Neuronal activators

Abstract

To provide a method of inducing the differentiation of pluripotent stem cells into neurons while suppressing the survival of undifferentiated cells in a biological environment that excludes the involvement of unidentified components, without going through the formation process of the germ layer body.SOLUTION: Provided is a method of inducing the differentiation of iPS cells into neurons in a xeno-free culture system without going through the formation process of the germ layer body, the method comprising: a) culturing iPS cells in which a human Neurogenin-2 gene is induced in a laminin-coated vessel; b) transiently inducing expression of the Neurogenin 2 gene to initiate differentiation; and c) culturing the iPS cells that have started differentiation in the laminin-coated vessel and adding, as a development promoting agent for differentiation, at least one of the six types of a small molecule TGFβ family inhibitor, an adenylate cyclase activator, a GSK3 inhibitor, a smoothened agonist, a rock inhibitor, and an indolinone compound.SELECTED DRAWING: Figure 7

Description

[Technical Field]

[0001] First aspect of the present invention The present invention relates to a neuron activator containing an indolinone compound as an active ingredient and used for treating neuropsychiatric disorders, etc. The present invention also relates to a neuron activator containing an indolinone compound as an active ingredient that promotes the expression of one or more of neurotrophic factors, namely, brain-derived neurotrophic factor (BDNF), the vascular endothelial growth factor (VEGF) family, the VEGF receptor family, and VEGF co-receptors, which were named after the BDNF family, which was discovered as a growth factor involved in angiogenesis but is now recognized as a neurotrophic factor as well.

[0002] Second Aspect of the Invention The present invention relates to a method for inducing differentiation of pluripotent stem cells into neurons, using a biological environment (xeno-free culture system) that does not contain feeder cells or unidentified components, such as animal-derived serum or Matrigel.The present invention also relates to a differentiation induction method that is simpler than conventional methods, leaves fewer undifferentiated cells, and facilitates analysis of mechanisms. [Background technology]

[0003] First aspect of the present invention In recent years, as society has become more sophisticated and complex, the number of patients suffering from depression, PTSD, panic disorder, and other conditions has increased, raising concerns. Against this backdrop, mental illnesses have been added to the four major diseases (cancer, stroke, myocardial infarction, and diabetes), which are considered to be the five major diseases that require priority countermeasures. Decreased brain function due to brain and nervous system disorders not only significantly reduces the quality of life of the patient themselves, but also significantly impacts the lives of those around them, such as family members involved in nursing care. Therefore, improving and preventing the decline in brain function caused by these diseases has become an important issue. It has been reported that decreased expression of brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) is observed in various neuropsychiatric disorders caused by a decline in brain and nerve function, including amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, Huntington's disease, age-related neurological disorders (dementia, etc.), depression, schizophrenia, bipolar disorder, post-traumatic stress disorder (PTSD), anxiety disorder, panic disorder, autism spectrum disorder, and developmental disorders (Non-Patent Documents 1-6, 8-15, 17, 18). Therefore, replacement therapy with BDNF and VEGF proteins has been attempted for brain and nervous system diseases. Because VEGF protein does not cross the blood-brain barrier, intraventricular administration has been attempted. Drugs or compounds that increase BDNF expression have also been investigated (Patent Documents 1 and 2). There are two main approaches to regenerative medicine. One is to utilize stem cells such as iPS cells. The other is to bring out the self-regenerative ability of cells that have become damaged and no longer function. As a functional substance to be used in this method of bringing out the self-repair ability of damaged and non-functional cells, the search for functional substances that activate cell growth factors, including BDNF, has been proposed (Non-Patent Document 1). Currently, there is discussion about the limitations of antidepressants that target monoamine substances, such as serotonin reuptake inhibitors. Substances that promote BDNF production are attracting attention as seeds for new antidepressants, and screening is being conducted on various herbal medicines (Non-Patent Document 1).

[0004] Meanwhile, increased expression of VEGF signaling pathway proteins is believed to be effective in promoting the proliferation and differentiation of neural stem cells. Therefore, the treatment of cognitive impairment has been investigated using recombinant VEGF-C, recombinant VEGF-D, and compounds activating VEGF receptors 2 and 3, which are receptors for VEGF-C and VEGF-D (Patent Documents 3-6). Furthermore, since increased VEGF expression using VEGF protein administration or viral vectors has shown significant improvement in ALS disease model animals (Non-Patent Documents 16, 17, and 18), VEGF protein has been investigated as a therapeutic agent for ALS. However, because VEGF protein does not cross the blood-brain barrier, direct intraventricular administration was used in clinical trials (Patent Document 7, Non-Patent Document 8, Non-Patent Document 14), but the clinical trials were discontinued in 2015. Ketamine, a powerful, fast-acting antidepressant approved by the US FDA in 2019, has been shown to induce re-extension of stress-induced atrophied neuronal dendrites via VEGF and BDNF, leading to symptom improvement (Non-Patent Document 7). Thus, the cooperative function of VEGF and BDNF is essential for the efficacy of the antidepressant ketamine. Although the use of VEGF protein agents has been investigated in the treatment of neuropsychiatric disorders, no successful studies have been conducted in humans, and no studies have been conducted to treat neurological disorders by increasing VEGF expression using small molecule compounds.

[0005] Second Aspect of the Invention Numerous methods for differentiating embryonic stem cells (ES cells) or induced pluripotent stem cells (iPS cells) into neurons have been reported (Patent Document 8 and Patent Document 9, Non-Patent Documents 19-24), and efforts are being made to utilize these methods in regenerative medicine and drug discovery for neurodegenerative diseases (Patent Document 8 and Patent Document 9) (Non-Patent Documents 19, 25, and 26).

[0006] However, these methods require the removal of undifferentiated cells remaining after differentiation induction, for example, by cell sorting (Non-Patent Document 19). Furthermore, they require the use of xenogeneic feeder cells (Patent Document 8, Non-Patent Documents 19, 20, and 24) or mouse sarcoma cell-derived Matrigel, which contains unidentified components (Patent Documents 8 and 9, Non-Patent Documents 19-26). Furthermore, they require the use of serum containing unidentified components derived from xenogeneic organisms or mouse glial cell co-culture (Non-Patent Documents 19, 21, and 25). Furthermore, they require a complex stepwise differentiation process, including the formation of embryoid somatic cells (cell aggregates) (Patent Documents 8 and 9, Non-Patent Documents 22, 24-26). Furthermore, they require the use of highly cytotoxic reagents (Patent Document 9, Non-Patent Documents 19, 20, and 24). [Prior art documents] [Patent documents]

[0007] [Patent Document 1] JP 2001-328947 A [Patent Document 2] WO2017 / 104706 publication [Patent Document 3] Publication EP2714063 [Patent Document 4] US2007 / 0082848 publication [Patent Document 5] US2008 / 0057028 publication [Patent Document 6] WO2012 / 163542 publication [Patent Document 7] Special publication 2008-500312 (P2008-500312A) [Patent Document 8] Special publication 2013-501502 [Patent Document 9] WO2016 / 063985 publication [Non-patent literature]

[0008] [Non-Patent Document 1] Mamoru Fukuchi, Journal of Pharmaceutical Sciences 137(9)1103-1115(2017) [Non-patent document 2] Kenji Hashimoto, Japanese Pharmacological Journal 127, 201-204 (2006) [Non-patent document 3] Gutierrez et al., Frontiers in Molecular Neuroscience.12:335(2020) [Non-patent document 4] Review: Angelucci et al., Molecular Psychiatry. 10:345-352 (2005) [Non-Patent Document 5] Lai et al., World J of Psychiatry.6:102-117(2016) [Non-patent document 6] Miller et al., J Clinical Medicine.6:108(2017) [Non-Patent Document 7] Deyama et al., Biol Psychiatry.86:143-152(2019) [Non-patent document 8] Review article: Keifer et al., Pharmacology & Therapeutics. 141:261-271 (2014) [Non-Patent Document 9] Lange et al., Nature Reviews Neurology 12:439-454 (2016) [Non-Patent Document 10] Hohman et al., JAMA 72:520-529 (2015) [Non-Patent Document 11] Martino et al., Nature Neurosci.10:1089-1093 (2007) [Non-Patent Document 12] Nieto et al., Frontiers in Psychiatry 12:662407 (2021) [Non-Patent Document 13] Saghazadeh et al., J Autism Dev Disord. 47:1018–1029 (2017) [Non-Patent Document 14] Dammaら、Brain Commun.2:fcaa160 (2020)

Non-licensed Document 15

Non-licensed Document 16

Non-licensed Document 17

Non-licensed Document 18

Non-licensed Document 19

Non-licensed Document 20

Non-licensed Document 21

Non-licensed Document 22

Non-licensed Document 23

Non-licensed Document 24

Non-licensed Document 25

Non-licensed Document 26

[0009] First aspect of the present invention An object of the present invention is to provide a neuronal activating agent for use in the treatment of neuropsychiatric disorders, etc. In particular, an object of the present invention is to provide a neuronal activating agent that activates neurons by promoting the expression of proteins related to the BDNF and / or VEGF signaling pathway. Second Aspect of the Invention The present invention aims to provide a method for inducing differentiation from pluripotent stem cells into neurons while suppressing the remaining undifferentiated cells, without going through the process of forming embryoid bodies (cell aggregates), in a biological environment (xeno-free culture system) that eliminates the involvement of unidentified components. [Means for solving the problem]

