1-3 alkyl group;
A compound in which X is O.

Further suitable phenylcyclopropylamine derivatives (I) are:
R ¹ is a hydrogen atom or a group represented by the formula —NHCO—R ⁴ (wherein R ⁴ is an optionally substituted phenyl);
R ² is ethylene which may be substituted,
R ³ is an optionally substituted C _6-10 aryl-C _1-3 alkyl group,
A compound in which X is O.

Even more preferred phenylcyclopropylamine derivatives (I) are:
R ¹ is a hydrogen atom or a group represented by the formula —NHCO—R ⁴ (wherein R ⁴ is an optionally substituted phenyl);
R ² is ethylene,
R ³ is an optionally substituted benzyl,
A compound in which X is O.

Particularly suitable phenylcyclopropylamine derivatives (I) are:
R ¹ is a hydrogen atom or a group represented by the formula —NHCO—R ⁴ (wherein R ⁴ is phenyl),
R ² is ethylene,
R ³ is benzyl,
A compound in which X is O.

Specifically, the formula

NCL-1, represented by the formula

NCL-2 represented by the formula

NCL-3 represented by the formula

NCL-4, more preferably NCL-1, NCL-3 and NCL-4, still more preferably NCL-1 and NCL-3, and particularly preferably NCL-3.

Examples of the pharmaceutically acceptable salt of the phenylcyclopropylamine derivative (I) include inorganic acid salts such as hydrochloride, hydrobromide, phosphate, sulfate, and nitrate, formate, and acetate. , Propionate, maleate, fumarate, succinate, lactate, malate, tartrate, citrate, ascorbate, malonate, oxalate, glycolate, phthalate And organic acid salts such as benzene sulfonate. These salts can also be used in combination. Hydrochloride is preferable.

The prodrug of the phenylcyclopropylamine derivative (I) is a compound that is hydrolyzed in vivo and converted to the phenylcyclopropylamine derivative (I). For example, the amino group is substituted with an alkanoyl group (acyl group). Derivatives (ie, amidated derivatives), hemiaminal ether derivatives, derivatives substituted with alkoxycarbonyloxymethyl groups, N-oxide derivatives, and the like.

The phenylcyclopropylamine derivative (I) can be synthesized by the method described in WO 2010/143582 and a method analogous thereto. Specifically, it can be synthesized by the following production method. In addition, each reaction condition in a scheme shows an example, and those skilled in the art can appropriately change and modify as desired. Unless otherwise specified, the definition of each symbol in the scheme is as defined above.
Hereinafter, R ¹ in the formula (I) is a group represented by the formula —NHCO—R ⁴ (wherein R ⁴ is as defined above), X is O, and R ² is ethylene. The case of the group will be described as an example.
Production Method 1 (Production of Compound of Formula (I) —Meta Position)
Step 1-1

The compound (9), which is a precursor, is synthesized by the above process.
Step 1-2

The compound (13), which is a precursor, is synthesized by the above process.
Step 1-3
Using the compound (9), which is the coupling precursor synthesized in Step 1-1, and the compound (13), which is the coupling precursor synthesized in Step 1-2, according to the synthesis route shown below, Mitsunobu reaction is performed. Perform the coupling reaction used.

Production Method 2 (Production of Compound of Formula (I)-Para position)
Step 2-1

The compound (21), which is a precursor, is synthesized by the above process.
Step 2-2
Using the compound (13), which is the coupling precursor synthesized in Step 1-2, and the compound (21), which is the coupling precursor synthesized in Step 2-1, according to the synthesis route shown below, Mitsunobu reaction is performed. Perform the coupling reaction used.

Production Method 3 (Production of Compound of Formula (I) —Meta Position)
Step 3-1

The compound (24) which is a precursor is synthesize | combined by the said process.
Step 3-2
Mitsunobu reaction was performed according to the synthesis route shown below using compound (9), which is the coupling precursor synthesized in step 1-1, and compound (24), which is the coupling precursor synthesized in step 3-1. Perform the coupling reaction used.

Production Method 4 (Production of Compound of Formula (I)-Para position)
Mitsunobu reaction was performed according to the synthesis route shown below using compound (21), which is the coupling precursor synthesized in step 2-1, and compound (24), which is the coupling precursor synthesized in step 3-1. Perform the coupling reaction used.

The second establishment efficiency improving factor of the present invention is a phenylalkylhydrazine compound represented by the formula (II) (hereinafter also referred to as phenylalkylhydrazine compound (II)), or a pharmaceutically acceptable salt or solvate thereof. Or it is a prodrug.

(Where
R is a hydrogen atom or a C _1-4 alkyl group, a C _1-3 alkoxy group, an aryl group, an aralkyl group, a phenylalkoxy group, a phenoxy group, a hydroxy group, an alkylenedioxy group, and a halogen atom. Represents 5 substituents,
R ′ represents a hydrogen atom, a C _1-3 alkyl group, a C _3-6 cycloalkyl group or an aralkyl group,
R ″ represents a hydrogen atom, a C _1-6 alkyl group, a C _1-6 hydroxyalkyl group, a C _2-4 alkenyl group, an optionally substituted aryl group, an optionally substituted aralkyl group, C _{3- 6 represents a} cycloalkyl group, a C _2-4 alkynyl group, a thienylmethyl group or a pyridylmethyl group,
Y represents a linear or branched C _2-5 alkylene group. )

Examples of the C _1-4 alkyl group for the substituent R include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, and the like. Among these, C _1-3 alkyl (group) is exemplified. preferable.
Examples of the C _1-4 alkoxy group for the substituent R include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy and the like, among which C _1-3 alkoxy (group) Is preferred.
Examples of the aryl group in the substituent R include C _6-12 aryl (group) such as phenyl, 1-naphthyl, 2-naphthyl, anthryl, phenanthryl, biphenyl, and the like, and among them, phenyl is preferable.
Examples of the aralkyl group in the substituent R include benzyl, 1-phenylethyl, 2-phenylethyl, 1-phenylpropyl, 1-phenylbutyl, etc. Among them, phenyl-C _1-3 alkyl (group) is preferable. Benzyl is more preferable.
Examples of the phenylalkoxy group in the substituent R include phenylmethoxy, phenylethoxy, phenylpropoxy and the like.
Examples of the alkylenedioxy group in the substituent R include methylenedioxy and ethylenedioxy.
Examples of the halogen atom in the substituent R include a fluorine atom, a chlorine atom, a bromine atom or an iodine atom, and a chlorine atom is preferable.

Examples of the C _1-3 alkyl group for the substituent R ′ include methyl, ethyl, propyl, isopropyl and the like.
Examples of the C _3-6 cycloalkyl group for the substituent R ′ include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.
Examples of the aralkyl group in the substituent R ′ include benzyl, 1-phenylethyl, 2-phenylethyl, 1-phenylpropyl and the like, and among them, benzyl is preferable.

Examples of the C _1-6 alkyl group for the substituent R ″ include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, 2, Examples include 2-dimethylbutyl, 3,3-dimethylbutyl, 2-ethylbutyl and the like, and among these, C _1-3 alkyl (group) is preferable.
Examples of the C _1-6 hydroxyalkyl group in the substituent R ″ include hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl, hydroxypentyl, hydroxyhexyl and the like.
Examples of the C _2-4 alkenyl group for the substituent R ″ include vinyl, allyl, 1- (2-propenyl), 1- (2-butenyl) and the like.
Examples of the aryl group which may be substituted in the substituent R ″ include phenyl, hydroxyphenyl, methoxyphenyl, chlorophenyl, acetoxyphenyl and the like.
Examples of the aralkyl group which may be substituted in the substituent R ″ include phenethyl, phenylpropyl, phenylisopropyl, p-chlorophenylpropyl and the like.
Examples of the C _3-6 cycloalkyl group for the substituent R ″ include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.
Examples of the C _2-4 alkynyl group in the substituent R ″ include ethynyl, propynyl, butynyl and the like.

