WO2012157612A1 - 細胞分化誘導剤および分化誘導方法 - Google Patents
細胞分化誘導剤および分化誘導方法 Download PDFInfo
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- C12N2501/30—Hormones
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- C12N2501/385—Hormones with nuclear receptors of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
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- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
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- the present invention relates to a cell differentiation inducer comprising a catechol derivative which is a low molecular compound, the use of the catechol derivative for inducing differentiation of an undifferentiated cell, and an undifferentiated cell using a catechol derivative as a neural crest cell, etc.
- the present invention relates to a method for inducing differentiation into nervous system cells.
- the cells constituting multicellular organisms are roughly classified into differentiated cells, TA cells (Transient Amplifying Cell) and stem cells.
- Differentiated cells are also called terminally differentiated cells and terminally differentiated cells, and they do not differentiate into different types of cells such as nerve cells and organ cells, and hardly proliferate.
- TA cells are intermediate between differentiated cells and stem cells, and after differentiation, actively proliferate to become differentiated cells.
- Stem cells are defined as cells having both self-renewal ability and differentiation ability, and can self-proliferate and differentiate into TA cells.
- stem cells can be differentiated into all types of cells, totipotent cells that can form solids, unipotent cells that can only differentiate into single cells, and many cells that can be differentiated into various cells even though they cannot become solid unless special operations are performed. Differentiate into competent cells. Examples of totipotent cells include fertilized eggs. Examples of unipotent cells include germ stem cells such as sperm. Examples of pluripotent cells include pluripotent stem cells.
- stem cells for example, it is possible to prepare regenerative medicine that does not show rejection by preparing skin tissue or organ tissue from the patient itself, or to proliferate problematic cells from patients with specific diseases such as genetic diseases. , May be able to help create new drugs.
- Patent Document 1 discloses sesamin compounds as agents for inducing differentiation into nerve cells.
- Patent Document 2 and Non-Patent Document 1 describe retinoic acid as an agent for inducing differentiation from undifferentiated cells to ectoderm cells.
- ectoderm By the way, early embryos of multicellular organisms are classified into ectoderm, mesoderm and endoderm.
- the ectoderm is a nerve or skin tissue
- the mesoderm is a muscle, bone, blood vessel or blood
- the endoderm is a specific organ such as the digestive tract or lung.
- Others are called neural crest cells.
- neural crest cells are classified as ectoderm, they migrate from the neural crest at the stage of neurogenesis and form melanocytes, peripheral nerve neurons, smooth muscle, cartilage and bones centered on the head, etc. And has an important role, it is also called the fourth germ layer.
- Patent Document 2 discloses a method for inducing differentiation of undifferentiated cells into neural crest cells without using feeder cells using a medium containing a specific component.
- Non-Patent Document 2 uses feeder cells, even if neural crest cells derived from patient-derived iPS cells are introduced into the patient, a rejection reaction may occur. There is also a problem that the differentiation induction rate varies greatly depending on the lot of feeder cells. Furthermore, BMP4, which is a secreted protein, is very expensive. When it is used as a differentiation inducer, there is a problem of cost. In the method described in Patent Document 3, feeder cells are not required, but unless bone morphogenetic protein (BMP4) or the like is used, the differentiation induction rate is very low, and there are still problems of cost and efficiency.
- BMP4 bone morphogenetic protein
- sesamin used in Patent Document 1 is a low molecule, this technique only induces differentiation of neural cell precursor cells into nerve cells, and induces differentiation of iPS cells. is not. Moreover, although it is a low molecular compound, sesamin must be extracted from sesame oil, and since it has four asymmetric carbons, chemical synthesis is also difficult. Furthermore, although retinoic acid described in Patent Document 2 and Non-Patent Document 1 is also a low molecular weight compound, for example, when retinoic acid is allowed to act on ES cells, cells other than the ectoderm system are also induced to induce differentiation. It cannot be said.
- the present invention provides a cell differentiation inducer capable of efficiently inducing differentiation of undifferentiated cells into neural cells with high selectivity while being a low-molecular compound that can be easily synthesized, and an undifferentiated cell in the nervous system.
- a cell differentiation inducer capable of efficiently inducing differentiation of undifferentiated cells into neural cells with high selectivity while being a low-molecular compound that can be easily synthesized, and an undifferentiated cell in the nervous system.
- the inventors of the present invention have made extensive studies to solve the above problems. As a result, the inventors have found that a catechol derivative having a specific structure can induce differentiation of undifferentiated cells into neural cells efficiently while having a very simple chemical structure, thereby completing the present invention.
- the cell differentiation inducer according to the present invention includes a catechol derivative represented by the following formula (I).
- R 1 has a carboxy group, a (C 1-6 alkoxy) carbonyl group, a C 1-7 alkanoyl group, a C 2-8 alkynyl group, a carbamoyl group, a cyano group, a nitro group or a halogen atom, or a substituent ⁇ .
- R 2 represents a hydrogen atom, a C 1-6 alkyl group or a benzyl group; n represents an integer of 2 or more and 5 or less;
- the substituent ⁇ represents one or more selected from a carboxy group, a (C 1-6 alkoxy) carbonyl group, a C 1-7 alkanoyl group, a carbamoyl group, a cyano group, a nitro group, and a halogen atom.
- the “C 1-6 alkyl group” refers to a linear or branched saturated aliphatic hydrocarbon group having 1 to 6 carbon atoms.
- a C 1-4 alkyl group is preferred, a C 1-2 alkyl group is more preferred, and a methyl group is most preferred.
- (C 1-6 alkoxy) carbonyl group refers to a C 1-6 alkyloxycarbonyl group (C 1-6 alkyl-O—C ( ⁇ O) — group).
- a (C 1-4 alkoxy) carbonyl group is preferred, a (C 1-2 alkoxy) carbonyl group is more preferred, and a methoxycarbonyl group is most preferred.
- the “C 1-7 alkanoyl group” means a carbonyl group substituted with a formyl group and the above C 1-6 alkyl group. Examples include formyl group, acetyl group, n-propionyl group, isopropionyl group, n-butyryl group, isobutyryl group, pentanoyl group, pivaloyl group, valeryl group and isovaleryl group.