[0010] First aspect of the present invention Due to the properties of iPS cells as pluripotent stem cells, research, development, and clinical trials have been conducted on them as one of the pillars of regenerative medicine. Clinical trials have been initiated for myocardial regeneration in ischemic heart disease by transplanting cardiomyocyte cell sheets created from iPS cells, Parkinson's disease treatment by transplanting dopaminergic neural progenitor cells created from iPS cells, and age-related macular degeneration treatment by transplanting retinal pigment epithelial cells created from iPS cells, and the development of platelet preparations created from iPS cells is also progressing. The availability of iPS cells has also been a major technological innovation in the field of drug discovery. Currently, it takes over 10 years and over 100 billion yen in R&D costs to bring a single drug to market. The drug discovery process involves (1) basic research, such as elucidating pathological mechanisms and exploring drug discovery seeds, (2) building screening systems and screening compounds, and (3) safety, efficacy, and kinetic testing of candidate drugs, followed by clinical trials. However, many drugs are dropped out after clinical trials or after being marketed due to toxicity or other reasons. To successfully develop safe and effective new drugs while reducing development costs, it is essential to improve the efficiency and accuracy of each step of the drug discovery process leading up to clinical trials. In recent years, therefore, there has been growing expectation for innovation and acceleration of the drug discovery process using hepatocytes, cardiomyocytes, nerve cells, etc., generated from human iPS cells. As a method for exploring therapeutic drugs, attempts have been made to evaluate (screen) the efficacy of existing drugs for other diseases using disease-specific iPS cells. Through this iPS drug discovery, it has been confirmed that bosutinib, an anticancer drug for chronic myeloid leukemia, can be used as a therapeutic drug for ALS; rapamycin, an immunosuppressant, can be used as a therapeutic drug for fibrodysplasia ossificans progressiva and Pendred syndrome; bromocriptine, an ergot alkaloid-derived Parkinson's disease drug, can be used as a therapeutic drug for Alzheimer's disease; and ropiniole, a dopamine D2 receptor agonist that improves Parkinson's disease, can be used as a therapeutic drug for ALS. At Kyoto University, clinical trials of bosutinib in ALS patients, rapamycin in patients with fibrodysplasia ossificans progressiva, and bromocriptine in patients with Alzheimer's disease have begun. At Keio University, clinical trials of ropiniol in ALS patients and rapamycin in patients with Pendred syndrome have begun. An interim report was released showing that ropiniol slows the progression of ALS by approximately seven months. On September 30, 2021, a Phase I trial of bosutinib reported its potential to slow the progression of ALS in some patients, but significant challenges remain, including liver damage.

[0011] Given the well-known utility of iPS cells as a human disease model, the present inventors developed a simple, xeno-free culture system that does not require animal-derived components, such as feeder cells, Matrigel, or serum, whose quantity and quality cannot be determined, from iPS cell culture to the completion of neuronal differentiation. Using three lines of iPS cells derived from healthy individuals, we first performed a detailed analysis of genes whose expression is enhanced or decreased during neuronal differentiation in iPS cells derived from healthy individuals. Furthermore, we performed a detailed analysis of the differences in gene expression dynamics between iPS cells derived from healthy individuals and iPS cells derived from ALS patients, using three lines of iPS cells derived from healthy individuals and four lines of iPS cells derived from familial or sporadic ALS patients. Furthermore, we elucidated the mechanisms involved in healthy neuronal development and survival, and aimed to develop disease-modifying drugs (DMDs) that suppress symptom progression or ameliorate neurodegenerative diseases by activating self-regenerative capacity, a pillar of regenerative medicine.

[0012] We tracked the gene expression dynamics of VEGF-A and its family members and their receptors, which are known to be involved in preventing motor neuron death in ALS, over time during the differentiation of iPS cells into motor neurons and in long-term neuronal culture after differentiation. We found that VEGF-A expression remained constant throughout the differentiation process, without any significant increase in neurons. However, expression of VEGF receptor-1, a negative regulator of VEGF-A signaling, was almost completely eliminated in terminally differentiated motor neurons. However, expression of VEGF receptor-2, which is thought to mediate neuronal survival through VEGF-A signaling, also tended to decrease. Based on these findings, we screened for compounds that bind to and modify the activity of VEGF receptor-2, and discovered that the indolinone compounds of the present invention are effective in preventing motor neuron death differentiated from iPS cells derived from sporadic ALS patients.

[0013] In addition, it was found that the indolinone compounds of the present invention can maintain the survival of neurons without supplementing neurotrophic factors in the screening evaluation system, and it was confirmed that the indolinone compounds of the present invention have a neuronal activation effect due to the autocrine action of neurotrophic factors (BDNF, VEGF family, VEGF family receptors) and have a survival-maintaining effect.

[0014] The present inventors have extensively investigated the mechanism of action of semaxanib, an indolinone compound of the present invention, and discovered a novel mechanism of action in which the simultaneous promotion of the expression of genes involved in BDNF and VEGF signaling pathways improves the maintenance of healthy nervous system functions, such as maintaining neuronal survival and neuroplasticity. The present inventors have found that the compound of the present invention promotes BDNF expression as well as the promotion and enhancement of the expression of VEGF signaling pathway proteins (VEGF-A, VEGF-C, VEGF-D, VEGF receptor-2, VEGF receptor-3, and the co-receptor for VEGF-A, VEGF-C, and VEGF-D (neuropilin 2)), which are involved in maintaining neuronal survival, promoting dendritic outgrowth and branching, and neuroplasticity, such as spine formation and synaptogenesis, and are involved in promoting the proliferation and differentiation of neural stem cells. The present inventors have discovered from the mechanism of action of the indolinone compounds of the present invention that the compounds of the present invention function as neuronal activators for preventing or treating neuropsychiatric disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, Huntington's disease, Rett syndrome, and age-related neurological disorders (dementia, etc.), as well as neuropsychiatric disorders such as depression, schizophrenia, bipolar disorder, autism spectrum disorder, developmental disorders, post-traumatic stress disorder (PTSD), anxiety disorder, and panic disorder, as well as symptom recovery after stroke and functional recovery after spinal cord injury, and are therefore believed to be effective in preventing or treating these neuropsychiatric disorders. These compounds are also believed to contribute to extending healthy lifespan.

[0015] Second Aspect of the Invention The present inventors have conducted extensive research into improving the method for inducing differentiation of induced pluripotent stem cells (iPS cells) into neural cells. In particular, they have investigated the optimization of the reagents used in culture and the associated differentiation induction method. As a result, they have gained many insights, such as the elimination of the need for the addition of retinoic acid (RA), which was previously thought to be necessary for inducing differentiation of neural cells. An overview of these insights is shown in Figure 7. The differentiation induction method of the present invention can be broadly divided into the following two stages.

[0016] a) Initiation of differentiation (step 2): To initiate differentiation of induced pluripotent stem cells (iPS cells) into neurons, conventional methods require the formation of embryoid bodies, but this requires a long period of time and is a complex process. The inventors discovered that the formation and control of embryoid bodies is difficult in a xeno-free culture system that does not use feeder cells. For this reason, we considered using the induction of expression of the neurogenin gene family (1, 2, and 3), a transcription factor that plays a crucial role in the initiation of neuronal differentiation. Using expression vectors, we performed transient expression induction experiments by introducing all combinations of the neurogenin gene family (1, 2, and 3) and each gene alone, and found that neurogenin 2 was necessary and sufficient for inducing differentiation of iPS cells into neurons. Transient expression of neurogenin 2 can be achieved by introducing the neurogenin 2 gene into iPS cells using RNA lipofection. Alternatively, the neurogenin 2 gene can be incorporated into a vector and then introduced into iPS cells, followed by transient induction of neurogenin 2 expression with, for example, doxycycline. The method can be selected appropriately depending on the intended use of the cells. For clinical applications, introducing the neurogenin 2 gene using RNA lipofection is desirable; however, this method has low gene transfer efficiency and is dependent on many factors, including culture conditions, making it difficult to obtain consistent results. Drug screening and analysis of drug mechanisms require stable induction of iPS cell differentiation. Therefore, it is desirable to introduce the neurogenin 2 gene into iPS cells using a vector and then induce expression with, for example, doxycycline. Neurogenin 2 is a switch that initiates differentiation. While induction of neurogenin 2 expression is necessary for a certain period of time at the beginning of differentiation, it is preferable to discontinue neurogenin 2 expression thereafter. We found that doxycycline treatment for 16 to 20 hours is preferable for neurogenin 2 expression induction. When iPS cells transfected with a neurogenin 2 gene whose expression can be induced by doxycycline were treated with doxycycline for 16 to 20 hours to express neurogenin 2, the cells began to extend processes, became smaller, and took on the morphology of neural progenitor cells (Figure 8a), as shown in Figure 8b and Figure 9b, compared to control cells not treated with doxycycline (Figure 9a). This state was defined as the initiation stage of differentiation (Figure 7). Furthermore, it was found that the iPS maintenance medium containing a Rock inhibitor is preferable as an environment for initiating differentiation of iPS cells.