Examples of the linear or branched C _2-5 alkylene group in the substituent Y include, for example, ethylene, 1-methylethylene, propylene, 2-methylmethylene, butylene, 1-methylpropylene, 2-methylpropylene, and 3-methylpropylene. , 3-methylbutylene and the like, and ethylene is preferable.

Particularly suitable phenylalkylhydrazine compounds (II) are:
R, R ′ and R ″ are hydrogen atoms;
A compound represented by the following formula in which Y is ethylene, that is, phenelzine (phenethylhydrazine).

Examples of the pharmaceutically acceptable salt of the phenylalkylhydrazine compound (II) include inorganic acid salts such as hydrochloride, hydrobromide, phosphate, sulfate, nitrate, formate, acetate, Propionate, maleate, fumarate, succinate, lactate, malate, tartrate, citrate, ascorbate, malonate, oxalate, glycolate, phthalate, Examples include organic acid salts such as benzene sulfonate. These salts can also be used in combination. Hydrochloride is preferable.

The prodrug of the phenylalkyl hydrazine compound (II) refers to a compound that is hydrolyzed in vivo and converted to the phenylalkyl hydrazine compound (II). For example, a derivative in which an amino group is substituted with an alkanoyl group (acyl group) (Ie, amidated derivatives), hemiaminal ether derivatives, derivatives substituted with alkoxycarbonyloxymethyl groups, N-oxide derivatives and the like.

The phenylalkylhydrazine compound (II) can be synthesized by the method described in US Pat. No. 3,000,903 and a method analogous thereto.

The establishment efficiency improving factor of the present invention can be used in a concentration range that is sufficient for improving the establishment efficiency of iPS cells and does not show cytotoxicity. For example, in the case of NCL-3, 0.1 to 50 μM, preferably Can be used at a concentration of 1 to 10 μM, in the case of phenelzine, 0.1 to 200 μM, preferably 1 to 100 μM.
The contact of the establishment efficiency improving factor of the present invention with somatic cells is achieved by dissolving the factor in a non-aqueous solvent such as DMSO at an appropriate concentration, and a medium suitable for culturing somatic cells isolated from humans or other mammals. (Eg, minimal essential medium (MEM), Dulbecco's modified Eagle medium (DMEM), RPMI1640 medium, 199 medium, F12 medium (which may contain about 5-20% fetal calf serum), etc.) It can be carried out by adding cells so that the concentration is in the above range and culturing the cells for a certain period. The contact period is not particularly limited as long as it is sufficient to achieve somatic cell nuclear reprogramming. For example, the contact period may be about 4 to about 14 days, preferably about 5 to 10 days. The timing of contact with the somatic cells is not particularly limited, and the somatic cells may be contacted simultaneously with the nuclear reprogramming substance, or may be contacted about 1 to about 10 days after the contact with the nuclear reprogramming substance. Preferably, the establishment efficiency improving factor of the present invention can be contacted with a somatic cell about 5 to about 10 days after contact with the nuclear reprogramming substance, more preferably about 6 to about 8 days.

(C) Nuclear reprogramming substance In the present invention, the “nuclear reprogramming substance” refers to an iPS cell derived from a somatic cell by introducing it into a somatic cell or by contacting the somatic cell together with the establishment efficiency improving factor of the present invention. As long as it is a substance (group) capable of inducing protein, it may be composed of any substance such as a protein factor or a nucleic acid encoding the same (including a form incorporated in a vector) or a low molecular weight compound. When the nuclear reprogramming substance is a protein factor or a nucleic acid encoding the same, the following combinations are preferably exemplified (in the following, only the name of the protein factor is described).
(1) Oct3 / 4, Klf4, c-Myc
(2) Oct3 / 4, Klf4, c-Myc, Sox2 (where Sox2 can be replaced with Sox1, Sox3, Sox15, Sox17 or Sox18. Klf4 can be replaced with Klf1, Klf2 or Klf5. Furthermore, c-Myc can be replaced with T58A (active mutant) or L-Myc.)
(3) Oct3 / 4, Klf4, c-Myc, Sox2, Fbx15, Nanog, ERas, TclI
(4) Oct3 / 4, Klf4, c-Myc, Sox2, TERT, SV40 Large T antigen (SV40LT)
(5) Oct3 / 4, Klf4, c-Myc, Sox2, TERT, HPV16 E6
(6) Oct3 / 4, Klf4, c-Myc, Sox2, TERT, HPV16 E7
(7) Oct3 / 4, Klf4, c-Myc, Sox2, TERT, HPV6 E6, HPV16 E7
(8) Oct3 / 4, Klf4, c-Myc, Sox2, TERT, Bmil
(Refer to WO 2007/069666 above (however, regarding the substitution of Sox2 to Sox18 and the substitution of Klf4 to Klf1 or Klf5 in the combination of (2) above, see Nature Biotechnology, 26, 101-106 (2008). For the combination of “Oct3 / 4, Klf4, c-Myc, Sox2,” see also Cell, 126, 663-676 (2006), Cell, 131, 861-872 (2007), etc. “Oct3 / 4 , Klf2 (or Klf5), c-Myc, Sox2 ”, see also Nat. Cell Biol., 11, 197-203 (2009)“ Oct3 / 4, Klf4, c-Myc, Sox2, hTERT, (See also Nature, 451, 141-146 (2008) for "SV40LT" combinations.)
(9) Oct3 / 4, Klf4, Sox2 (see Nature Biotechnology, 26, 101-106 (2008))
(10) Oct3 / 4, Sox2, Nanog, Lin28 (see Science, 318, 1917-1920 (2007))
(11) Oct3 / 4, Sox2, Nanog, Lin28, hTERT, SV40LT (see Stem Cells, 26, 1998-2005 (2008))
(12) Oct3 / 4, Klf4, c-Myc, Sox2, Nanog, Lin28 (see Cell Research (2008) 600-603)
(13) Oct3 / 4, Klf4, c-Myc, Sox2, SV40LT (see also Stem Cells, 26, 1998-2005 (2008))
(14) Oct3 / 4, Klf4 (see Nature 454: 646-650 (2008), Cell Stem Cell, 2: 525-528 (2008))
(15) Oct3 / 4, c-Myc (see Nature 454: 646-650 (2008))
(16) Oct3 / 4, Sox2 (see Nature, 451, 141-146 (2008), WO2008 / 118820)
(17) Oct3 / 4, Sox2, Nanog (see WO2008 / 118820)
(18) Oct3 / 4, Sox2, Lin28 (see WO2008 / 118820)
(19) Oct3 / 4, Sox2, c-Myc, Esrrb (where Essrrb can be replaced by Esrrg; see Nat. Cell Biol., 11, 197-203 (2009))
(20) Oct3 / 4, Sox2, Esrrb (see Nat. Cell Biol., 11, 197-203 (2009))
(21) Oct3 / 4, Klf4, L-Myc (See Proc. Natl. Acad. Sci. USA., 107, 14152-14157 (2010))
(22) Oct3 / 4, Nanog
(23) Oct3 / 4 (See Cell 136: 411-419 (2009), Nature, 08436, doi: 10.1038 published online (2009))
(24) Oct3 / 4, Klf4, c-Myc, Sox2, Nanog, Lin28, SV40LT (see Science, 324: 797-801 (2009))
(25) Nr5a2, Sox2, Klf4, c-Myc (where Nr5a2 can be replaced by Nr5a1, see Cell Stem Cell. 6: 167-74 (2010))
(26) Oct3 / 4, Sox2, Klf4, Tbx3 (see Nature. 463: 1096-100 (2010))
(27) Nr5a2, Sox2, c-Myc, GLISl (see WO 2010/098419)

In the above (1)-(27), when Oct3 / 4 is included, other Oct family members such as Oct1A and Oct6 can be used instead of Oct3 / 4. When Sox2 is included, other Sox family members such as Sox7 can be used instead of Sox2 (or Sox1, Sox3, Sox15, Sox17, Sox18). Further, when c-Myc or Lin28 is included as a nuclear reprogramming substance in the above (1)-(27), L-Myc or Lin28B can be used instead of c-Myc or Lin28, respectively.