- a C 1-5 alkanoyl group is preferable, a C 1-3 alkanoyl group is more preferable, and a formyl group or an acetyl group is most preferable.
- the “C 2-8 alkynyl group” refers to a linear or branched unsaturated aliphatic hydrocarbon group having a carbon-carbon triple bond and having 2 to 8 carbon atoms.
- the group is preferably one in which the carbon at the 1-position, that is, the carbon atom bonded to the benzene ring of the catechol derivative (I) has a carbon-carbon triple bond.
- Examples include ethynyl group, 1-propynyl group, 1-butynyl group, 1-pentynyl group, 3-methyl-1-butynyl group, and 4-methyl-1-pentynyl group.
- a C 2-4 alkynyl group is preferable, and an ethynyl group or a 1-propynyl group is more preferable.
- halogen atom examples include a fluorine atom, a chlorine atom, a bromine atom and an iodine atom, preferably a chlorine atom, a bromine atom or an iodine atom, and more preferably a chlorine atom or a bromine atom.
- the cell differentiation inducer according to the present invention includes those in which R 2 is a hydrogen atom, those in which at least two —OR 2 groups are adjacent to each other, and those in which R 1 is a cyano group or a carbamoyl group. preferable. According to the experimental findings by the present inventors, these can induce differentiation of undifferentiated cells into neural cells with high efficiency, particularly at a relatively low concentration and with high selectivity.
- n is preferably an integer of 2 or more and 4 or less, more preferably 2 or 3.
- a benzene compound has a higher number of substituents, which makes it difficult to synthesize and is expensive.
- Two or more R 2 O groups may be the same or different from each other, but are preferably the same because they are easier to produce.
- the compound (I) When the compound (I) has a carboxy group as a substituent, it may be a salt.
- the salt is preferably pharmaceutically acceptable.
- examples of such salts include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt and magnesium salt; inorganic amine salts such as ammonium salt; organic compounds such as trimethylamine salt, triethylamine salt and pyridine salt. Mention may be made of amine salts.
- the number of substituents ⁇ in the thiazolyl group is not particularly limited as long as substitution is possible, but is preferably 1 or more and 2 or less, and more preferably 1.
- the cell differentiation inducer according to the present invention preferably further contains retinoic acid.
- retinoic acid In combination with retinoic acid, the induction effect on nervous system cells is further improved.
- the cell differentiation inducer As the cell differentiation inducer according to the present invention, those for inducing differentiation of undifferentiated cells into nervous system cells are preferable.
- the method for inducing differentiation of undifferentiated cells according to the present invention into neural cells includes a step of allowing the above-mentioned cell differentiation inducer according to the present invention to act on undifferentiated cells.
- the catechol derivative (I) according to the present invention is an active ingredient of the cell differentiation inducer and is used for inducing differentiation of undifferentiated cells.
- the method for inducing differentiation of undifferentiated cells into neural cells includes a step of culturing undifferentiated cells in a medium containing a catechol derivative (I) which is an active ingredient of the cell differentiation inducer. It is characterized by that.
- the catechol derivative (I), which is an active ingredient of the cell differentiation inducer according to the present invention has a very simple chemical structure, it can be obtained at low cost or can be easily synthesized. Therefore, the cost is lower than that of expensive proteins.
- the catechol derivative (I), which is an active ingredient of the cell differentiation inducer of the present invention is capable of treating undifferentiated cells with high selectivity and good efficiency without using feeder cells even at a relatively low concentration. It becomes possible to induce differentiation into cell lines. Therefore, the present invention is very useful industrially as it can further promote the realization of regenerative medicine and drug discovery research using iPS cells.
- FIG. 1 is an enlarged photograph of a mouse ES cell treated with the cell differentiation inducer of the present invention and an enlarged photograph of a control treated similarly without using the cell differentiation inducer of the present invention.
- FIG. 2 is an enlarged photograph of mouse ES cells treated with the cell differentiation inducer of the present invention.
- FIG. 3 is an enlarged photograph of mouse ES cells treated with the cell differentiation inducer of the present invention.
- FIG. 4 is an enlarged photograph of mouse ES cells differentiated using the cell differentiation inducer of the present invention or retinoic acid when stained with an ectoderm stem cell marker and a mesoderm stem cell marker.
- FIG. 1 is an enlarged photograph of a mouse ES cell treated with the cell differentiation inducer of the present invention and an enlarged photograph of a control treated similarly without using the cell differentiation inducer of the present invention.
- FIG. 2 is an enlarged photograph of mouse ES cells treated with the cell differentiation inducer of the present invention.
- FIG. 3 is an enlarged photograph of mouse ES cells treated
- FIG. 5 is an electrophoresis photograph showing the results of analyzing the gene expression of mouse ES cells, their embryoid bodies, and mouse ES cells treated with the differentiation inducer of the present invention.
- FIG. 6 is an enlarged photograph of human iPS cells treated with the cell differentiation inducer of the present invention and an enlarged photograph of a control treated in the same manner without using the cell differentiation inducer of the present invention.
- FIG. 7 is a graph showing the combined effect of the cell differentiation inducer of the present invention and retinoic acid.
- FIG. 8 is an enlarged photograph of mouse ES cells treated with the cell differentiation inducer of the present invention.
- the cell differentiation inducer according to the present invention contains a catechol derivative represented by the formula (I) and has a very simple chemical structure, and therefore, if it is commercially available, it may be purchased, or Those skilled in the art can easily synthesize them from commercially available compounds by a conventional method.
- benzene compounds having two or more hydroxyl groups.
- Those skilled in the art can easily derive such a phenolic hydroxyl group to an alkoxy group or a benzyloxy group.
- R 1 group or convert a functional group depending on the substitution position of the phenolic hydroxyl group.
- functional group conversion is possible as follows.
- the compound (I) having an oxazolinyl group or a thiazolinyl group can be synthesized by reacting a benzonitrile compound with serine or cysteine and closing the ring as follows.