[0017] b) Differentiation development stage (step 3): In order to further induce differentiation of iPS cells whose differentiation was initiated by transient expression of Neurogenin 2 due to doxycycline treatment, we investigated culture methods and culture reagents. As shown in Figure 7, differentiation was advanced in primary and secondary cultures in laminin-coated containers using five appropriate reagents (Rock inhibitor, TGFβ family inhibitor, GSK3 inhibitor, adenylate cyclase activator, and Smoothened agonist) in stages. In the tertiary culture, a Rock inhibitor and neurotrophic factors (BDNF, GDNF, and IGF-1) were added, making it possible to induce neuronal differentiation in a xeno-free culture system (Figure 7: Developmental stages of differentiation). In the case of iPS cells derived from non-healthy patients (such as ALS patients), the cells die during the differentiation induction process, making it impossible to continue the long-term culture required for complete differentiation into neurons. In such cases, it was found that adding an indolinone compound makes long-term culture possible and allows the creation of patient-derived fully differentiated neurons. Thus, the present inventors discovered that it is possible to generate desired nerve cells from iPS cells of healthy individuals and patients. The present inventors have investigated the reagents necessary for differentiation induction at the above-mentioned differentiation development stage and found that differentiation into neural cells can be induced without the addition of retinoic acid (RA) or the BMP signal inhibitor LDN193189, which were thought to be necessary in conventional differentiation induction methods. Furthermore, they have found that the addition of RA or LDN193189, which are conventional differentiation inducers for neural cells, causes cell death in the differentiation induction method of the present invention, contrary to general knowledge.

[0018] When the method of the present invention for inducing differentiation of iPS cells into neurons is compared with conventional differentiation culture methods, it has become clear that it has the following features. 1) By introducing neurogenin 2 into iPS cells, it became possible to precisely induce differentiation. 2) It is now possible to induce differentiation into neural cells in a xeno-free culture system, which avoids contamination with substances derived from different organisms and makes it possible to apply this technology to human regenerative medicine. 3) Since differentiation into neurons can be induced without forming embryoid bodies (cell aggregates), the process of transferring the cells to a container for suspension cells and culturing them can be omitted, making it possible to culture the cells for stable differentiation induction. 4) Only a maximum of six types of differentiation promoters are required, and when neurotrophic factors are added during final differentiation, a maximum of nine types of reagents can be used for culture, making process management easier and reducing impurities. 5) Generally, when using materials derived from different organisms, the type and amount of constituents cannot be identified, and they vary greatly depending on the lot, making it difficult to distinguish between artifacts and meaningful data. The differentiation method of the present invention avoids the influence of contaminants, which vary in their state, and ensures higher reproducibility. This means that it provides a means to further elucidate function and pathology at the neuronal level. 6) By adding indolinone compounds to the differentiation induction process, it became possible to produce terminally differentiated neurons without the cells dying during the differentiation process, even if the iPS cells had low cell activity, such as those derived from patients with neurological diseases. 7) We found that indolinone compounds promote the gene expression of the apoptosis-inhibiting protein survivin in iPS cells. As a result, survivin gene expression is significantly reduced in iPS cells derived from ALS patients. However, by adding indolinone compounds to the differentiation of such iPS cells, survivin expression was improved, and differentiated neurons could be produced without early cell death during differentiation induction. The present inventors have completed the present invention based on the above findings.

[0019] That is, the gist of the present invention is as follows. First aspect of the present invention (1) General formula (1) [ka] [In the formula, R1, R2, and R3 each independently represent a hydrogen atom, a halogen atom, a substituted or unsubstituted lower alkyl group having 1 to 4 carbon atoms, a formyl group, an acetyl group, a carboxyl group, a substituted or unsubstituted lower alkoxycarbonyl group having 1 to 4 carbon atoms, or a substituted or unsubstituted lower alkylamido group having 1 to 4 carbon atoms, and the substituent is a halogen atom, a carboxyl group, a lower alkoxycarbonyl group having 1 to 4 carbon atoms, or a di-lower alkylamino group having 1 to 4 carbon atoms.] R4, R5, R6, and R7 are each independently selected from the group consisting of a hydrogen atom, a halogen atom, a nitro group, a substituted or unsubstituted lower alkyl group having 1 to 4 carbon atoms, a lower alkoxy group having 1 to 4 carbon atoms, a lower alkylcarbonyl group having 1 to 4 carbon atoms, a lower alkoxycarbonyl group having 1 to 4 carbon atoms, a carboxyl group, and a hydroxyl group, and the substituent is a halogen atom, a carboxyl group, or a lower alkoxycarbonyl group having 1 to 4 carbon atoms. (2) The neuron activating agent according to (1) above, wherein R1, R2, and R3 are each independently a hydrogen atom, a methyl group, an ethyl group, a halogen atom, a formyl group, an acetyl group, a carboxyl group, an ethyloxycarbonyl group, a methoxycarbonylmethyl group, an ethoxycarbonylmethyl group, a 2-carboxyethyl group, a 2-methoxycarbonylethyl group, a 2-ethoxycarbonylethyl group, or a 2-diethylaminoethylamide group, and R4, R5, R6, and R7 are each independently a hydrogen atom, a halogen atom, or a nitro group. (3) The nerve cell activating agent according to (1) or (2) above, wherein the halogen atoms in R1, R2 and R3 are chlorine atoms, bromine atoms, or iodine atoms, and the halogen atoms in R4, R5, R6 and R7 are fluorine atoms or chlorine atoms. (4) The nerve cell activating agent according to (1) or (2) above, wherein R1 and R3 are methyl groups, R2 is a hydrogen atom, and R4, R5, R6 and R7 are hydrogen atoms. (5) The nerve cell activating agent according to any one of (1) to (4) above, wherein the nerve cell activation is activation of nerve cell survival. (6) The nerve cell activating agent according to any one of (1) to (5) above, wherein the nerve cells are nerve cells of the central or peripheral nervous system. (7) The nerve cell activating agent according to any one of (1) to (6) above, wherein the nerve cells are transplantable neural stem cells for regenerative medicine. (8) The nerve cell activating agent according to any one of (1) to (6) above, wherein the nerve cell activation is mediated by promoting the expression of BDNF and / or VEGF signaling proteins.

[0020] (9) A therapeutic agent for neuropsychiatric disorders, comprising the neuron activating agent according to any one of (1) to (8) above as an active ingredient. (10) The therapeutic agent for neuropsychiatric disorders according to (9) above, wherein the neuropsychiatric disorder is amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, Huntington's disease, age-related neurological disorders (dementia, etc.), depression, schizophrenia, bipolar disorder, post-traumatic stress disorder (PTSD), anxiety disorder, panic disorder, autism spectrum disorder, developmental disorder, Rett syndrome, Down syndrome, cerebral ischemic disorder, or spinal cord injury.

[0021] (11) General formula (1) [ka] [In the formula, R1, R2, and R3 each independently represent a hydrogen atom, a halogen atom, a substituted or unsubstituted lower alkyl group having 1 to 4 carbon atoms, a formyl group, an acetyl group, a carboxyl group, a substituted or unsubstituted lower alkoxycarbonyl group having 1 to 4 carbon atoms, or a substituted or unsubstituted lower alkylamido group having 1 to 4 carbon atoms, and the substituent is a halogen atom, a carboxyl group, a lower alkoxycarbonyl group having 1 to 4 carbon atoms, or a di-lower alkylamino group having 1 to 4 carbon atoms.] wherein R4, R5, R6, and R7 are each independently selected from the group consisting of a hydrogen atom, a halogen atom, a nitro group, a substituted or unsubstituted lower alkyl group having 1 to 4 carbon atoms, a lower alkoxy group having 1 to 4 carbon atoms, a lower alkylcarbonyl group having 1 to 4 carbon atoms, a lower alkoxycarbonyl group having 1 to 4 carbon atoms, a carboxyl group, and a hydroxyl group, and the substituent is a halogen atom, a carboxyl group, or a lower alkoxycarbonyl group having 1 to 4 carbon atoms. (12) The expression promoter described in (11) above, wherein R1, R2, and R3 are each independently a hydrogen atom, a methyl group, an ethyl group, a halogen atom, a formyl group, an acetyl group, a carboxyl group, an ethyloxycarbonyl group, a methoxycarbonylmethyl group, an ethoxycarbonylmethyl group, a 2-carboxyethyl group, a 2-methoxycarbonylethyl group, a 2-ethoxycarbonylethyl group, or a 2-diethylaminoethylamide group, and R4, R5, R6, and R7 are each independently a hydrogen atom, a halogen atom, or a nitro group. (13) An expression promoter according to (11) or (12) above, wherein the halogen atoms in R1, R2 and R3 are chlorine atoms, bromine atoms, or iodine atoms, and the halogen atoms in R4, R5, R6 and R7 are fluorine atoms or chlorine atoms. (14) The expression promoter according to (11) or (12) above, wherein R1 and R3 are methyl groups, R2 is a hydrogen atom, and R4, R5, R6 and R7 are hydrogen atoms. (15) The expression promoter according to any one of (11) to (14) above, wherein the expression promotion is promotion of expression of a BDNF or VEGF signaling pathway protein. (16) The expression promoter according to any one of (11) to (14) above, wherein the expression promotion is simultaneous promotion of the expression of BDNF and VEGF signaling pathway proteins. (17) An expression promoter according to any one of (11) to (16) above, characterized in that the VEGF signal transduction system protein is at least one of VEGF-A, VEGF-C, VEGF-D, VEGF receptor-2, VEGF receptor-3, and neuropilin 2, a co-receptor for VEGF-A, VEGF-C, and VEGF-D. (18) The expression promoter according to any one of (11) to (17) above, wherein the VEGF signal transduction proteins are VEGF-A, VEGF-C, and VEGF-D. (19) An expression promoter according to any one of (11) to (17) above, wherein the VEGF signal transduction system proteins are VEGF-A, VEGF-C, VEGF-D, VEGF receptor-2, VEGF receptor-3, and neuropilin 2, a co-receptor for VEGF-A, VEGF-C, and VEGF-D.