A combination that does not fall under the above (1)-(27) but includes all of the components in any of them and further includes any other substance is also included in the category of “nuclear reprogramming substance” in the present invention. Can be included. In addition, the condition that the somatic cells subject to nuclear reprogramming endogenously express some of the components in any of the above (1)-(27) at a sufficient level for nuclear reprogramming. In this case, a combination of only the remaining components excluding the component can also be included in the category of “nuclear reprogramming substance” in the present invention.

Among these combinations, at least one selected from Oct3 / 4, Sox2, Klf4, c-Myc or L-Myc, Nanog, Lin28 or Lin28B and SV40LT, preferably two or more, more preferably three or more Are examples of preferred nuclear reprogramming materials.
In particular, when the obtained iPS cells are used for therapeutic purposes, a combination of reprogramming factors not using c-Myc is preferable. For example, a combination of three factors Oct3 / 4, Sox2 and Klf4 (ie (9) above), a combination of four factors Oct3 / 4, Sox2, Klf4 and L-Myc (ie (2) above), or A combination including a combination and not including c-Myc can be exemplified. On the other hand, when iPS cells are not used for therapeutic purposes (for example, as a research tool for drug discovery screening), the three factors Oct3 / 4, Sox2 and Klf4, Oct3 / 4, Sox2, In addition to 4 factors of Klf4 and L-Myc, 4 factors of Oct3 / 4, Sox2, Klf4 and c-Myc, Oct3 / 4, Sox2, Klf4, c-Myc / L-Myc and / or Nanog and / or Lin28 / Examples include 5 or 6 factors of Lin28B, and 6 or 7 factors added with SV40 Large T.

Mouse and human cDNA sequence information of each of the above protein factors can be obtained by referring to NCBI accession numbers described in WO 2007/069666 or WO 2010/098419 (Nanog is “ECAT4” in the publication) The mouse and human cDNA sequence information of Lin28, Lin28B, Esrrb, Esrrg, L-Myc, Nr5a2, Nr5a1, and Tbx3 can be obtained by referring to the following NCBI accession numbers, respectively. ), Those skilled in the art can easily isolate these cDNAs.
Gene name mouse human
Lin28 NM_145833 NM_024674
Lin28b NM_001031772 NM_001004317
Esrrb NM_011934 NM_004452
Esrrg NM_011935 NM_001438
L-Myc NM_008506 NM_001033081
Nr5a2 NM_030676 NM_205860
Nr5a1 NM_139051 NM_004959
Tbx3 NM_011535 NM_005996

When proteinaceous factor itself is used as a nuclear reprogramming substance, the obtained cDNA is inserted into an appropriate expression vector, introduced into a host cell, and cultured from the resulting culture. Can be prepared by recovering. On the other hand, when a nucleic acid encoding a protein factor is used as a nuclear reprogramming substance, the obtained cDNA is inserted into a viral vector, plasmid vector, episomal vector or the like to construct an expression vector, which is then used for the nuclear reprogramming step. Is done.

(D) Method of introducing nuclear reprogramming substance into somatic cells When the substance is a proteinaceous factor, the nuclear reprogramming substance is introduced into a somatic cell using a known method for introducing protein into cells. be able to. Examples of such methods include a method using a protein introduction reagent, a method using a protein introduction domain (PTD) or a cell-penetrating peptide (CPP) fusion protein, and a microinjection method. Protein introduction reagents include cationic lipid-based BioPOTER Protein Delivery Reagent (Gene Therapy Systmes), Pro-Ject ^™ Protein Transfection Reagent (PIERCE) and ProVectin (IMGENEX), and lipid-based Profect-1 (Targeting Systems) ), Penetrain Peptide (Q biogene) and Chariot Kit (Active Motif) based on a membrane-permeable peptide, GenomONE (Ishihara Sangyo) using HVJ envelope (inactivated Sendai virus), and the like are commercially available. The introduction can be carried out according to the protocol attached to these reagents, but the general procedure is as follows. Dilute the nuclear reprogramming substance in an appropriate solvent (for example, buffer solution such as PBS, HEPES, etc.), add the introduction reagent and incubate at room temperature for about 5-15 minutes to form a complex. Add to the exchanged cells and incubate at 37 ° C for 1 to several hours. Thereafter, the medium is removed and replaced with a serum-containing medium.

As PTD, Drosophila-derived AntP, HIV-derived TAT (Frankel, A. et al, Cell 55, 1189-93 (1988); Green, M. & Loewenstein, PM Cell 55, 1179-88 (1988)), Penetratin (Derossi, D. et al, J. Biol. Chem. 269, 10444-50 (1994)), Buforin II (Park, C. B. et al. Proc. Natl Acad. Sci. USA 97, 8245-50 (2000)), Transportan (Pooga, M. et al. FASEB J. 12, 67-77 (1998)), MAP (model amphipathic peptide) (Oehlke, J. et al. Biochim. Biophys. Acta. 1414, 127 -39 (1998)), K-FGF (Lin, Y. Z. et al. J. Biol. Chem. 270, 14255-14258 (1995)), Ku70 (Sawada, M. et al. Nature Cell Biol. 5 , 352-7 (2003)), Prion (Lundberg, P. et al. Biochem. Biophys Res. Commun. 299, 85-90 2002 (2002)), pVEC (Elmquist, A. et al. Exp. 269, 237-44 (2001)), Pep-1 (Morris, M. C. et al. Nature Biotechnol. 19, 1173-6 (2001)), Pep-7 (Gao, C. et al. Bioorg. Med . Chem. 10, 4057-65 (2002)), SynBl (Rousselle, C. et al. Mol. Pharmacol. 57, 679-86 (2000)), HN-I (Hong, F. D. & Clayman, G L. Cancer Res. 60, 6551 -6 (2000)), those using cell-passing domains of proteins such as VP22 derived from HSV have been developed. Examples of CPP derived from PTD include polyarginine such as 11R (Cell Stem Cell, 4: 381-384 (2009)) and 9R (Cell Stem Cell, 4: 472-476 (2009)).

A fusion protein expression vector incorporating a nuclear reprogramming substance cDNA and a PTD or CPP sequence is prepared and recombinantly expressed, and the fusion protein is recovered and used for introduction. Introduction can be performed in the same manner as described above except that no protein introduction reagent is added.

Microinjection is a method in which a protein solution is put into a glass needle having a tip diameter of about 1 μm and puncture is introduced into a cell, and the protein can be reliably introduced into the cell.

The protein introduction operation can be performed any number of one or more times (for example, 1 to 10 times, or 1 to 5 times, etc.), and preferably the introduction operation is performed twice or more (for example, 3 or 4 times). ) Can be done repeatedly. Examples of the interval when the introduction operation is repeated include 6 hours to 7 days, preferably 12 to 48 hours or 7 days.