- the absolute configuration at the carbon atom such as the oxazolinyl ring substituted by the carboxy group can be controlled by using optically active serine or the like.
- a carboxy group such as an oxazolinyl ring can be converted into a functional group in the same manner as described above.
- the reaction of introducing a thiazolinyl group using cysteine is shown as a representative.
- the benzonitrile compound and cysteine may be reacted in the presence of a base.
- a base As the solvent, cysteine and the like are dissolved in water, but some benzonitrile compounds are difficult to dissolve in water. Therefore, it is preferable to use a mixed solution of water and a water-miscible organic solvent.
- the water-miscible organic solvent include alcohols such as methanol and ethanol; amides such as dimethylformamide and dimethylacetamide; sulfoxides such as dimethyl sulfoxide.
- a buffer solution may be used as water.
- the base examples include alkali metal hydrogen carbonates such as sodium hydrogen carbonate; alkali metal carbonates such as sodium carbonate; alkali metal hydroxides such as sodium hydroxide, and the like.
- the reaction temperature is not particularly limited and may be adjusted as appropriate, but can usually be about 30 ° C. or higher and 100 ° C. or lower.
- the compound (I) having an oxazolyl group or a thiazolyl group can be synthesized by reacting a benzamide compound or a benzothioamide compound with a halogenated pyruvate compound and ring-closing as follows.
- An alkoxycarbonyl group (ester group) such as an oxazolyl ring can be converted into a functional group in the same manner as described above.
- a reaction for introducing a thiazolyl group using a benzothioamide compound is shown as a representative.
- Hal represents a halogen atom
- R represents a C 1-6 alkyl group, particularly a C 1-2 alkyl group.
- a benzothioamide compound and a halogenated pyruvate compound are reacted.
- the solvent include alcohols such as methanol and ethanol; halogenated hydrocarbons such as dichloromethane and chloroform; amides such as dimethylformamide and dimethylacetamide.
- the reaction temperature is not particularly limited and may be adjusted as appropriate, but can usually be about 30 ° C. or higher and 100 ° C. or lower.
- the catechol derivative according to the present invention may further contain retinoic acid.
- retinoic acid By using retinoic acid in combination, the induction effect on nervous system cells is further improved.
- the content of retinoic acid may be appropriately adjusted. For example, it is preferably about 0.5 to 2.0 mol per mol of the catechol derivative according to the present invention. If the ratio is 0.5 times mol or more, a synergistic effect by retinoic acid can be expected. On the other hand, if the ratio is too high, cells other than the ectoderm system may be induced to be differentiated by retinoic acid and the selectivity may be lowered. Therefore, the ratio is preferably 2.0 times mol or less. The ratio is more preferably 0.7 times mol or more, further preferably 0.8 times mol or more, more preferably 1.5 times mol or less, and further preferably 1.2 times mol or less.
- the catechol derivative which is the main component of the cell differentiation inducer according to the present invention, has a very simple chemical structure and is very easy to synthesize, and is highly capable of transforming undifferentiated cells into nervous system cells. Differentiation can be induced efficiently with selectivity.
- the differentiation induction method will be described.
- the undifferentiated cells used in the method of the present invention are not particularly limited as long as they have pluripotency to differentiate into neural cells.
- ES cells iPS cells
- mesenchymal cells Those that have the ability to differentiate into neural cells and have the ability to self-replicate, such as stem cells, are particularly useful for application to regenerative medicine.
- embryoid bodies obtained from these cells are also included in the undifferentiated cells in the present invention. Embryoid bodies generally include ectoderm, mesoderm and endoderm, and nervous system cells are ectodermal cells. Therefore, by using embryoid bodies, it is easier to induce differentiation into nervous system cells. Become.
- the origin of the undifferentiated cells is not particularly limited, and may be derived from fish, amphibians, birds, mammals, but in view of application to regenerative medicine, those derived from mammals are preferable, those derived from humans More preferred.
- the undifferentiated cells can be prepared by a conventional method.
- ES cells can be isolated as an undifferentiated stem cell population by culturing in vitro an inner cell mass inside a blastocyst stage embryo and repeating dissociation and passage of the cell mass.
- iPS cells are prepared by introducing specific genes into somatic cells such as fibroblasts.
- Mesenchymal stem cells can be used after culturing those isolated from umbilical cord blood, bone marrow, placenta and the like.
- An embryoid body can be obtained by suspension culture of these cells in a medium for embryoid body formation.
- the cell differentiation inducing agent according to the present invention can act on undifferentiated cells by adding it to the culture medium of the undifferentiated cells.
- a medium for embryoid body formation when an embryoid body is used as an undifferentiated cell, it is preferable to change from a medium for embryoid body formation to a medium suitable for induction of differentiation.
- a medium include inorganic salts such as sodium chloride, potassium chloride, calcium chloride, iron nitrate, magnesium sulfate, sodium bicarbonate, and sodium dihydrogen phosphate; amino acids such as natural amino acids and unnatural amino acids; calcium pantothenate, folic acid, Examples include vitamins such as inositol, nicotinamide, pyridoxine hydrochloride and thiamine hydrochloride; and other nutrients such as glucose and sodium pyruvate.
- basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), leukocyte migration inhibitory factor (LIF), serum and the like may be added.
- the cell differentiation-inducing agent according to the present invention may be added and mixed as it is into the medium of undifferentiated cells, but it may be impossible to mix sufficiently due to problems such as solubility, so it is preferable to prepare a solution in advance.
- a solvent for such a solution distilled water or the like can be used if the cell differentiation inducer according to the present invention has sufficient water solubility, but if the water solubility is not sufficient, the cell differentiation inducer can be mixed with water. You may add, after melt
- water-miscible organic solvent examples include alcohol solvents such as methanol and ethanol; amide solvents such as dimethylformamide and dimethylacetamide; sulfoxide solvents such as dimethyl sulfoxide.
- alcohol solvents such as methanol and ethanol
- amide solvents such as dimethylformamide and dimethylacetamide
- sulfoxide solvents such as dimethyl sulfoxide.