[0022] Second Aspect of the Invention (20) A promoter of survivin expression in iPS cells, containing an indolinone compound as an active ingredient. (21) The survivin expression promoter according to (20) above, wherein the iPS cells are derived from an ALS patient. (22) The survivin expression promoter according to (20) or (21) above, wherein the iPS cells are iPS cells undergoing differentiation induction. (23) The survivin expression promoter according to any one of (20) to (22) above, wherein the indolinone compound is a compound according to (1) to (4) above. (24) The survivin expression promoter according to (23) above, wherein the indolinone compound is semaxanib. (25) The survivin expression promoter according to any one of (20) to (24) above, wherein the iPS cells are capable of inducing transient expression of neurogenin 2.

[0023] (26) A survivin expression promoter in neurons induced to differentiate from iPS cells, comprising an indolinone compound as an active ingredient. (27) The survivin expression promoter according to (26) above, wherein the nerve cells are motor nerve cells. (28) The survivin expression promoter according to (26) or (27) above, wherein the nerve cells are nerve cells induced to differentiate from iPS cells derived from an ALS patient. (29) The survivin expression promoter according to any one of (26) to (28) above, wherein the indolinone compound is a compound according to (1) to (4) above. (30) The survivin expression promoter according to (29) above, wherein the indolinone compound is semaxanib. (31) The survivin expression promoter according to any one of (26) to (30) above, wherein the iPS cells are capable of inducing transient expression of neurogenin 2. In (20) to (31), the indolinone compound may be contacted with iPS cells or nerve cells differentiated from iPS cells at a concentration of 1 M to 1 pM, particularly 10 mM to 10 pM, particularly 100 μM to 100 pM. In the present invention, the term "transient" in the transient induction of neurogenin 2 expression can be 16 to 20 hours.

[0024] (32) A method for inducing differentiation of iPS cells into neurons in a xeno-free culture system without going through a process of forming embryoid bodies (cell aggregates), comprising: 1) culturing iPS cells into which the human neurogenin 2 gene has been introduced in a laminin-coated container; 2) transiently inducing the expression of the neurogenin 2 gene to initiate differentiation; 3) culturing the iPS cells that have begun to differentiate in a laminin-coated container, and adding at least one of six differentiation development promoting agents: a small molecule TGFβ family inhibitor, an adenylate cyclase activator, a GSK3 inhibitor, a Smoothened agonist and Rock inhibitor, and an indolinone compound; A method for inducing differentiation of iPS cells into nerve cells, comprising: In (32), step 2) is a step of inducing differentiation of iPS cells into neural progenitor cells, and step 3) is a step of inducing differentiation of neural progenitor cells into neural cells. In (32), the small molecule TGFβ family inhibitor may be contacted with iPS cells or iPS cells that have begun to differentiate at a concentration of 1 mM to 1 nM, particularly 100 μM to 10 nM. In (32), the adenylate cyclase activator may be contacted with iPS cells or iPS cells that have begun to differentiate at a concentration of 1 mM to 1 nM, particularly 100 μM to 10 nM. In (32), the GSK3 inhibitor may be contacted with iPS cells or iPS cells that have begun to differentiate at a concentration of 1 mM to 1 nM, particularly 100 μM to 10 nM. In (32), the Smoothened agonist may be contacted with iPS cells or iPS cells that have begun to differentiate at a concentration of 1 mM to 1 nM, particularly 100 μM to 10 nM. In (32), the Rock inhibitor may be contacted with iPS cells or iPS cells that have begun to differentiate at a concentration of 1 mM to 1 nM, particularly 100 μM to 10 nM. In (32), the indolinone compound may be contacted with iPS cells or iPS cells that have begun to differentiate at a concentration of 1 M to 1 pM, particularly 10 mM to 10 pM, particularly 100 μM to 100 pM. (33) The differentiation induction method described in (32) above, wherein the laminin coating is a coating with a human recombinant laminin fragment. (34) The differentiation induction method described in (32) or (33) above, in which the human neurogenin 2 gene is introduced by RNA lipofection or by using a vector in which the neurogenin 2 gene is carried in a plasmid vector or a viral vector and whose expression can be transiently induced by doxycycline treatment or the like. (35) The differentiation induction method according to any one of (32) to (34) above, wherein the culture is a two-dimensional culture on a culture dish. (36) The differentiation induction method according to any one of (32) to (35) above, wherein the initiation of differentiation, i.e., step 2), is carried out in an iPS cell maintenance medium to which a Rock inhibitor has been added and, if necessary, an indolinone compound has been added. In (36), the concentration of the Rock inhibitor in the medium can be 1 mM to 1 nM, preferably 100 μM to 100 nM, and the concentration of the indolinone compound can be 1 M to 1 pM, preferably 10 mM to 10 pM, preferably 100 μM to 100 pM. (37) In the step 2), when transient induction of neurogenin 2 expression is performed using iPS cells into which the neurogenin 2 gene has been introduced using a vector, the transient induction is for 16 to 20 hours, and an indolinone compound is added as necessary. (38) In step 3), as a differentiation development promoter, corresponding to the stage of differentiation induction, a) In the first stage (primary culture), a Rock inhibitor, an adenylate cyclase activator, a GSK3 inhibitor, and a small molecule TGFβ family inhibitor are added, and an indolinone compound is added as needed. b) In the second stage (secondary culture), a Rock inhibitor, an adenylate cyclase activator, a Smoothened agonist, and an indolinone compound are added as needed. c) In the third step (tertiary culture), a Rock inhibitor is added, and a neurotrophic factor and / or an indolinone compound are added as needed. In (38), the first stage (primary culture) refers to a stage in which cells further differentiate from neural progenitor cells, the second stage (secondary culture) refers to a stage in which cells differentiate into neurons, and the third stage (tertiary culture) refers to a stage in which cells complete their differentiation into neurons. For example, from the start of culturing iPS cells with the addition of a neurogenin 2 expression inducer (i.e., the start of step 2), the first stage (primary culture) can be days 1 to 14, the second stage (secondary culture) can be days 14 to 6, and the third stage (tertiary culture) can be days 6 to 40, particularly days 6 to 22. (39) The method for inducing differentiation into neural cells according to any one of (32) to (38) above, wherein retinoic acid (RA) and LDN193189 are not used in culturing iPS cells after the initiation of differentiation. In (39), the initiation of differentiation refers to the initiation of step 2). (40) A method for inducing differentiation into neural cells according to (38) above, in which an indolinone compound is added to the differentiation induction medium in step 2) and / or step 3) for iPS cells that have low cellular activity and stagnate or die during differentiation. (41) The method for inducing differentiation into neurons according to (40) above, wherein the iPS cells are derived from a patient with a neurological disease. (42) The method for inducing differentiation into neurons according to (41) above, wherein the neurological patient is an amyotrophic lateral sclerosis (ALS) patient. (43) The method for inducing differentiation into nerve cells according to any one of (41) to (42) above, wherein the indolinone compound is semaxanib. (44) The method for inducing differentiation into nerve cells according to any one of (32) to (43) above, wherein the nerve cells are motor nerve cells.

[0025] (45) A neural cell derived from an iPS cell, produced by the differentiation induction method according to any one of (32) to (44) above. (46) The iPS cell-derived nerve cell according to (45) above, wherein the nerve cell is a motor nerve cell. (47) The iPS cell-derived nerve cells according to (45) or (46) above, which can be maintained in a xeno-free culture system for a long period of time. (48) The iPS cell-derived neurons according to (46) above, wherein the long-term culture maintenance is culture maintenance for at least 4 months. (49) The iPS cell-derived nerve cell according to any one of (45) to (48) above, wherein the iPS cell is derived from a patient with a neurological disease. (50) The iPS cell-derived nerve cells according to (49) above, wherein the patient with a neurological disease is an ALS patient.