If importance is attached to the establishment efficiency of iPS cells, it is preferable to use the nuclear reprogramming substance in the form of a nucleic acid that encodes it rather than as a protein factor itself. The nucleic acid may be DNA or RNA, or may be a DNA / RNA chimera. The nucleic acid may be double-stranded or single-stranded. Preferably the nucleic acid is double stranded DNA, in particular cDNA.
The cDNA of the nuclear reprogramming substance is inserted into an appropriate expression vector containing a promoter that can function in a host somatic cell. Examples of expression vectors include retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpes viruses, Sendai virus and other viral vectors, animal cell expression plasmids (eg, pA1-11, pXT1, pRc / CMV, pRc / RSV). , PcDNAI / Neo) or the like.

The type of vector to be used can be appropriately selected according to the intended use of the iPS cells obtained. For example, adenovirus vectors, plasmid vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, Sendai virus vectors, episomal vectors and the like can be used.

Examples of the promoter used in the expression vector include EF1α promoter, CAG promoter, SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (rous sarcoma virus) promoter, MoMuLV (Molone murine leukemia virus) LTR. HSV-TK (herpes simplex virus thymidine kinase) promoter and the like are used. Of these, EF1α promoter, CAG promoter, MoMuLV LTR, CMV promoter, SRα promoter and the like are preferable.

In addition to the promoter, the expression vector may contain an enhancer, a poly A addition signal, a selection marker gene, an SV40 replication origin, and the like as desired. Examples of the selection marker gene include a dihydrofolate reductase gene, a neomycin resistance gene, a puromycin resistance gene, and the like.

Nucleic acid that is a nuclear reprogramming substance (reprogramming gene) may be incorporated on separate expression vectors, or two or more, preferably 2-3 types of genes may be incorporated into one expression vector. It is preferable to select the former when using a retrovirus or lentiviral vector with high gene transfer efficiency, and the latter when using a plasmid, adenovirus, episomal vector, or the like. Furthermore, an expression vector incorporating two or more types of genes and an expression vector incorporating only one gene can be used in combination.

When a plurality of reprogramming genes (for example, two or more selected from Oct3 / 4, Sox2, Klf4, and c-Myc, preferably 2 to 3 genes) are incorporated into one expression vector, the plurality of genes are Can be incorporated into the expression vector, preferably via a sequence allowing polycistronic expression. By using a sequence that allows polycistronic expression, a plurality of genes incorporated in one type of expression vector can be expressed more efficiently. Examples of sequences enabling polycistronic expression include 2A sequences of foot-and-mouth disease virus (PLoS ONE3, e2532, 2008, Stem Cells 25, 1707, 2007), IRES sequences (US Patent No. 4,937,190), preferably 2A An array can be used.

The expression vector containing the reprogramming gene can be introduced into cells by a technique known per se, depending on the type of vector. For example, in the case of a viral vector, a virus produced in the culture supernatant by introducing a plasmid containing the nucleic acid into an appropriate packaging cell (eg, Plat-E cell) or a complementary cell line (eg, 293 cell) The vector is collected and cells are infected with the vector by an appropriate method according to each viral vector. For example, specific means using a retroviral vector as a vector are disclosed in WO2007 / 69666, Cell, 126, 663-676 (2006) and Cell, 131, 861-872 (2007). The case of use is disclosed in Science, 318, 1917-1920 (2007) 2007. When iPS cells are used for medical purposes such as transplantation therapy, the expression (reactivation) of reprogramming genes may increase the risk of carcinogenesis in transplanted cells differentiated from iPS cells. It is preferable that it is transiently expressed without being integrated into the chromosome. From this point of view, it is preferable to use an adenovirus vector that rarely integrates into the chromosome. Specific means using an adenoviral vector is disclosed in Science, 322, 945-949 (2008). In addition, adeno-associated virus also has a low frequency of integration into chromosomes, and has lower cytotoxicity and inflammation-inducing action than adenovirus vectors, and thus can be mentioned as another preferred vector. The Sendai virus vector can exist stably outside the chromosome, and can be preferably used in the same manner because it can be decomposed and removed by siRNA as necessary. As the Sendai virus vector, those described in J. Biol. Chem., 282, 27383-27391 (2007) and Japanese Patent No. 3602058 can be used.

When using a retrovirus vector or lentivirus vector, even if silencing of the transgene occurs, it may be reactivated later, so it became unnecessary, for example, using the Cre / loxP system A method of excising a nucleic acid encoding a nuclear reprogramming substance at a time point can be preferably used. That is, loxP sequences are arranged at both ends of the nucleic acid, and after iPS cells are induced, Cre recombinase is allowed to act on the cells using a plasmid vector or an adenovirus vector to cut out the region sandwiched between the loxP sequences. be able to. In addition, the enhancer-promoter sequence in the LTR 領域 U3 region may up-regulate nearby host genes by insertion mutation. Therefore, the 3′-self is deleted or replaced with a polyadenylation sequence such as SV40. More preferably, an inactivated (SIN) LTR is used to avoid expression control of the endogenous gene by an LTR outside the loxP sequence that is not excised and remains in the genome. Specific means using the Cre-loxP system and the SIN LTR are disclosed in Chang et al., Stem Cells, 27: 1042-1049 (2009).

On the other hand, in the case of a plasmid vector which is a non-viral vector, the vector is transferred to cells using lipofection method, liposome method, electroporation method, calcium phosphate coprecipitation method, DEAE dextran method, microinjection method, gene gun method, etc. Can be introduced. Specific means using a plasmid as a vector are described in, for example, Science, 322, 949-953 (2008).

When a plasmid vector, adenovirus vector, or the like is used, gene transfer can be performed any number of times of 1 or more (for example, 1 to 10 times, or 1 to 5 times). When two or more types of expression vectors are introduced into a somatic cell, it is preferable to introduce all these types of expression vectors into the somatic cell at the same time. The number of times (for example, 1 or more and 10 or less, or 1 or more and 5 or less, etc.) can be performed, and the introduction operation can be preferably repeated by 2 or more times (for example, 3 or 4 times).

Even when an adenovirus or a plasmid is used, since the transgene may be integrated into the chromosome, it is necessary to finally confirm that there is no gene insertion into the chromosome by Southern blotting or PCR. Therefore, it may be advantageous to use a means for removing the gene after the transgene has been once integrated into the chromosome, as in the Cre-loxP system. In another preferred embodiment, there is a method for completely removing a transgene from a chromosome by incorporating a transgene into a chromosome using a transposon and then allowing a transferase to act on the cell using a plasmid vector or an adenovirus vector. Can be used. Preferred transposons include, for example, piggyBac, which is a transposon derived from a lepidopteran insect. Specific means using the piggyBac transposon are disclosed in Kaji, K. et al., Nature, 458: 771-775 (2009), Woltjen et al., Nature, 458: 766-770 (2009).
Another preferred non-integrated vector is an episomal vector capable of autonomous replication outside the chromosome. Specific means using an episomal vector is disclosed in Yu et al., Science, 324, 797-801 (2009).

Examples of the episomal vector used in the present invention include a vector containing a sequence necessary for autonomous replication derived from EBV, SV40, etc. as a vector element. Specifically, vector elements necessary for autonomous replication include a replication origin and a gene encoding a protein that binds to the replication origin and controls replication. For example, in EBV, the replication origin oriP And EBNA-1 gene, SV40 includes the origin of replication ori and SV40 large T antigen gene.

The episomal expression vector also contains a promoter that controls transcription of the reprogramming gene. As the promoter, the same promoter as described above can be used. Moreover, the episomal expression vector may further contain an enhancer, a poly A addition signal, a selection marker gene, and the like as desired, as described above. Examples of the selection marker gene include a dihydrofolate reductase gene and a neomycin resistance gene.