- the addition amount of the cell differentiation inducer according to the present invention may be appropriately adjusted depending on the activity of the compound, and can be, for example, about 10 ⁇ M or more and 300 ⁇ M or less with respect to the whole medium.
- concentration is 10 ⁇ M or more, differentiation induction into nervous system cells can be more reliably promoted.
- concentration is preferably 300 ⁇ M or less.
- the concentration is more preferably 15 ⁇ M or more, further preferably 20 ⁇ M or more, more preferably 200 ⁇ M or less, and further preferably 150 ⁇ M or less.
- the culture conditions after adding the cell differentiation inducer according to the present invention may be appropriately adjusted according to the type of undifferentiated cells used.
- the atmosphere may be a 5% carbon dioxide atmosphere and cultured at about 25 ° C. or more and 45 ° C. or less for about 1 day or more and 10 days or less.
- the cell differentiation inducer according to the present invention can induce differentiation of undifferentiated cells into neural cells.
- neural cells in the present invention include neural cells such as neural cells and glial cells, as well as neural progenitor cells such as neural stem cells, neural progenitor cells, and neural crest cells.
- the differentiation-induced neural cell can be determined by morphological observation, identification of expressed protein, gene expression analysis, and the like.
- a general nerve cell has a unique form having a process.
- neural crest cells are ectodermal cells, they also express Flk1, which is a mesoderm stem cell marker, together with Nestin, which is an ectoderm stem cell marker.
- examples of neural crest cell markers include Sox9, Sox10, and Slug. Therefore, it is possible to determine whether differentiation-induced cells are neural crest cells by ELISA using antibodies specific to these proteins as primary antibodies, or by expression analysis of genes encoding these proteins.
- nervous system cells can be identified by morphological observation and separated from other cells.
- the isolated neural cells can be applied to treatment of diseases related to nerve cells or regenerative medicine.
- neural crest cells are induced to be differentiated and isolated according to the present invention, they are applied to treatment or regenerative medicine for diseases caused by neural crest induction or formation defects, or neural crest cell migration defects, etc. It can be done.
- the neural crest cells obtained are induced to differentiate into glial cells, pigment cells, corneal parenchymal cells, smooth muscle cells, chondrocytes, bone cells, etc. by conventional methods, and can be applied to the treatment of diseases related to these cell groups and regenerative medicine. obtain.
- Example 7 Mouse ES Cell Differentiation Induction Experiment (1) Mouse ES Cell Differentiation Induction Formation of embryoid bodies by mixing an ES medium and NP medium having the composition shown in Table 1 in a ratio of 1: 1 to a 60 mm diameter Petri dish. Medium (4 mL) was added, and 1 ⁇ 10 6 mouse ES cells (obtained from Dr. Alan Bradley, The Wellcome Trust Sanger Institute, AB2-2) were seeded at 37 ° C. in a 5% CO 2 atmosphere for 1 day. Cultured. Next, after detaching the cells by pipetting using a glass pipette, the cells were seeded again in the same petri dish and cultured at 37 ° C. for 3 days in a 5% CO 2 atmosphere to form embryoid bodies. The obtained cell mass was collected and sedimented, and then suspended in NP medium (10 mL) having the composition shown in Table 1.
- the cultured cell suspension (100 ⁇ L) was then added to a type I collagen-coated 96-well plate and incubated overnight at 37 ° C. in a 5% CO 2 atmosphere. Separately, each test compound was dissolved in a differentiation medium (medium obtained by removing 2-mercaptomethanol and leukemia inhibitory factor from ES medium) and added to each well so that the final concentration was 25 to 100 ⁇ M.
- a differentiation medium medium obtained by removing 2-mercaptomethanol and leukemia inhibitory factor from ES medium
- compounds synthesized according to the methods of Examples 1-6 were also used. The structure of the compound is shown in Table 2.
- bFGF basic fibroblast growth factor
- FIG. 1 (1) shows a 200x optical micrograph of a control in which only dimethyl sulfoxide was added to a final concentration of 0.5% without adding a compound.
- FIGS. 1 (2) to 3 show optical micrographs of 50 times, 100 times, or 200 times the minimum density. Table 2 summarizes the minimum concentration of each test compound when morphological changes were observed.
- the differentiation-inducing agent according to the present invention can induce differentiation of mouse ES cells into cells having protrusions at a relatively low concentration.
- almost all of the test compounds Nos. 12 to 13 were able to significantly change the cell morphology.
- retinoic acid which has an action of inducing differentiation of undifferentiated cells into neuroectodermal.
- photographs using retinoic acid are shown in the left two columns of FIG. 4
- photographs using the differentiation inducer according to the present invention are shown in the two right columns of FIG. 4, and FITC labeling.
- a photograph in which the anti-mouse antibody was detected is shown in the upper part of FIG. 4, and a photograph in which the rabbit IgG antibody was detected is shown in the lower part of FIG.
- cells induced to differentiate using retinoic acid express Nestin but do not express Flk1. Since Nestin is an ectoderm stem cell marker and Flk1 is a mesoderm stem cell marker, it was confirmed that retinoic acid induces differentiation of undifferentiated cells into ectoderm stem cells.
- the cells induced to differentiate with the differentiation inducer according to the present invention were positive for Nestin and Flk1. From this experimental result, it becomes clear that the differentiation inducer according to the present invention can induce differentiation of undifferentiated cells into neural crest cells that are classified as ectodermal cells but also have properties as mesoderm cells. It was.
- Example 8 Differentiation Induction Experiment of Human iPS Cells Differentiation was induced in the same manner as in Example 7 (1) except that human iPS cells (obtained from RIKEN, hPS0001 201B7 lot3) were used instead of mouse ES cells.
- the cells induced to differentiate by the test compound are fixed with paraformaldehyde, washed with PBS, 5% goat serum / 1% BSA / PBS is added at 100 ⁇ L / well, and incubated at room temperature for 60 minutes. Blocking. Then, anti-Nestin mouse IgG (manufactured by SIGMA, final concentration 5 ⁇ g / mL) was added and incubated at 4 ° C. overnight. After washing each well with PBS-0.1% Tween 20, FITC-labeled anti-rabbit IgG antibody (Biosource International, final concentration 1 ⁇ g / mL) was added as a secondary antibody and incubated at room temperature for 2 hours.