[0026] (51) iPS cells derived from ALS patients into which the human neurogenin 2 gene had been introduced were subjected to transient induction of neurogenin 2 expression in a xeno-free culture system, which initiated differentiation induction, and the resulting neurons were then cultured in the presence of an indolinone compound.

[0027] (52) A method for screening a therapeutic agent for neuropsychiatric disorders, which uses neurons derived from iPS cells according to any one of (45) to (51) above. (53) The method for screening a therapeutic agent for neurological diseases according to (52), wherein the iPS cell-derived neurons are neurons derived from iPS cells of an ALS patient, and the therapeutic agent for neurological diseases is a therapeutic agent for ALS. The screening methods of (52) and (53) can be, for example, methods comprising the steps of contacting a test substance with iPS cell-derived neurons described in any of (45) to (51) above, comparing biomarkers for neuropsychiatric disorders between when the test substance is contacted and when it is not contacted, or comparing biomarkers after contact with the test substance with those before contact, and selecting a test substance that increases biomarkers that are decreased in neuropsychiatric disorders by contact with the test substance, or selecting a test substance that decreases biomarkers that are increased in neuropsychiatric disorders by contact with the test substance. As biomarkers for neuropsychiatric disorders, changes in gene expression or changes in the type and / or amount of produced proteins that are specific to the disorder can be used. In the case of neurodegenerative diseases such as ALS and Alzheimer's disease, increases in neuronal breakdown products, such as neurofilament light chains, can be used as biomarkers of the severity of the disease state.

[0028] (54) A cell activator containing an indolinone compound as an active ingredient for cell populations committed to neuronal differentiation through differentiation induction from iPS cells of ALS patients. That is, a cell activator containing an indolinone compound as an active ingredient for cell populations committed to neuronal differentiation through differentiation induction from iPS cells of ALS patients. (55) The cell activating agent according to (54) above, which activates differentiation-induced nerve cells so that they can be maintained in culture for at least one month. (56) The cell activating agent according to (54) above, which activates differentiation-induced nerve cells so that they can be maintained in culture for 4 months. (57) The nerve cell activating agent according to any one of (54) to (56) above, wherein the nerve cells are motor nerve cells. (58) The cell activating agent according to any one of (54) to (57) above, wherein the indolinone compound is semaxanib. (59) The cell activating agent according to any one of (54) to (58) above, wherein the iPS cells are capable of inducing transient expression of neurogenin 2. [Effects of the Invention]

[0029] First aspect of the present invention The indolinone compounds of the present invention have excellent neuron activating activity and are therefore useful for activating damaged or dysfunctional central or peripheral neurons or transplanted neural stem cells. As a result, the neuron activating agents of the present invention are useful as preventive and / or therapeutic agents for neuropsychiatric disorders. Furthermore, since the compounds of the present invention are low-molecular-weight compounds, they are less expensive and more stable than BDNF or VEGF signaling protein drugs. Furthermore, while VEGF protein drugs do not cross the blood-brain barrier and therefore require intraventricular administration, posing significant challenges for clinical use, the compounds of the present invention do not present such challenges and can be suitably used for central neurons or transplanted neural stem cells. They are also effective against peripheral neurons such as lower motor neurons. The indolinone compounds of the present invention simultaneously promote the expression of genes involved in the BDNF and / or VEGF signaling pathways, promote the proliferation and differentiation of neural stem cells, promote the re-elongation and branching of dendrites atrophied by stress or disease, and enhance spine formation, thereby improving neuroplasticity. As a result, neuronal activation occurs. Furthermore, the autocrine expression of BDNF and VEGF signaling neurotrophic factors in neurons improves neuronal maintenance, neuroplasticity, and the proliferation and differentiation of neural stem cells, thereby promoting memory reconstitution in the hippocampus. Due to these effects, the indolinone compounds of the present invention are useful as therapeutic or preventive agents for various neuropsychiatric disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, Huntington's disease, age-related neurological disorders (dementia, etc.), depression, schizophrenia, bipolar disorder, post-traumatic stress disorder (PTSD), anxiety disorder, panic disorder, autism spectrum disorder, developmental disorder, Rett syndrome, Down syndrome, cerebral ischemic injury, and spinal cord injury, as well as therapeutic or preventive agents in regenerative medicine and neuronal activators. Furthermore, by suppressing age-related brain atrophy, the indolinone compounds are thought to contribute to extending healthy lifespan.

[0030] The neuroactivator of the present invention simultaneously activates multiple neurotrophic factors that are the basis for maintaining normal nervous system function and that must act in conjunction under physiological conditions to maintain normal nervous system function. The scientific evidence that the neuroactivator of the present invention can be a future therapeutic agent for not only ALS, the effectiveness of which has been demonstrated in the Examples, but also the various psychodegenerative diseases and psychiatric disorders exemplified above, is explained below.

[0031] The neuroactivator of the present invention prevented cell death in ALS patient-derived neurons, maintained their survival, and maintained the neurons even after cessation of BDNF, which is considered essential for maintaining neuronal culture. This suggests that the neuroactivator has the activity of promoting the autocrine production of neurotrophic factors essential for maintaining the survival of various neurons. Measurements demonstrated that the neuroactivator activated the expression of BDNF, as well as the VEGF family, VEGF receptor family, and VEGF co-receptor neuropilin-2.

[0032] VEGF and its receptor family were discovered as growth factors involved in angiogenesis, but have since been shown to have important neurotrophic activity and to play an important role in maintaining neuronal survival, neurogenesis, and axonal outgrowth (Jin K., et al., Proc. Natl. Acad. Sci. USA(2002)99(18);11946-11950 "Vascular endothelial growth factor (VEGF) stimulates neurogenesis in vitro and in vivo" (Sondel M., et al., J Neurosci (1999)19(14);5761-5740 "Vascular endothelial growth factor has neurotrophic activity and stimulates axonal outgrowth, enhancing cell survival and Schwann cell proliferation in the peripheral nervous system" (Almodovar CR., et al., Physiological Reviews (2009) 89(2);607-648 "Role and potential of VEGF In addition, VEGF has been shown to support neuroplasticity, which is essential for maintaining normal brain functions such as memory and learning (Licht T., et al., Proc. Natl. Acad. Sci. USA (2011) 108:5081-5086 "Reversible modulations of neuronal plasticity by VEGF"). Furthermore, due to its neuroprotective properties, it has been applied to the treatment of neurodegenerative diseases such as ALS and Alzheimer's disease (Lange C., et al., Nature Reviews Neurology (2016) 12; 439-454 "Vascular endothelial growth factor: a neurovascular target in neurological diseases").Additionally, Yale University reported that the cooperative function of BDNF and VEGF is essential for the effects of ketamine, a powerful, fast-acting antidepressant drug approved by the US FDA in 2019 (News releases: www.nih.gov Philadelphia, January 31, 2019 BDNF-VEGF interplay key to rapid antidepressant actions) (Deyama S., et al., Biological Psychiatry (2019) 86; 143-152 “Neurotrophic and antidepressant actions of brain-dereived neurotrophic factor require vascular endothelial growth factor”). Autocrine regulation occurs in motor neurons (Hohman TJ., et al., JAMA (2015)72(5); 520-529 “The role of vascular endothelial growth factor in neurodegeneration and cognitive decline”). Vascular endothelial growth factor has been shown to be involved not only in neurodegenerative diseases but also in the pathology and improvement of psychiatric disorders.