Episomal vectors can be introduced into cells using, for example, lipofection method, liposome method, electroporation method, calcium phosphate coprecipitation method, DEAE dextran method, microinjection method, gene gun method and the like. Specifically, for example, the method described in Science, 324: 797-801 (2009) can be used.

Whether or not the episomal vector has been removed from the iPS cell is determined by performing Southern blot analysis or PCR analysis using a part of the vector as a probe or primer and the episomal fraction isolated from the iPS cell as a template. It can be carried out by examining the presence or absence of the light or the length of the detection band. The episomal fraction may be prepared by a method well known in the art, for example, a method described in Science, 324: 797-801 (2009) or the like.

(E) Other iPS cell establishment efficiency improving substances Since the establishment efficiency of conventional iPS cells is low, various substances for improving the efficiency have recently been proposed. Therefore, in addition to the establishment efficiency improving factor of the present invention, it is expected that the establishment efficiency of iPS cells can be further enhanced by bringing other establishment efficiency improving substances into contact with somatic cells.
Other iPS cell establishment efficiency improving substances include, for example, histone deacetylase (HDAC) inhibitors [for example, valproic acid (VPA), trichostatin A (TSA), sodium butyrate (Cell Stem Cell, 7: 651- 655 (2010)), small molecule inhibitors such as MC 1293, M344, siRNA and shRNA against HDAC (eg, HDAC1 siRNA Smartpool ^TM (Millipore), HuSH 29mer shRNA Constructs against HDAC1 (OriGene), etc.) Agents], DNA methyltransferase inhibitors (eg 5'-azacytidine (5'azaC)) (Nat. Biotechnol., 26 (7): 795-797 (2008)), G9a histone methyltransferase inhibitors [eg BIX -01294 (Cell Stem Cell, 2: 525-528 (2008)) and other small molecule inhibitors, G9a siRNA and shRNA (eg, G9a siRNA (human) (Santa Cruz Biotechnology) etc.) and other nucleic acid expression inhibitors Etc.], L-channel calcium agonist (eg Bayk8644) (Cell Stem Cell, 3, 568-574 (2008)), p53 inhibitor (eg p5 SiRNA against shRNA, shRNA, dominant negative etc. (Cell Stem Cell, 3, 475-479 (2008); Nature 460, 1132-1135 (2009))), Wnt Signaling (eg soluble Wnt3a) (Cell Stem Cell, 3, 132-135 (2008)), 2i / LIF (2i is an inhibitor of mitogen-activated protein kinase signaling and glycogen synthase kinase-3; PLoS Biology, 6 (10), 2237-2247 (2008)), ES cell specific miRNA (eg miR-302-367 cluster (Mol. Cell. Biol. doi: 10.1128 / MCB.00398-08), miR-302 (RNA (2008) 14: 1-10), miR-291-3p, miR -294 and miR-295 (Nat. Biotechnol. 27: 459-461 (2009))), 3'-phosphoinositide-dependent kinase-1 (PDK1) activator (eg, PS48 (Cell Stem Cell, 7: 651- 655 (2010))), neuropeptide Y (WO 2010/147395), prostaglandins (for example, prostaglandin E2 and prostaglandin J2) (WO 2010/068955), and the like. Not. The nucleic acid expression inhibitor may be in the form of an expression vector containing DNA encoding siRNA or shRNA.

Among the components of the nuclear reprogramming substance, for example, SV40 large T is not an essential factor for somatic cell nuclear reprogramming, but is an auxiliary factor. It can also be included in a category. In the current situation where the mechanism of nuclear reprogramming is not clear, whether auxiliary factors other than those essential for nuclear reprogramming are positioned as nuclear reprogramming substances or substances that improve the establishment efficiency of iPS cells. It may be convenient. In other words, the somatic cell nuclear reprogramming process is regarded as an overall event caused by the contact of the somatic cell with the nuclear reprogramming substance and the iPS cell establishment efficiency improving substance. There will be no gender.

The contact of the other iPS cell establishment efficiency improving substance with the somatic cell may be performed depending on whether the substance is (a) protein factor or (b) 核酸 nucleic acid encoding the protein factor. Each of the chemical substances can be carried out by the same method as described above. On the other hand, when the substance is (c) a low molecular weight compound, contact of the substance with somatic cells can be achieved by dissolving the factor in an aqueous or non-aqueous solvent at an appropriate concentration and isolating it from a human or other mammal. Medium suitable for culturing cultured somatic cells (eg, minimal essential medium (MEM), Dulbecco's modified Eagle medium (DMEM), RPMI1640 medium, 199 medium, F12 medium (may contain about 5-20% fetal calf serum) Etc.) and the cells can be cultured for a certain period of time. The factor concentration varies depending on the type of establishment efficiency improving substance used, but is appropriately selected within the range of about 0.1 nM to about 100 μM. The contact period is not particularly limited as long as it is a time sufficient for the nuclear reprogramming of the cells to be achieved, but it is usually sufficient that the contact period coexists in the medium until a positive colony appears.

Other iPS cell establishment efficiency improving substances can be contacted with somatic cells at the same time as the nuclear reprogramming substance as long as iPS cell establishment efficiency from somatic cells is significantly improved compared to the absence of the substance. Either may be contacted first. In one embodiment, for example, when the nuclear reprogramming substance is a nucleic acid encoding a proteinous factor, and the substance that improves the establishment efficiency of iPS cells is a chemical inhibitor, the former removes the proteinous factor from the gene transfer treatment. Since there is a lag of a certain period until large-scale expression, the latter can act on the cells quickly, so after culturing the cells for a certain period from the gene transfer treatment, a substance that improves the establishment efficiency of iPS cells is added to the medium can do. In another embodiment, for example, when both a nuclear reprogramming substance and an iPS cell establishment efficiency improving substance are used in the form of a viral vector or a plasmid vector, both may be introduced into a cell simultaneously.

(F) Improving the establishment efficiency by culturing conditions The iPS cell establishment efficiency can be further improved by culturing the cells under hypoxic conditions in the somatic cell nuclear reprogramming step (Cell Stem Cell., 5 ( 3): 237-241 (2009); see WO2010 / 013845). In the present specification, the “hypoxic condition” means that the oxygen concentration in the atmosphere when cells are cultured is significantly lower than that in the air. Specifically, the oxygen concentration condition is lower than the oxygen concentration in the atmosphere of 5-10% CO ₂ / 95-90% air generally used in normal cell culture. For example, oxygen in the atmosphere Conditions with a concentration of 18% or less apply. Preferably, the oxygen concentration in the atmosphere is 15% or less (eg, 14% or less, 13% or less, 12% or less, 11% or less, etc.), 10% or less (eg, 9% or less, 8% or less, 7% or less) 6% or less), or 5% or less (eg, 4% or less, 3% or less, 2% or less, etc.). The oxygen concentration in the atmosphere is preferably 0.1% or more (eg, 0.2% or more, 0.3% or more, 0.4% or more), 0.5% or more (eg, 0.6% or more, 0.7% or more, 0.8% or more, 0.9 % Or more), or 1% or more (eg, 1.1% or more, 1.2% or more, 1.3% or more, 1.4% or more, etc.).

A method for creating a hypoxic state in the cell environment is not particularly limited, but a method of culturing the cells in a CO ₂ incubator in which the oxygen concentration can be adjusted is the easiest and is a preferable example. CO ₂ incubators with adjustable oxygen concentration are sold by various equipment manufacturers (for example, CO for low oxygen culture by manufacturers such as Thermo scientific, Ikemoto Rika Kogyo, Toji Field, and Waken Pharmaceutical Co., Ltd.) ₂ incubators can be used).