- the number of cells per se was small in the control cells, and the ratio of Nestin positive cells to the whole cells was also clearly small.
- the compound of the present invention when used, it was confirmed that the whole cell was sufficiently positive for Nestin and differentiated into ectoderm stem cells. From these results, the differentiation-inducing effect of the compound of the present invention was also demonstrated in human iPS cells. Note that the difference between Hoechst 33258 positive cells and Nestin positive cells is that the green color of the cells in the upper part of the photograph has become strong due to auto-contrast by image analysis software, and that Nestin strongly stains the protrusions of the cells. However, it is considered that the staining of the cell body itself has become weak.
- Example 9 Combined Effect with Retinoic Acid
- an embryoid body-forming medium (4 mL) in which an ES medium and an NP medium having the composition shown in Table 1 were mixed at a ratio of 1: 1 was added, and the mouse was further added.
- 1 ⁇ 10 6 ES cells obtained from Dr. Alan Bradley, The Wellcome Trust Sanger Institute, AB2-2
- ES cells were seeded and cultured at 37 ° C. in a 5% CO 2 atmosphere for 1 day.
- the cells were seeded again in the same petri dish and cultured at 37 ° C.
- the number of nervous system cells when the compound of the present invention was used was 1.71 ⁇ 0.51 ⁇ 10 6 , whereas it was 3.23 ⁇ 0.42 in combination with retinoic acid.
- ⁇ 10 6 markedly increased to about 1.89 times (P value: 0.0023). From the above results, it was revealed that the effect of inducing differentiation into nervous system cells can be further improved by the combined use of the compound of the present invention and retinoic acid.
- Example 10 Experiment for Inducing Differentiation of Mouse ES Cell
- the medium for embryoid body formation (4 mL) used in Example 7 was added, and 1 ⁇ 10 6 mouse ES cells (AB2-2) were further added.
- the seeds were plated and cultured at 37 ° C. for 3 days in a 5% CO 2 atmosphere.
- the obtained embryoid body was collected in a 15 mL tube.
- 1 ⁇ PBS (5 mL) was added, and the same PBS was collected in the tube.
- Half of the collected cells were transferred to another tube and allowed to stand at room temperature for 5 minutes. After removing the supernatant, NP medium (10.5 mL) used in Example 7 was added.
- 3,4-dihydroxybenzonitrile (manufactured by Tokyo Chemical Industry Co., Ltd.) was dissolved in DMSO and added to each well so that the final concentration was 12.5 ⁇ M, 25 ⁇ M or 50 ⁇ M, and the total amount was 200 ⁇ L. Further, serum was added to each well at a final concentration of 5% and bFGF (basic fibroblast growth factor) was added to a final concentration of 10 ng / mL, and cultured at 37 ° C. for 4 days in a 5% CO 2 atmosphere. did.
- bFGF basic fibroblast growth factor
- FIG. 8 (1) shows an enlarged photograph of 50 times when the compound of the present invention is added at a concentration of 50 ⁇ M