[0033] ALS is a motor neuron degenerative disease that progresses relatively rapidly. It often results in death within 3–5 years of diagnosis due to bulbar paralysis, limb muscle atrophy, and respiratory muscle paralysis. Approved therapeutic agents include riluzole, which has a life-prolonging effect, and edaravone, which has been shown to further improve quality of life. However, their effectiveness is unfortunately limited. Familial ALS, for which genetic mutations have been identified, accounts for 5–10% of cases, while sporadic cases account for 90–95%. In 2001, Osthuyse et al. demonstrated a relationship between the pathology of neurodegenerative disease and decreased VEGF expression (Oosthuyse B, et al. Nat Genet (2001) 28; 131–8 “Deletion of the hypoxia-response element in the vascular endothelial growth factor promoter causes motor neuron degeneration”), demonstrating the protective role of VEGF on motor neurons. Subsequently, an international meta-analysis comparing the genes of ALS patients and healthy controls revealed that reduced VEGF expression increases the risk of developing ALS in many cases of ALS, including sporadic cases (Lambrechts D, S et al., Nat Genet (2003) 34; 383-94 "VEGF is a modifier of amyotrophic lateral sclerosis in mice and humans and protects motoneurons against ischemic death"). In 2006, Brocking et al. reported cases in which VEGFR-2 mRNA was reduced to below the detection limit in motor neurons of the anterior horn of the spinal cord of ALS patients (Brockington A., et al., NeuroPathol Exp. Neurol (2006) 65; 26-36 "Expression of vascular endothelial growth factor and its receptors in the central nervous system in amyotrophic lateral sclerosis"). Based on these findings, the use of VEGF in ALS treatments has been actively investigated (Orion P Keifer Jr, et al., Pharmacol Ther. (2014)141(3); 261-71 “Gene and protein therapies utilizing VEGF for ALS”; Tover-y-Romo L., et al., frontiers in cellular neuroscience (2014)8; 61 “Trophic factors as modulators of motor neuron physiology and survival: implications for ALS therapy”). Because VEGF protein cannot cross the blood-brain barrier, it must be administered intracerebroventricularly when used as a drug. In ALS model animals, VEGF administration delayed the onset of disease and prolonged survival (Storkebaum E., et al., Nature Neuroscience (2005) 8(1); 85-92 “Treatment of motoneuron degeneration by intracerebroventricular delivery of VEGF in a rot model of ALS”). Significant effects have been observed in animal experiments using viral vectors (Azzouz M, et al., Nature (2004) 429; 413-7 “VEGF delivery with retrogradely transported lentivector prolongs survival in a mouse ALS model”), but there are significant obstacles to applying this to human medicine.Currently, several clinical trials using intraventricular VEGF administration in ALS patients are being conducted (Orion P. Keifer Jr. et al., Pharmacol Ther. (2014) 141(3): 261-271 "Gene and protein therapies utilizing VEGF for ALS") (Patent Document JP2008-500312 (P2008-500312A) (Damma P. et al., Brain Commun. (2020) 2(2); fcaa160 "Intracerebroventricular delivery of vascular endothelial growth factor in patients with amyotrophic lateral sclerosis, a phase I study"). However, these trials require a pump-equipped device to be permanently placed in the brain, which places a physical burden on the patient, requires advanced technology, can only be performed in specialized facilities, and is expensive. From these perspectives, the neuron activating agent of the present invention clearly has significant advantages.

[0034] In the case of Alzheimer's disease, the Alzheimer's Disease neuroimaging initiative (ADNI), an international clinical research organization whose mission is to establish methods for predicting the onset of Alzheimer's disease and assessing the effectiveness of therapeutic drugs, has shown that VEGF is a biomarker for Alzheimer's disease and age-related dementia, and that higher concentrations of VEGF in cerebrospinal fluid are associated with higher cognitive ability and larger hippocampal volume (Hohman TJ., et al., JAMA (2015) 72(5); 520-529 "The role of vascular endothelial growth factor in neurodegeneration and cognitive decline"). It has also been reported that VEGF has a neuroprotective effect in Huntington's disease (Ellison SM, et al., Mol Ther. (2013)21:1862-75 “Dose-dependent neuroprotection of VEGF165 in Huntington's disease striatum”). In addition to neurodegenerative diseases (ALS, Alzheimer's disease, Huntington's disease, etc.), the VEGF family, VEGF receptor family, and VEGF co-receptors have neuroprotective effects in peripheral neuropathy and epilepsy, suggesting their potential as therapeutic agents (Lange C., et al., Nature Reviews in Neurology (2016)12(8): 439-54 “Vascular endothelial growth factor: A new target in neurological diseases”). Research led by Yale University has also suggested that VEGF may play a fundamental role in the treatment of depression. Although the molecular mechanism of antidepressant action remains unclear, one hypothesis suggests that this effect may be related to growth factor signaling in the hippocampus and promotion of adult neurogenesis. Warner-Schmidt and Duman investigated the effects of different antidepressants on the expression of vascular endothelial growth factor (VEGF), a neurotrophic and pro-angiogenic factor, in the hippocampus. This factor was previously shown to be enhanced by electroconvulsive seizure therapy (ECS). VEGF mRNA levels, as well as VEGF levels in hippocampal homogenates, were increased in the hippocampal granule cell layer of rats treated with fluoxetine (a serotonin reuptake inhibitor) or desipramine (a norepinephrine reuptake inhibitor) for 14 days. Pharmacological inhibition of the VEGF receptor Flk-1 (also known as VEGF receptor-2) prevented the increased cell proliferation in the hippocampal subgranular zone (SGZ) produced by chronic exposure to ECS or fluoxetine or desipramine, whereas intracerebroventricular administration of VEGF isoforms promoted SGZ cell proliferation. Furthermore, pharmacological inhibition of Flk-1 (also known as VEGF receptor-2) prevented the effects of desipramine on behavioral responses in rats treated with chronic and subchronic antidepressant drugs, whereas VEGF exhibited antidepressant-like effects. These findings suggest that VEGF plays an important role in mediating both the behavioral effects of several antidepressants and their effects on SGZ cell proliferation (JL Warner-Schmidt, RS Duman, Proc. Natl. Acad. Sci. USA (2007)104; 4647-4652 "VEGF is an essential mediator of the neurogenic and behavioral actions of antidepressants").

[0035] Ketamine, a potent, fast-acting antidepressant drug approved by the US FDA in 2019, is reported to exert its effects through VEGF (Deyama S., et al., Am J Psychiatry. (2019)176(5); 388-400 “Role of Neuronal VEGF Signaling in the Prefrontal Cortex in the Rapid Antidepressant Effects of Ketamine”), and it has been reported that the cooperative function of BDNF and VEGF is essential (News releases: www.nih.gov Philadelphia, January 31, 2019 “BDNF-VEGF interplay key to rapid antidepressant actions”) (Deyama S., et al., Biological Psychiatry (2019) 86; 143-152 “Neurotrophic and antidepressant actions of brain-derived neurotrophic factor require vascular endothelial growth factor”).

[0036] Additionally, a group at Yale University has shown that VEGF-C, VEGF-D, and their receptors, VEGF receptor-2 and VEGF receptor-3 (VEGF receptor-2 is a receptor for VEGF-A, -C, and -D, and VEGF receptor-3 is a receptor for VEGF-C and -D), promote the proliferation and differentiation of neural progenitor cells in the hippocampus and are essential for neurogenesis in the adult hippocampus (Han J., et al., Cell Reports (2015) 10; 1158-1172 "Vascular Endothelial Growth Factor Receptor 3 Controls Neural Stem Cell Activation in Mice and Humans"). Patent Documents 2 and 3 describe that activators of VEGF-C and VEGF-D proteins and their receptors, VEGF receptor-2 / 3, are useful as therapeutic agents for aging and neurodegenerative diseases.

[0037] Therefore, it is clear that the neuroactivator of the present invention has great potential as a future therapeutic agent for many neuropsychiatric disorders.

[0038] Next, BDNF is a neurotrophic factor that controls the generation of new neurons, the differentiation and growth of neural progenitor cells, and the promotion of neurite outgrowth. It is essential for the survival of brain nerves, the maintenance of neurites, and the re-extension of neurites that have atrophied due to disease (Deyama S, et al., Biological Psychiatry (2019) 86;143-152 Neurotrophic and antidepressant actions of Brain-dereived neurotrophic factor require vascular endothelial growth factor), and supports the construction of complex networks of neurons found in the central nervous system. In mature neurons, BDNF controls the formation and disappearance of synapses, as well as neural plasticity related to learning and memory, maintaining normal brain function (Mattson and Wan, Neuromolecular Med (2008) 10; 157-158) (Miranda M. et al., Front Cell Neurosci. (2019) 13; 363 “Brain-Derived Neurotrophic Factor: A Key Molecule for Memory in the Healthy and the Pathological Brain”). BDNF has been shown to be a biomarker for many neurodegenerative and psychiatric disorders, and to have therapeutic effects on neuropsychiatric disorders and spinal cord injury. In 2019, a group from Tokyo Medical and Dental University demonstrated that administering BDNF mRNA to the injury site of spinal cord injury model mice, thereby enhancing BDNF expression at the injury site, resulted in early motor function recovery (Crowley ST., et al., Molecular Therapy Nucleic Acids (2019) 17: 465-476 “Enhancement of Motor Function Recovery after Spinal Cord Injury in Mice by Delivery of Brain-Derived Neurotrophic Factor mRNA”).

[0039] BDNF is a biomarker for Alzheimer's disease and age-related cognitive impairment (Beeri MS. and Sonnen J., Neurology. (2016)86(8);702-3 “Brain BDNF expression as a biomarker for cognitive reserve against Alzheimer's disease progression”), a key molecule for maintaining healthy memory (Magdalena Miranda, frontiers in Cellular Neuroscience (2019) 13; 363 “Brain-Derived Neurotrophic Factor: A Key Molecule for Memory in the Healthy and the Pathological Brain”) (Tapia-Arancibia et al. Brain Res Rev(2008)59:201-220 New insights into brain BDNF function in normal aging and Alzheimer's disease“), and is considered an effective treatment for Alzheimer's disease and Parkinson's disease (Sampaio, T.et al., Neural Regen. Res. (2017)12; 549-557 “Neurotrophic factors in Alzheimer's and Parkinson's diseases: Implications for pathogenesis and therapy”). BDNF is also a biomarker for Huntington's disease (frontiers in Molecular Neuroscience (2020) 12: article 335 “Evaluation of Biochemical and Epigenetic Measures of Peripheral Brain-Derived Neurotrophic Factor (BDNF) as a Biomarker in Huntington's Disease Patients”).