The time when cell culture is started under hypoxic conditions is not particularly limited as long as it does not prevent the iPS cell establishment efficiency from being improved compared to the case of normal oxygen concentration (20%). Although it may be before contact with the establishment efficiency improving factor of the invention and the nuclear reprogramming substance, simultaneously with the contact, or after the contact, Incubate under hypoxic conditions immediately after contact or after a period of time (eg 1 to 10 (eg, 2,3,4,5,6,7,8 or 9) days) after contact. preferable.

The period for culturing cells under hypoxic conditions is not particularly limited as long as it does not prevent the establishment efficiency of iPS cells from being improved compared to the case of normal oxygen concentration (20%). Examples include, but not limited to, a period of not less than 7 days, not less than 10 days, not more than 50 days, not more than 40 days, not more than 35 days, or not more than 30 days. A preferable culture period under low oxygen conditions varies depending on the oxygen concentration in the atmosphere, and those skilled in the art can appropriately adjust the culture period according to the oxygen concentration used. In one embodiment, when selection of iPS cell candidate colonies is performed using drug resistance as an index, it is preferable to return from a low oxygen condition to a normal oxygen concentration before drug selection is started.

Furthermore, the preferred timing and preferred culture period for starting cell culture under hypoxic conditions vary depending on the type of nuclear reprogramming substance used, iPS cell establishment efficiency under normoxic conditions, and the like.

(G) Selection and confirmation of iPS cells After contacting the establishment efficiency improving factor of the present invention with a nuclear reprogramming substance (and other iPS cell establishment efficiency improving substances), the cells are suitable for, for example, culturing ES cells. Can be cultured under different conditions. In the case of mouse cells, Leukemia Inhibitory Factor (LIF) is added to a normal medium as a differentiation inhibitory factor and cultured. On the other hand, in the case of human cells, it is usually desirable to add basic fibroblast growth factor (bFGF) and / or stem cell factor (SCF) instead of LIF.

Ordinarily, the cells are cultured as feeder cells in the presence of mouse embryonic fibroblasts (MEFs) that have been treated with radiation or antibiotics to stop cell division. STO cells are usually used as MEFs, but SNL cells (McMahon, A. P. & Bradley, A. Cell 62, 1073-1085 (1990)) are often used to induce iPS cells. ing. Co-culture with feeder cells may be started before contact with the nuclear reprogramming substance, or may be started at the time of contact or after the contact (for example, 1-10 days later).

The selection of iPS cell candidate colonies includes a method using drug resistance and reporter activity as indicators and a method using visual morphological observation. Examples of the former include a drug resistance gene and / or a gene locus that is specifically highly expressed in differentiated pluripotent cells (for example, Fbx15, Nanog, Oct3 / 4, etc., preferably Nanog or Oct3 / 4). A recombinant cell targeted with a reporter gene is used to select colonies that are drug resistant and / or reporter activity positive. Such recombinant cells include, for example, mice (Takahashi & Yamanaka, Cell, 126, 663-676) in which the βgeo (encoding a fusion protein of β-galactosidase and neomycin phosphotransferase) gene is knocked in at the Fbx15 locus. 2006)) derived from transgenic mice (Okita et al., Nature, 448, 313-317 (2007)) in which a green fluorescent protein (GFP) gene and a puromycin resistance gene are incorporated into the MEF or TTF or Nanog locus MEF, TTF, etc. On the other hand, examples of a method for selecting candidate colonies by visual morphological observation include the methods described in Takahashi et al., Cell, 131, 861-872-8 (2007). Although a method using a reporter cell is simple and efficient, when iPS cells are produced for the purpose of human therapeutic use, visual colony selection is desirable from the viewpoint of safety.

Confirmation that the cells of the selected colony are iPS cells can be performed by the above-mentioned Nanog (or Oct3 / 4) reporter positive (puromycin resistance, GFP positive, etc.) and visual formation of ES cell-like colonies. To be more accurate, tests such as alkaline phosphatase staining, expression of various ES cell-specific genes, and transplantation of selected cells to mice to confirm teratoma formation can also be performed. .

(H) Use of iPS cells The iPS cells established in this way can be used for various purposes. For example, differentiation induction methods reported for pluripotent stem cells such as ES cells (eg, differentiation induction methods for neural stem cells are disclosed in JP-A-2002-291469, and differentiation induction methods for pancreatic stem-like cells are Examples of methods for inducing differentiation into hematopoietic cells include those described in JP-T-2003-505006, and other methods for inducing differentiation by formation of embryoid bodies. The method described in Table 2003-523766 is exemplified.) Differentiation from iPS cells into various cells (eg, cardiomyocytes, blood cells, nerve cells, vascular endothelial cells, insulin secreting cells, etc.) Can be induced. Therefore, if iPS cells are induced using somatic cells collected from the patient or another person with the same or substantially the same type of HLA, the desired cells (ie, the organs in which the patient is affected) Stem cell therapy by autotransplantation is possible, in which cells and cells that exhibit therapeutic effects on diseases are differentiated and transplanted into the patient. Furthermore, since functional cells differentiated from iPS cells (eg, hepatocytes) are considered to reflect the actual state of functional cells in vivo more than the corresponding existing cell lines, drug candidates It can also be suitably used for in vitro screening of the efficacy and toxicity of compounds.

Hereinafter, the present invention will be described more specifically with reference to examples, but it goes without saying that the present invention is not limited thereto.

Transgenic mice ecotropic virus receptor Slc7a1 gene was expressed adult skin-derived fibroblasts (aHDF-Slc7a1) previously reported method (Cell, 131, 861-872, 2007 ) was prepared according to. This aHDF-Slc7a1 was sprinkled at a rate of 3 × 10 ⁵ pieces / 60 mm dish, and the next day, pMXs-hOCT3 / 4, pMXs-hKLF4, pMXs-hSOX2 and pMXs-Hu-L- Gene transfer was performed with a virus-containing solution prepared using Myc (all available from Addgene).

Examination of drug addition time 1st to 5th day (d1-d5), 4th to 9th day (d4-d9), and 7th to 14th day after gene transfer to cells obtained by the above method ( d7-d14), DMSO (base only), 10 μM phenelzine (Sigma) and each LSD1 inhibitor (NCL-1 (Formula I-1), NCL-2 (Formula I-2), NCL-3 (Formula I-3) and NCL-4 (Formula I-4); J. AM. CHEM. SOC. 131, 17536-17537, 2009) were added to the medium.

In all conditions, the cells were reseeded to a new dish on the 6th day after gene transfer. The number of ES cell-like colonies was measured from 25 to 30 days after gene introduction, and the results are shown in FIG. It was confirmed that the condition for adding the drug in d7-d14 has the highest colony formation efficiency.

Examination of drug concentration Cells obtained by gene transfer as described above were repopulated on the 6th day after gene transfer to a new dish, and between 7 and 14 days (d7-d14) after gene transfer, 1 μM to 500 μM Phenelzine and NCL-3, or DMSO, 10 μM phenelzine, NCL-1, NCL-2, NCL-3 and NCL-4 were added and cultured. On the 30th day after gene transfer, the number of ES cell-like colonies and the number of non-ES cell-like colonies were counted. The results are shown in FIG. In addition, cells were killed with any drug when 500 μM was added. In NCL-3, the number of colonies formed was high by addition of 1 μM and 10 μM, and in phenelzine, the number of colonies formed was high by addition of 50 μM or 100 μM.