- FIGS. 8 (2) to (3) show enlarged photographs of 100 times.
- nervous system cells having protrusions as a whole were observed.
- other forms of cells were not observed, and it was proved that the compound of the present invention can induce differentiation of undifferentiated cells into neural cells with high selectivity.
Abstract
Description
R1は、カルボキシ基、(C1-6アルコキシ)カルボニル基、C1-7アルカノイル基、C2-8アルキニル基、カルバモイル基、シアノ基、ニトロ基もしくはハロゲン原子、または、置換基αを有していてもよいオキサゾリル基、置換基αを有していてもよいチアゾリル基、置換基αを有していてもよいオキサゾリニル基、もしくは置換基αを有していてもよいチアゾリニル基を示し;
R2は、水素原子、C1-6アルキル基またはベンジル基を示し;
nは2以上、5以下の整数を示し;
置換基αは、カルボキシ基、(C1-6アルコキシ)カルボニル基、C1-7アルカノイル基、カルバモイル基、シアノ基、ニトロ基およびハロゲン原子から選択される1以上を示す。
本発明において「C1-6アルキル基」は、炭素数1~6の直鎖状または分枝鎖状の飽和脂肪族炭化水素基をいう。例えば、メチル基、エチル基、n-プロピル基、イソプロピル基、n-ブチル基、イソブチル基、t-ブチル基、n-ペンチル基、n-ヘキシル基等である。好ましくはC1-4アルキル基であり、より好ましくはC1-2アルキル基であり、最も好ましくはメチル基である。
さらに、オキサゾリニル基またはチアゾリニル基を有する化合物(I)は、以下のとおり、ベンゾニトリル化合物とセリンまたはシステインを反応させ、閉環することにより合成することができる。この際、カルボキシ基が置換しているオキサゾリニル環等の炭素原子における絶対配置は、光学活性なセリン等を用いることで制御可能である。また、オキサゾリニル環等のカルボキシ基は、上記と同様に官能基変換が可能である。なお、以下ではシステインを用いてチアゾリニル基を導入する反応を代表して示す。
本発明方法で用いる未分化細胞としては、神経系細胞へ分化する多能性を有するものであれば特に制限されないが、例えば、ES細胞、iPS細胞、間葉系幹細胞のように、神経系細胞への分化能を有すると共に自己複製能を有するものが、特に再生医療への適用のために有用である。また、これら細胞から得られる胚様体も本発明における未分化細胞に含めるものとする。胚様体は一般的に外胚葉、中胚葉および内胚葉を含み、神経系細胞は外胚葉系の細胞であるので、胚様体を用いることによって、神経系細胞への分化誘導がより容易となる。
本発明に係る細胞分化誘導剤は、上記未分化細胞の培養液に添加することにより、未分化細胞へ作用させることができる。
2,3-ジヒドロキシ安息香酸(5g,32.4mmol)をメタノール(50mL)に溶解し、得られた溶液を0℃に冷却した。同温にて濃硫酸(3mL)を滴下し、15時間加熱還流した。反応溶液を減圧濃縮し、飽和炭酸水素ナトリウム水溶液を発泡が収まるまで加えた後、酢酸エチル(100mL)で3回抽出した。有機層を無水硫酸マグネシウムで乾燥後、濾過し、濾液を減圧濃縮した。残渣に28%アンモニア水(92mL)を加え、50℃で16時間撹拌した。次いで反応溶液をおよそ20mLまで減圧濃縮し、酢酸エチル(50mL)で5回抽出した。有機層を無水硫酸マグネシウムで乾燥後、濾過し、濾液を減圧濃縮することで、淡桃色固体の目的化合物(3.92g,収率:80%)を得た。
2,3-ジヒドロキシベンズアミド(2g,13.06mmol)をジメチルホルムアミド(13.06mL)に溶解し、得られた溶液に室温にて炭酸カリウム(9.03g,65mmol)と臭化ベンジル(3.26mL,27.41mmol)を加え、18時間撹拌した。次いで反応溶液に水(20mL)を加えた後、酢酸エチル(100mL)で3回抽出した。有機層を無水硫酸マグネシウムで乾燥後、濾過し、濾液を減圧濃縮して粗生成物を得た。シリカゲル(関東化学社製,Silica Gel 60N)を用いたカラムクロマトグラフィー(溶離液:クロロホルム)により精製し、淡桃色固体の目的化合物(3.59g,収率:83%)を得た。
2,3-ビス(ベンジルオキシ)ベンズアミド(393mg,1.2mmol)を無水ジクロロメタン(4.8mL)に溶解し、得られた溶液に室温にてLawesson試薬(291mg,0.719mmol)を加え、10時間加熱還流した。次いで反応溶液を減圧濃縮して粗生成物を得た。シリカゲル(関東化学社製,Silica Gel 60N)を用いたカラムクロマトグラフィー(溶離液:クロロホルム)により精製し、黄色固体の目的化合物(371mg,収率:86%)を得た。
2,3-ビス(ベンジルオキシ)ベンゾチオアミド(371mg,1.06mmol)をエタノール(5.3mL)に溶解し、得られた溶液に室温にて臭化ピルビン酸エチル(160μL,1.27mmol)を加え、4時間加熱還流した。反応溶液を室温まで冷却し、飽和炭酸水素ナトリウム水溶液(10mL)を加えた後、酢酸エチル(50mL)で3回抽出した。有機層を無水硫酸マグネシウムで乾燥後、濾過し、濾液を減圧濃縮して粗生成物を得た。シリカゲル(関東化学社製,Silica Gel 60N)を用いたカラムクロマトグラフィー(溶離液:クロロホルム)により精製し、紫色油状の目的化合物(432mg,収率:92%)を得た。
2-{2,3-ビス(ベンジルオキシ)フェニル}チアゾール-4-カルボン酸エチル(432mg,0.97mmol)をメタノール(9.7mL)に溶解し、得られた溶液へ4N水酸化ナトリウム水溶液(2.43mL,9.7mmol)を室温にて加え、3時間撹拌した。次いで反応溶液をおよそ10mLまで減圧濃縮し、6N塩酸(10mL)を加えてクロロホルム(50mL)で3回抽出した。有機層を無水硫酸マグネシウムで乾燥後、濾過し、濾液を減圧濃縮することで、褐色不定形(アモルファス)の目的化合物(324mg,収率:80%)を得た。
2,3-ビス(ベンジルオキシ)ベンズアミド(800mg,2.4mmol)を水-アセトニトリルの混合溶媒(1:1,40mL)に溶解し、得られた溶液へ塩化パラジウム(64mg,0.36mmol)を室温にて加え、50℃で6時間撹拌した。次いで反応溶液をおよそ10mLまで減圧濃縮した後、酢酸エチル(100mL)で3回抽出した。有機層を無水硫酸マグネシウムで乾燥後、濾過し、濾液を減圧濃縮して粗生成物を得た。シリカゲル(関東化学社製,Silica Gel 60N)を用いたカラムクロマトグラフィー(溶離液:n-ヘキサン/酢酸エチル=5/1)により精製し、淡黄色固体の目的化合物(692.3mg,収率:91%)を得た。