[0040] The lifetime prevalence of depression is extremely high at approximately 10-20%, and it is said that 60-70% of suicides are caused by depression. Therefore, analyzing the pathology of depression and developing treatments are urgent social issues (Hashimoto Kenji, Brain and Psychiatric Medicine (2009) 20(1); 55-60 "Brain-derived neurotrophic factor and depression"). Decreased BDNF levels are associated with depression and anxiety disorders (Martinowich K., et al., Nature Neurosci.(2007)10; 1089-1093 “New insights into BDNF function in depression and anxiety”). There are numerous reports showing that BDNF levels increase in patients who respond to antidepressant or electrical stimulation treatments for depression, and that BDNF administration can also improve depression (Sen S., et al., Biol Psychiatry.( 2008)64(6);527-32 “Serum brain-derived neurotrophic factor, depression, and antidepressant medications: meta-analyses and implications”) (Shirayama Y., et al., J Neurosci.(2002)22(8);3251-61 “Brain-derived neurotrophic factor produces antidepressant effects in behavioral models of depression”). There is currently debate about the limitations of antidepressants that target monoamine compounds, such as serotonin reuptake inhibitors. Substances that promote BDNF production are attracting attention as seeds for new antidepressants, and various herbal medicines are being screened (Fukuchi Mamoru, Pharmaceutical Journal (2017) 137(9); 1103-1115).

[0041] Decreased BDNF is a biomarker for bipolar disorder (Fernandes et al. BMC Medicine (2015) 13;289 “Peripheral brain-derived neurotrophic factor (BDNF) as a biomarker in bipolar disorder: a meta-analysis of 52 studies”), a biomarker for severe psychosis accompanied by delusions and hallucinations, such as schizophrenia (Nieto et al. Frontiers in Psychiatry(2021)12;662407 “BDNF as a Biomarker of Cognition in Schizophrenia / Psychosis: An Updated Review”), and a biomarker for autism spectrum disorder (Saghazadeh A. and Rezaei N., J Autism Dev Disord. (2017) 47(4):1018-1029 “Brain-Derived Neurotrophic Factor Levels in Autism: A Systematic Review and Meta-Analysis”).

[0042] It has been suggested that the lack of efficacy in clinical trials using BDNF alone may be due to the need for the synergistic action of other cytotrophic factors (Shanmukha H., et al., Neurodegener Dis (2017) 17: 44-58). In fact, Yale University reports that the synergistic action of BDNF and VEGF is essential for the effectiveness of ketamine, a powerful, fast-acting antidepressant approved by the US FDA in 2019 (News releases: www.nih.gov Philadelphia, January 31, 2019 “BDNF-VEGF interplay key to rapid antidepressant actions”) (Deyama S. et al., Biological Psychiatry (2019)86;143-152 “Neurotrophic and antidepressant actions of brain-dereived neurotrophic factor require vascular endothelial growth factor”).

[0043] In summary, for a therapeutic agent for neuropsychiatric disorders to function physiologically, multiple neurotrophic factors must work together. However, conventional drug development has focused on a single target substance, which is clearly one of the reasons why drugs developed using conventional methods have not demonstrated the expected effects. Furthermore, compared to protein preparations, the neuron activators of the present invention are low-molecular-weight compounds, offering the advantages of low cost, easy storage, and potential blood-brain barrier penetration. By activating multiple neurotrophic factors physiologically required for normal nervous system function, the neuron activators of the present invention provide future drugs with physiological functionality not previously considered in drug discovery. These drugs may contribute to the treatment of not only ALS, but also Alzheimer's disease, Parkinson's disease, dementia, and depression—all of which have a significant social impact. Therefore, they will bring about enormous social and economic contributions.

[0044] Second Aspect of the Invention The differentiation induction method of the present invention enables highly efficient differentiation of induced pluripotent stem cells (iPS cells) into neurons in a xeno-free culture system without the formation of embryoid bodies (cell aggregates). This avoids contamination with xenogeneic materials, enabling application in human regenerative medicine. Furthermore, because differentiation into neurons can be induced without the formation of embryoid bodies (cell aggregates), the process of transferring cells to suspension cell containers for culture can be omitted, enabling stable culture for differentiation induction. Furthermore, the addition of only up to six differentiation promoters is sufficient, and up to nine reagents, including the addition of neurotrophic factors or indolinone compounds for terminal differentiation, can be used for culture, simplifying process management and reducing contaminants. In particular, in the case of iPS cells derived from patients with neurological disorders, even with low cell activity, the addition of indolinone compounds can promote survivin gene expression, enabling the generation of terminally differentiated neurons without cell death during the process.

[0045] Furthermore, it has become possible to induce differentiation of iPS cells derived from human disease patients into neurons without using retinoic acid (RA) or LDN193189, which are differentiation inducers used in conventional differentiation culture of neural cells, and to maintain the resulting neurons for a long period of time. In particular, the cultures of motor neurons derived from ALS patients obtained using a xeno-free culture system are unprecedented. They are free of contaminants derived from biological culture aids (serum, feeder cells, and Matrigel), which have significant problems due to the large number of unidentifiable components and the inability to control the amount of each component. Instead, they simplify the process by using only materials of known origin and whose amounts can be consistently controlled. This provides a useful tool for drug discovery screening. Furthermore, by avoiding the influence of contaminants with variable contaminant content, higher reproducibility can be ensured. This provides a means for further elucidating function and pathology at the neuronal level. Furthermore, neurons derived from iPS cells differentiated using RNA lipofection are now suitable for clinical applications. Thus, the motor neurons of the present invention are considered to be useful for regenerative medicine and cell therapy. Furthermore, the differentiation culture method of the present invention can easily induce differentiation of mature neurons from iPS cells derived from healthy individuals or diseased patients, making it easy to use these mature neurons to screen for therapeutic agents for neurological diseases, thereby providing a useful tool for drug discovery. [Brief explanation of the drawings]