While the invention has been described with emphasis on preferred embodiments, it will be apparent to those skilled in the art that the preferred embodiments can be modified. The present invention contemplates that the present invention may be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications encompassed within the spirit and scope of the appended claims.
The contents of all publications, including the patents and patent application specifications mentioned herein, are hereby incorporated by reference herein to the same extent as if all were specified. .
This application is based on a patent application No. 2011-066827 filed in Japan on March 24, 2011, the entire contents of which are hereby incorporated by reference.

According to the present invention, since the efficiency of iPS cell establishment can be remarkably improved, human iPS cell transplantation therapy such as induction of human iPS cells by three factors other than c-Myc, which has been low in the establishment efficiency, has been proposed. It is particularly useful for applications.

Claims

In the nuclear initialization step, the formula (I):

(Where
R 1 represents a hydrogen atom, an optionally substituted alkyl group, an optionally substituted aryl group, an optionally substituted heterocyclic group, or a formula —NHCO—R 4 (wherein R 4 is An optionally substituted alkyl group, an optionally substituted aryl group, an optionally substituted heterocyclic group, an optionally substituted alkoxy group, an optionally substituted aryloxy group, a substituted A mono- or di-alkylamino group which may be substituted, or a mono- or di-arylamino group which may be substituted;
R 2 represents an optionally substituted alkylene group,
R 3 represents an alkyl group that may be substituted, an aryl group that may be substituted, a heterocyclic group that may be substituted, or an aralkyl group that may be substituted;
X represents O, NH, NHCO, CONH, S or CH 2 . ) And formula (II):

(Where
R is a hydrogen atom or a C 1-4 alkyl group, a C 1-3 alkoxy group, an aryl group, an aralkyl group, a phenylalkoxy group, a phenoxy group, a hydroxy group, an alkylenedioxy group, and a halogen atom. Represents 5 substituents,
R ′ represents a hydrogen atom, a C 1-3 alkyl group, a C 3-6 cycloalkyl group or an aralkyl group,
R ″ represents a hydrogen atom, a C 1-6 alkyl group, a C 1-6 hydroxyalkyl group, a C 2-4 alkenyl group, an optionally substituted aryl group, an optionally substituted aralkyl group, C 3- 6 represents a cycloalkyl group, a C 2-4 alkynyl group, a thienylmethyl group or a pyridylmethyl group,
Y represents a linear or branched C 2-5 alkylene group. )
A method for improving iPS cell establishment efficiency, which comprises contacting a somatic cell with one or more compounds selected from the group consisting of a compound represented by the formula: and a pharmaceutically acceptable salt, solvate and prodrug thereof.
The compound represented by formula (I) is represented by the formula

NCL-1, represented by the formula

NCL-2 represented by the formula

NCL-3 represented by

The method according to claim 1, which is NCL-4 represented by:
The method according to claim 1, wherein the compound represented by the formula (II) is phenelzine (phenethylhydrazine).
Formula (I):

(Where
R 1 represents a hydrogen atom, an optionally substituted alkyl group, an optionally substituted aryl group, an optionally substituted heterocyclic group, or a formula —NHCO—R 4 (wherein R 4 is An optionally substituted alkyl group, an optionally substituted aryl group, an optionally substituted heterocyclic group, an optionally substituted alkoxy group, an optionally substituted aryloxy group, a substituted A mono- or di-alkylamino group which may be substituted, or a mono- or di-arylamino group which may be substituted;
R 2 represents an optionally substituted alkylene group,
R 3 represents an alkyl group that may be substituted, an aryl group that may be substituted, a heterocyclic group that may be substituted, or an aralkyl group that may be substituted;
X represents O, NH, NHCO, CONH, S or CH 2 . ) And formula (II):

(Where
R is a hydrogen atom or a C 1-4 alkyl group, a C 1-3 alkoxy group, an aryl group, an aralkyl group, a phenylalkoxy group, a phenoxy group, a hydroxy group, an alkylenedioxy group, and a halogen atom. Represents 5 substituents,
R ′ represents a hydrogen atom, a C 1-3 alkyl group, a C 3-6 cycloalkyl group or an aralkyl group,
R ″ represents a hydrogen atom, a C 1-6 alkyl group, a C 1-6 hydroxyalkyl group, a C 2-4 alkenyl group, an optionally substituted aryl group, an optionally substituted aralkyl group, C 3- 6 represents a cycloalkyl group, a C 2-4 alkynyl group, a thienylmethyl group or a pyridylmethyl group,
Y represents a linear or branched C 2-5 alkylene group. )
And an iPS cell establishment efficiency improving agent comprising at least one compound selected from the group consisting of a compound represented by formula (I) and a pharmaceutically acceptable salt, solvate and prodrug thereof.
The compound represented by formula (I) is represented by the formula

NCL-1, represented by the formula

NCL-2 represented by the formula

NCL-3 represented by

The agent of Claim 4 which is NCL-4 represented by these.
The agent according to claim 4, wherein the compound represented by the formula (II) is phenelzine (phenethylhydrazine).
Formula (I):

(Where
R 1 represents a hydrogen atom, an optionally substituted alkyl group, an optionally substituted aryl group, an optionally substituted heterocyclic group, or a formula —NHCO—R 4 (wherein R 4 is An optionally substituted alkyl group, an optionally substituted aryl group, an optionally substituted heterocyclic group, an optionally substituted alkoxy group, an optionally substituted aryloxy group, a substituted A mono- or di-alkylamino group which may be substituted, or a mono- or di-arylamino group which may be substituted;
R 2 represents an optionally substituted alkylene group,
R 3 represents an alkyl group that may be substituted, an aryl group that may be substituted, a heterocyclic group that may be substituted, or an aralkyl group that may be substituted;
X represents O, NH, NHCO, CONH, S or CH 2 . ) And formula (II):

(Where
R is a hydrogen atom or a C 1-4 alkyl group, a C 1-3 alkoxy group, an aryl group, an aralkyl group, a phenylalkoxy group, a phenoxy group, a hydroxy group, an alkylenedioxy group, and a halogen atom. Represents 5 substituents,
R ′ represents a hydrogen atom, a C 1-3 alkyl group, a C 3-6 cycloalkyl group or an aralkyl group,
R ″ represents a hydrogen atom, a C 1-6 alkyl group, a C 1-6 hydroxyalkyl group, a C 2-4 alkenyl group, an optionally substituted aryl group, an optionally substituted aralkyl group, C 3- 6 represents a cycloalkyl group, a C 2-4 alkynyl group, a thienylmethyl group or a pyridylmethyl group,
Y represents a linear or branched C 2-5 alkylene group. )
And a step of bringing a nuclear reprogramming substance into contact with one or more compounds selected from the group consisting of a compound represented by the formula (I) and pharmaceutically acceptable salts, solvates and prodrugs thereof: iPS cell production method.
The compound represented by formula (I) is represented by the formula

NCL-1, represented by the formula

NCL-2 represented by the formula

NCL-3 represented by

The method according to claim 7, which is NCL-4 represented by:
The method according to claim 7, wherein the compound represented by the formula (II) is phenelzine (phenethylhydrazine).
The nuclear reprogramming substance is selected from the group consisting of Oct family members, Sox family members, Klf4 family members, Myc family members, Lin family members and Nanog, and nucleic acids encoding them. The method according to any one of 1 to 9.
The method according to any one of claims 7 to 9, wherein the nuclear reprogramming substance is Oct3 / 4, Klf4 and Sox2, or a nucleic acid encoding them.
10. The nuclear reprogramming substance is Oct3 / 4, Klf4, Sox2, and c-Myc or L-Myc and / or Nanog and / or Lin28 or Lin28B, or a nucleic acid encoding them. The method described in 1.
Formula (I):