2,3-ビス(ベンジルオキシ)ベンゾニトリル(624mg,1.98mmol)を1/15Mリン酸緩衝液(pH6.4)-メタノールの混合溶媒(1:1,28mL)に懸濁させ、得られた懸濁液へL-システイン塩酸塩・一水和物(469mg,2.97mmol)と炭酸水素ナトリウム(286mg,3.4mmol)を室温にて加え、65℃で20時間撹拌した。反応溶液を氷冷した後、濾過した。残渣を水(10mL)、エタノール(10mL)、ジエチルエーテル(10mL)で洗浄することで黄色固体の目的化合物(185mg,収率:22%)を得た。
2,3-ジヒドロキシベンゾニトリル(1.43g,10.58mmol)を1/15Mリン酸緩衝液(pH6.4)-メタノールの混合溶媒(1:1,151mL)に懸濁し、得られた懸濁液へL-システイン塩酸塩・一水和物(2.5g,15.87mmol)と炭酸水素ナトリウム(1.53g,18.20mmol)を室温にて加え、65℃で20時間撹拌した。反応溶液を室温とした後、溶媒を減圧留去した。残渣を酢酸エチル(50mL)にて洗浄し、0.5M塩酸(20mL)を加え、酢酸エチル(100mL)で3回抽出した。有機層を無水硫酸マグネシウムで乾燥後、濾過し、濾液を減圧濃縮することで黄色固体の目的化合物(2.231g,収率:88%)を得た。
(R)-2-(2,3-ジヒドロキシフェニル)-4,5-ジヒドロチアゾール-4-カルボン酸(530mg,2.215mmol)をアセトン(4.5mL)に溶解し、得られた溶液へ硫酸ジメチル(723μL,7.64mmol)と炭酸カリウム(1g,7.64mmol)を室温にて加え、70℃で24時間撹拌した。反応懸濁液をセライトで濾過した後、濾液を減圧濃縮して粗生成物を得た。シリカゲル(関東化学社製,Silica Gel 60N)を用いたカラムクロマトグラフィー(溶離液:n-ヘキサン/酢酸エチル=10/1)により精製し、淡黄色油状の目的化合物(199mg,収率:32%)を得た。
(R)-2-(2,3-ジメトキシフェニル)-4,5-ジヒドロチアゾール-4-カルボン酸メチル(199mg,0.707mmol)を1,4-ジオキサン(3.5mL)に溶解し、得られた溶液へ二酸化マンガン(1.2g,14.15mmol)を室温にて加え、室温で25時間撹拌した。反応懸濁液をセライトで濾過した後、濾液を減圧濃縮することで白色固体の目的化合物(193.3mg,収率:98%)を得た。
2-(2,3-ジメトキシフェニル)チアゾール-4-カルボン酸メチル(193mg,0.691mmol)を無水ジクロロメタン(0.691mL)に溶解し、得られた溶液を-78℃に冷却した。アルゴン雰囲気下、同温にて三臭化ホウ素(1Mジクロロメタン溶液,2.1mL,2.073mmol)を滴下し、室温にて1時間撹拌した。次いで反応溶液を冷却し、水(5mL)を加えた後、酢酸エチル(50mL)で5回抽出した。有機層を無水硫酸マグネシウムで乾燥後、濾過し、濾液を減圧濃縮することで黄色固体の目的化合物(153.2mg,収率:93%)を得た。
3,4,5-トリヒドロキシ安息香酸(500mg,2.94mmol)をアセトン(6mL)に溶解し、得られた溶液に室温にて炭酸カリウム(1.8g,13.2mmol)と硫酸ジメチル(1.25mL,13.2mmol)を加え、13.5時間加熱還流した。反応懸濁液をセライトで濾過した後、濾液を減圧濃縮して粗生成物を得た。シリカゲル(関東化学社製,Silica Gel 60N)を用いたカラムクロマトグラフィー(溶離液:クロロホルム/メタノール=30/1)により精製し、白色固体の目的化合物(630mg,収率:95%)を得た。
3,4,5-トリメトキシ安息香酸メチル(300mg,1.33mmol)に28%アンモニア水溶液(3.8mL)を室温で加え、50℃で10時間撹拌した。次いで、反応溶液を減圧濃縮して粗生成物を得た。シリカゲル(関東化学社製,Silica Gel 60N)を用いたカラムクロマトグラフィー(溶離液:クロロホルム/メタノール=10/1およびn-ヘキサン/酢酸エチル=1/2→0/100)により精製し、白色固体の目的化合物(175mg,収率:63%)を得た。
3,4,5-トリメトキシベンズアミド(95mg,0.45mmol)を無水ジクロロメタン(0.45mL)に溶解し、得られた溶液を-78℃に冷却した。アルゴン雰囲気下、同温にて三臭化ホウ素(1Mジクロロメタン溶液,1.35mL,1.35mmol)を滴下し、室温にて18時間撹拌した。次いで反応溶液を氷冷し、水(5mL)を加えた後、酢酸エチル(50mL)で3回抽出した。得られた有機層を無水硫酸ナトリウムで乾燥後、濾過し、濾液を減圧濃縮して粗生成物を得た。シリカゲル(関東化学社製,Silica Gel 60N)を用いたカラムクロマトグラフィー(溶離液:酢酸エチル/メタノール=100/0→10/1→5/1)により精製し、淡茶色固体の目的化合物(27.4mg,36%)を得た。
(1) マウスES細胞の分化誘導
直径60mmのペトリディッシュへ、表1に示す組成のES培地とNP培地を1:1の割合で混合した胚様体形成用培地(4mL)を加え、さらにマウスES細胞(The Wellcome Trust Sanger InstituteのDr.Alan Bradleyから入手,AB2-2)を1×106個蒔き、5%CO2雰囲気下、37℃で1日間培養した。次いで、ガラスピペットを用いたピペッティングにより細胞を剥離した後、同じペトリディッシュに蒔き直し、5%CO2雰囲気下、37℃で3日間培養して胚様体を形成させた。得られた細胞塊を回収し、沈降させた後に表1に示す組成のNP培地(10mL)に懸濁した。
上記(1)のとおり被検化合物1により分化誘導された細胞をパラホルムアルデヒドで固定した後、PBSで洗浄し、5%ヤギ血清/1%BSA/PBSを100μL/ウェルの最終濃度となるように添加し、室温で60分間インキュベートすることによりブロッキングした。次いで、抗NestinマウスIgG(American Research Products社製,最終濃度:5μg/mL)または抗Flk1ウサギIgG(Millipore社製,最終濃度:5μg/mL)を添加し、4℃で一晩インキュベートした。各ウェルをPBS-0.1% Tween20で洗浄した後、二次抗体として、FITC標識抗マウス抗体(Biosource International社製,最終濃度:1μg/mL)またはウサギIgG抗体(Biosource International社製,最終濃度:1μg/mL)を添加し、室温で2時間インキュベートした。各ウェルをPBS-0.1% Tween20で洗浄した後、IN Cell Analyzer 1000(GE Healthcare社製)を用いて蛍光写真を撮影した。
80μMの被検化合物4を用いて5日間処理した以外は上記(1)と同様にして、マウスES細胞を分化誘導した。得られた細胞からトリゾル試薬を用いて全RNAを抽出し、得られた全RNAから常法に従ってcDNAを合成した。合成したcDNAを鋳型として、Oct3/4・Nanog(未分化細胞マーカー)、Nestin(神経幹細胞マーカー)、Sox9・Sox10・Slug(神経堤細胞マーカー)、GATA2(中胚葉マーカー)、Sca-1(幹細胞マーカー)、c-kit(造血幹細胞マーカー)の各プライマーを用いてPCRを行い、各マーカーの発現解析を行った。また、比較のために、未分化マウスES細胞と、マウスES細胞を3日間培養した胚様体についても同様に発現解析を行った。結果を図5に示す。
実施例7(1)において、マウスES細胞の代わりにヒトiPS細胞(理化学研究所から入手,hPS0001 201B7 lot3)を用いた以外は同様にして分化誘導した。
直径60mmのペトリディッシュへ、表1に示す組成のES培地とNP培地を1:1の割合で混合した胚様体形成用培地(4mL)を加え、さらにマウスES細胞(The Wellcome Trust Sanger InstituteのDr.Alan Bradleyから入手,AB2-2)を1×106個蒔き、5%CO2雰囲気下、37℃で1日間培養した。次いで、ガラスピペットを用いたピペッティングにより細胞を剥離した後、同じペトリディッシュに蒔き直し、5%CO2雰囲気下、37℃で3日間培養して胚様体を形成させた。得られた細胞塊を回収し、沈降させた後に表1に示す組成のNP培地(10mL)に懸濁した。次いで、培養細胞懸濁液(5mL)をI型コラーゲンコートされた96ウェルプレートに添加し、5%CO2雰囲気下、37℃で一晩インキュベートした。翌日、被検化合物5を単独で、或いは被検化合物とレチノイン酸を併用して各最終濃度10nMで添加し、5%CO2雰囲気下、37℃で4日間接着培養した。培養後、PBSで洗浄し、0.25%Trypsinを用いて細胞を剥がし、細胞数を計数した。実験は4回行い、その平均を求めた。結果を図7に示す。なお、図7中、「RA」はレチノイン酸を示す。
直径60mmのペトリディッシュへ、実施例7で用いた胚様体形成用培地(4mL)を加え、さらにマウスES細胞(AB2-2)を1×106個蒔き、5%CO2雰囲気下、37℃で3日間培養した。得られた胚葉体を15mL容チューブに回収した。上記ペトリディッシュを洗浄するために1×PBS(5mL)を加え、同PBSを上記チューブに回収した。回収された細胞の半量を別のチューブに移し、室温で5分間静置した。上清を除去した後、実施例7で用いたNP培地(10.5mL)を加えた。よく懸濁した後、I型コラーゲンコートされた96ウェルプレートの各ウェルに100μLずつ添加し、5%CO2雰囲気下、37℃で一晩インキュベートした。各ウェルへ、表3の組成を有するDifferentiation mediumを適量添加した。
Claims (10)
- 下記式(I)で表されるカテコール誘導体を含むことを特徴とする細胞分化誘導剤。
[式中、
R1は、カルボキシ基、(C1-6アルコキシ)カルボニル基、C1-7アルカノイル基、C2-8アルキニル基、カルバモイル基、シアノ基、ニトロ基もしくはハロゲン原子、または、置換基αを有していてもよいオキサゾリル基、置換基αを有していてもよいチアゾリル基、置換基αを有していてもよいオキサゾリニル基、もしくは置換基αを有していてもよいチアゾリニル基を示し;
R2は、水素原子、C1-6アルキル基またはベンジル基を示し;
nは2以上、5以下の整数を示し;
置換基αは、カルボキシ基、(C1-6アルコキシ)カルボニル基、C1-7アルカノイル基、カルバモイル基、シアノ基、ニトロ基およびハロゲン原子から選択される1以上を示す。
但し、2以上のR2O基は、互いに同一であっても異なっていてもよい。] - R2が水素原子である請求項1に記載の細胞分化誘導剤。
- 少なくとも2つの-OR2基が互いに隣り合っている請求項1または2に記載の細胞分化誘導剤。
- R1がシアノ基またはカルバモイル基である請求項1~3のいずれかに記載の細胞分化誘導剤。
- さらにレチノイン酸を含む請求項1~4のいずれかに記載の細胞分化誘導剤。
- 未分化細胞を神経系細胞へ分化誘導するためのものである請求項1~5のいずれかに記載の細胞分化誘導剤。
- 未分化細胞を神経堤細胞へ分化誘導するためのものである請求項1~5のいずれかに記載の細胞分化誘導剤。
- 請求項1~5のいずれかに記載の細胞分化誘導剤を含有することを特徴とする培養液。
- 未分化細胞の分化を誘導するために用いられる、下記式(I)で表されるカテコール誘導体。
[式中、
R1は、カルボキシ基、(C1-6アルコキシ)カルボニル基、C1-7アルカノイル基、C2-8アルキニル基、カルバモイル基、シアノ基、ニトロ基もしくはハロゲン原子、または、置換基αを有していてもよいオキサゾリル基、置換基αを有していてもよいチアゾリル基、置換基αを有していてもよいオキサゾリニル基、もしくは置換基αを有していてもよいチアゾリニル基を示し;
R2は、水素原子、C1-6アルキル基またはベンジル基を示し;
nは2以上、5以下の整数を示し;
置換基αは、カルボキシ基、(C1-6アルコキシ)カルボニル基、C1-7アルカノイル基、カルバモイル基、シアノ基、ニトロ基およびハロゲン原子から選択される1以上を示す。
但し、2以上のR2O基は、互いに同一であっても異なっていてもよい。] - 未分化細胞を神経系細胞へ分化誘導するための方法であって、
下記式(I)で表されるカテコール誘導体を含む培地で未分化細胞を培養する工程を含むことを特徴とする方法。
[式中、
R1は、カルボキシ基、(C1-6アルコキシ)カルボニル基、C1-7アルカノイル基、C2-8アルキニル基、カルバモイル基、シアノ基、ニトロ基もしくはハロゲン原子、または、置換基αを有していてもよいオキサゾリル基、置換基αを有していてもよいチアゾリル基、置換基αを有していてもよいオキサゾリニル基、もしくは置換基αを有していてもよいチアゾリニル基を示し;
R2は、水素原子、C1-6アルキル基またはベンジル基を示し;
nは2以上、5以下の整数を示し;
置換基αは、カルボキシ基、(C1-6アルコキシ)カルボニル基、C1-7アルカノイル基、カルバモイル基、シアノ基、ニトロ基およびハロゲン原子から選択される1以上を示す。
但し、2以上のR2O基は、互いに同一であっても異なっていてもよい。]
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CN111492052A (zh) | 2017-11-30 | 2020-08-04 | 国立大学法人京都大学 | 细胞的培养方法 |
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