(Where
R 1 represents a hydrogen atom, an optionally substituted alkyl group, an optionally substituted aryl group, an optionally substituted heterocyclic group, or a formula —NHCO—R 4 (wherein R 4 is An optionally substituted alkyl group, an optionally substituted aryl group, an optionally substituted heterocyclic group, an optionally substituted alkoxy group, an optionally substituted aryloxy group, a substituted A mono- or di-alkylamino group which may be substituted, or a mono- or di-arylamino group which may be substituted;
R 2 represents an optionally substituted alkylene group,
R 3 represents an alkyl group that may be substituted, an aryl group that may be substituted, a heterocyclic group that may be substituted, or an aralkyl group that may be substituted;
X represents O, NH, NHCO, CONH, S or CH 2 . ) And formula (II):

(Where
R is a hydrogen atom or a C 1-4 alkyl group, a C 1-3 alkoxy group, an aryl group, an aralkyl group, a phenylalkoxy group, a phenoxy group, a hydroxy group, an alkylenedioxy group, and a halogen atom. Represents 5 substituents,
R ′ represents a hydrogen atom, a C 1-3 alkyl group, a C 3-6 cycloalkyl group or an aralkyl group,
R ″ represents a hydrogen atom, a C 1-6 alkyl group, a C 1-6 hydroxyalkyl group, a C 2-4 alkenyl group, an optionally substituted aryl group, an optionally substituted aralkyl group, C 3- 6 represents a cycloalkyl group, a C 2-4 alkynyl group, a thienylmethyl group or a pyridylmethyl group,
Y represents a linear or branched C 2-5 alkylene group. )
IPS from a somatic cell, comprising one or more compounds selected from the group consisting of compounds represented by the formula (I) and pharmaceutically acceptable salts, solvates and prodrugs thereof, and a nuclear reprogramming substance. Cell inducer.
The compound represented by formula (I) is represented by the formula

NCL-1, represented by the formula

NCL-2 represented by the formula

NCL-3 represented by

The agent of Claim 13 which is NCL-4 represented by these.
The agent according to claim 13, wherein the compound represented by the formula (II) is phenelzine (phenethylhydrazine).
14. The nuclear reprogramming agent is selected from the group consisting of Oct family members, Sox family members, Klf4 family members, Myc family members, Lin family members and Nanog, and nucleic acids encoding them. The agent according to any one of 1 to 15.
The agent according to any one of claims 13 to 15, wherein the nuclear reprogramming substance is Oct3 / 4, Klf4 and Sox2, or a nucleic acid encoding them.
The nuclear reprogramming substance is Oct3 / 4, Klf4, Sox2, and c-Myc or L-Myc and / or Nanog and / or Lin28 or Lin28B, or a nucleic acid that encodes them. The agent described in 1.
The following steps:
(1) a step of producing iPS cells by the method according to any one of claims 7 to 12, and (2) subjecting the iPS cells obtained in the above step (1) to differentiation induction treatment, Differentiation step,
A method for producing somatic cells, comprising:
Formula (I) for improving iPS cell establishment efficiency:

(Where
R 1 represents a hydrogen atom, an optionally substituted alkyl group, an optionally substituted aryl group, an optionally substituted heterocyclic group, or a formula —NHCO—R 4 (wherein R 4 is An optionally substituted alkyl group, an optionally substituted aryl group, an optionally substituted heterocyclic group, an optionally substituted alkoxy group, an optionally substituted aryloxy group, a substituted A mono- or di-alkylamino group which may be substituted, or a mono- or di-arylamino group which may be substituted;
R 2 represents an optionally substituted alkylene group,
R 3 represents an alkyl group that may be substituted, an aryl group that may be substituted, a heterocyclic group that may be substituted, or an aralkyl group that may be substituted;
X represents O, NH, NHCO, CONH, S or CH 2 . ) And formula (II):

(Where
R is a hydrogen atom or a C 1-4 alkyl group, a C 1-3 alkoxy group, an aryl group, an aralkyl group, a phenylalkoxy group, a phenoxy group, a hydroxy group, an alkylenedioxy group, and a halogen atom. Represents 5 substituents,
R ′ represents a hydrogen atom, a C 1-3 alkyl group, a C 3-6 cycloalkyl group or an aralkyl group,
R ″ represents a hydrogen atom, a C 1-6 alkyl group, a C 1-6 hydroxyalkyl group, a C 2-4 alkenyl group, an optionally substituted aryl group, an optionally substituted aralkyl group, C 3- 6 represents a cycloalkyl group, a C 2-4 alkynyl group, a thienylmethyl group or a pyridylmethyl group,
Y represents a linear or branched C 2-5 alkylene group. )
And one or more compounds selected from the group consisting of pharmaceutically acceptable salts, solvates and prodrugs thereof.
The compound represented by formula (I) is represented by the formula

NCL-1, represented by the formula

NCL-2 represented by the formula

NCL-3 represented by

21. Use according to claim 20, which is NCL-4 represented by
The use according to claim 20, wherein the compound represented by the formula (II) is phenelzine (phenethylhydrazine).
Formula (I) for improving iPS cell establishment efficiency:

(Where
R 1 represents a hydrogen atom, an optionally substituted alkyl group, an optionally substituted aryl group, an optionally substituted heterocyclic group, or a formula —NHCO—R 4 (wherein R 4 is An optionally substituted alkyl group, an optionally substituted aryl group, an optionally substituted heterocyclic group, an optionally substituted alkoxy group, an optionally substituted aryloxy group, a substituted A mono- or di-alkylamino group which may be substituted, or a mono- or di-arylamino group which may be substituted;
R 2 represents an optionally substituted alkylene group,
R 3 represents an alkyl group that may be substituted, an aryl group that may be substituted, a heterocyclic group that may be substituted, or an aralkyl group that may be substituted;
X represents O, NH, NHCO, CONH, S or CH 2 . ) And formula (II):

(Where
R is a hydrogen atom or a C 1-4 alkyl group, a C 1-3 alkoxy group, an aryl group, an aralkyl group, a phenylalkoxy group, a phenoxy group, a hydroxy group, an alkylenedioxy group, and a halogen atom. Represents 5 substituents,
R ′ represents a hydrogen atom, a C 1-3 alkyl group, a C 3-6 cycloalkyl group or an aralkyl group,
R ″ represents a hydrogen atom, a C 1-6 alkyl group, a C 1-6 hydroxyalkyl group, a C 2-4 alkenyl group, an optionally substituted aryl group, an optionally substituted aralkyl group, C 3- 6 represents a cycloalkyl group, a C 2-4 alkynyl group, a thienylmethyl group or a pyridylmethyl group,
Y represents a linear or branched C 2-5 alkylene group. )
And one or more compounds selected from the group consisting of pharmaceutically acceptable salts, solvates and prodrugs thereof, wherein the compound is contacted with a somatic cell together with a nuclear reprogramming substance. Use, characterized by letting
The compound represented by formula (I) is represented by the formula

NCL-1, represented by the formula

NCL-2 represented by the formula

NCL-3 represented by

24. Use according to claim 23, which is NCL-4 represented by
The use according to claim 23, wherein the compound represented by the formula (II) is phenelzine (phenethylhydrazine).
24. The nuclear reprogramming agent is selected from the group consisting of Oct family members, Sox family members, Klf4 family members, Myc family members, Lin family members and Nanog, and nucleic acids encoding them. The use according to any one of 1 to 25.
The use according to any one of claims 23 to 25, wherein the nuclear reprogramming substance is Oct3 / 4, Klf4 and Sox2, or a nucleic acid encoding them.
The nuclear reprogramming substance is Oct3 / 4, Klf4, Sox2, and c-Myc or L-Myc and / or Nanog and / or Lin28 or Lin28B, or a nucleic acid that encodes them. Use as described in.