[0046] [Figure 1] A. Microscopic image showing the successful adaptation of iPS cell lines to a feeder-free and xeno-free culture system. B. Microscopic image of iPS cell clumps cultured using feeder cells, provided by the iPS Research Institute (https: / / www.jiji.com / jc / d4?p=yns002-05728491&d=d4_news). [Figure 2]This figure shows the establishment of a method for differentiating iPS cells into motor neurons. A. A phase-contrast microscope image of iPS cells derived from a healthy individual. B. A phase-contrast microscope image of motor neurons differentiated from iPS cells derived from a healthy individual (33 days after induction of differentiation). C. A schematic diagram showing the expression kinetics of marker genes during differentiation of iPS cells into motor neurons. As iPS cells differentiate, the neural progenitor marker (PAX6) is first expressed, but its expression subsequently decreases as differentiation progresses. Next, the motor neuron marker (HB9) is expressed, and its expression increases as differentiation into motor neurons occurs. Finally, a mature motor neuron marker is expressed. The neurotransmitter receptor GluA1 was used as a marker for mature motor neurons. [Figure 3] This figure shows the changes in differentiation marker mRNA expression during differentiation of iPS cells into motor neurons, as described in Figure 2C. Expression of the neural progenitor cell marker (PAX6) increased approximately 3,200-fold compared to the control on day 15 of differentiation induction, and subsequently decreased to approximately 1,500-fold by day 33 of differentiation (top panel). Expression of the motor neuron marker (HB9) increased approximately 2,200-fold compared to the control on day 15 of differentiation induction, reaching approximately 6,600-fold by day 33 (middle panel). Expression of the mature neuron marker (neurotransmitter receptor GluA1) remained almost unchanged until day 15 of differentiation induction, but increased approximately 600-fold by day 33 (bottom panel). Based on these findings, we concluded that differentiation of iPS cells into motor neurons had progressed, and the dynamics of this gene expression were used as an indicator to confirm subsequent differentiation of iPS cells into motor neurons. [Figure 4]These are microscopic images showing the effect of adding the indolinone compound (semaxanib) on maintaining the survival of neurons derived from sporadic ALS patients. A. These are microscopic images showing that the addition of semaxanib had no negative morphological effects when iPS cells derived from healthy individuals were differentiated into motor neurons. B. These are microscopic images showing that the addition of semaxanib prevented motor neurons from dying when iPS cells derived from sporadic ALS patients were differentiated into motor neurons. No difference was observed with the addition of semaxanib in the early stages of differentiation (day 1 of differentiation induction) (left column). However, by day 15 of differentiation induction, cells had died in the control group, while cells in the semaxanib-treated group were observed to have extended neurites (middle column). By day 27 of differentiation induction, phase-contrast microscopic images comparable to motor neurons derived from healthy individuals were observed (right panel). [Figure 5] This figure shows the effect of adding semaxanib on promoting BDNF expression. With the addition of semaxanib, BDNF expression in neurons derived from healthy individuals increased approximately 4.6-fold compared to controls, and in neurons derived from sporadic ALS patients, it increased approximately 3.4-fold compared to iPS cells. This indicates that the addition of semaxanib results in autocrine BDNF production by the neurons themselves. Left: derived from a healthy individual; right: derived from a sporadic ALS patient. [Figure 6A]The effects of adding semaxanib on the expression of genes involved in maintaining neuronal survival, neuroplasticity (promoting dendrite outgrowth and branching), and promoting neural stem cell proliferation and differentiation were examined using cells derived from healthy individuals. VEGF-A, which is involved in neuroplasticity (promoting dendrite outgrowth and branching) and maintaining survival, increased expression approximately three-fold over control (upper left). VEGF-C, which is involved in promoting neural stem cell proliferation and differentiation, increased expression approximately 9.3-fold over control (upper middle). VEGF-D, which is involved in promoting neural stem cell proliferation and differentiation, increased expression approximately 18-fold over control (upper right). VEGF receptor-2, the receptor for VEGF-A, VEGF-C, and VEGF-D, decreased expression to 50% in iPS cells as they differentiated into neurons, but the addition of semaxanib increased expression to approximately 2.4-fold over control (lower left). VEGF receptor-3, a receptor for VEGF-C and VEGF-D involved in promoting neural stem cell proliferation and differentiation, showed an approximately four-fold increase in expression compared to the control (bottom center figure). VEGF receptor-2, involved in promoting neural stem cell proliferation and differentiation, and neuropilin 2, a co-receptor for VEGF receptor-3, showed an approximately four-fold increase in expression compared to the control (bottom right figure). As shown above, semaxanib was shown to increase the expression of genes involved in the VEGF signaling pathway. [Figure 6B]The effects of semaxanib on the expression of genes involved in maintaining neuronal survival, neuroplasticity (promoting dendrite outgrowth and branching), and promoting neural stem cell proliferation and differentiation were examined using cells derived from sporadic ALS patients. Without semaxanib, cells died on day 15, so we compared the results with iPS cells. VEGF-A, which promotes neuroplasticity (promoting dendrite outgrowth and branching) and survival, was expressed approximately twice as much in iPS cells (top left). VEGF-C, which promotes neural stem cell proliferation and differentiation, was expressed approximately 5.5-fold as much in iPS cells (top center). VEGF-D, which promotes neural stem cell proliferation and differentiation, was expressed approximately four-fold as much in iPS cells (top right). VEGF receptor-2, the receptor for VEGF-A, VEGF-C, and VEGF-D, decreased to approximately 30% of the expression levels in iPS cells during differentiation into neurons (bottom left). Expression of VEGF receptor-3, a receptor for VEGF-C and VEGF-D involved in promoting neural stem cell proliferation and differentiation, was reduced by approximately 73% in iPS cells (bottom center figure). Expression of VEGF receptor-2, involved in promoting neural stem cell proliferation and differentiation, and neuropilin 2, a co-receptor for VEGF receptor-3, was increased approximately five-fold in iPS cells (bottom right figure). As mentioned above, semaxanib was shown to increase the expression of genes involved in the VEGF signaling pathway, with the exception of VEGF receptor-2 and VEGF receptor-3. [Figure 7] FIG. 1 is a diagram showing an outline of the method for inducing neuronal differentiation of the present invention. [Figure 8] These are phase-contrast micrographs of iPS cells derived from healthy individuals transfected with the human neurogenin 2 gene, whose expression can be transiently induced with doxycycline, cultured for 16 hours in the presence of doxycycline (DOX) (right) and for 16 hours in the absence of DOX (left). In the right image, most of the iPS cells began to extend processes, showing a morphological change to that of neural progenitor cells. In contrast, in the absence of DOX, no neural progenitor-like cells were observed. This shows that the presence of DOX induced differentiation in iPS cells that expressed human neurogenin 2, resulting in their transformation into neural progenitor-like cells. [Figure 9]This figure shows the changes that occurred when the neural stem cell-like cells in Figure 8 were further cultured. Even when iPS cells were cultured in the presence of DOX for a total of 24 hours, the neural progenitor cell-like morphology was maintained, as shown in the right figure. [Figure 10] These phase-contrast micrographs show the progression of morphological changes from neural progenitor-like cells to neurons on days 1, 3, and 32 after the start of differentiation induction of iPS cells derived from healthy individuals. Neurite outgrowth is observed from day 1 of differentiation induction (upper right, figure b). By day 3 of differentiation induction, further neurite outgrowth has progressed from all cells (lower left, figure c). By day 32 of differentiation induction, neurons with well-developed neurites were observed (lower right, figure d). [Figure 11] This figure shows the changes in differentiation marker mRNA expression during differentiation of iPS cells derived from healthy volunteers into motor neurons. Expression of the neural progenitor cell marker PAX6 mRNA increased on day 4 of differentiation induction, reached a maximum on day 15 of differentiation induction (approximately 3,200-fold higher than expression in iPS cells), and then gradually decreased (upper panel a). Expression of the motor neuron marker HB9 rapidly increased on day 15 of differentiation induction (approximately 2,200-fold higher than expression in iPS cells), and continued to increase as neurons matured (middle panel b). Expression of GluA1, a subunit of the neurotransmitter glutamate receptor, rapidly increased on day 18 of differentiation induction (approximately 660-fold higher than expression in iPS cells), and maintained high expression thereafter (lower panel c). These results suggest that differentiation of iPS cells using this method into neural stem cells and then into mature motor neurons progresses, with mature motor neurons being established by day 33 of differentiation induction. [Figure 12]These phase-contrast micrographs show the progression of morphological changes from neural stem cells to neurons 16 hours, 15 days, and 26 days after the start of differentiation induction of iPS cells derived from sporadic ALS patients, with and without the addition of an indolinone compound. b) 16 hours after differentiation induction, neurite outgrowth was observed in both the cells without (left) and with (right) the addition of an indolinone compound, similar to iPS cells derived from normal individuals (Figures 8-10). c) By the 15th day of differentiation induction, cells without the addition of an indolinone compound (left) had died, but with the addition of an indolinone compound (right), further neurite outgrowth progressed. By the 26th day of differentiation induction, neurons with well-developed neurites were observed in the cells with the addition of an indolinone compound. Meanwhile, neurons differentiated from iPS cells derived from healthy individuals were maintained for a long period of time (up to 4 months). [Figure 13] This figure shows the relative mRNA expression levels of the apoptosis inhibitor protein survivin in iPS cells derived from healthy individuals and ALS patients, and in differentiated neurons. Comparing survivin expression in iPS cells derived from healthy individuals and ALS patients, we found that survivin expression was reduced in ALS patient-derived iPS cells, approximately 18% of that in healthy individual-derived iPS cells. In neurons differentiated from iPS cells derived from healthy individuals, the addition of indolinone compounds increased survivin mRNA expression three-fold compared to neurons without indolinone compounds. Although ALS patient-derived iPS cells die upon differentiation into neurons, ALS patient-derived neurons differentiated with indoline compounds showed approximately three-fold increased survivin expression compared to ALS patient-derived iPS cells, reaching 80% of that in healthy individual-derived neurons (without indolinone compounds). DETAILED DESCRIPTION OF THE INVENTION

[0047] First aspect of the present invention The "indolinone compound" of the present invention is a compound represented by the general formula (1)

[0048] [ka]

[0049] [In the formula, R1, R2, and R3 each independently represent a hydrogen atom, a halogen atom, a substituted or unsubstituted lower alkyl group having 1 to 4 carbon atoms, a formyl group, an acetyl group, a carboxyl group, a substituted or unsubstituted lower alkoxycarbonyl group having 1 to 4 carbon atoms, or a substituted or unsubstituted lower alkylamido group having 1 to 4 carbon atoms, and the substituent is a halogen atom, a carboxyl group, a lower alkoxycarbonyl group having 1 to 4 carbon atoms, or a di-lower alkylamino group having 1 to 4 carbon atoms.] R4, R5, R6, and R7 are each independently selected from the group consisting of a hydrogen atom, a halogen atom, a nitro group, a substituted or unsubstituted lower alkyl group having 1 to 4 carbon atoms, a lower alkoxy group having 1 to 4 carbon atoms, a lower alkylcarbonyl group having 1 to 4 carbon atoms, a lower alkoxycarbonyl group having 1 to 4 carbon atoms, a carboxyl group, and a hydroxyl group, and the substituents are a halogen atom, a carboxyl group, or a lower alkoxycarbonyl group having 1 to 4 carbon atoms. The present invention refers to an indolinone compound represented by the formula (I), or a pharmaceutically acceptable salt thereof. Examples of the lower alkyl group having 1 to 4 carbon atoms include a methyl group, an ethyl group, an n-propyl group, an i-propyl group, and an n-butyl group. Preferred examples include a methyl group and an ethyl group. Examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom. Preferred examples include a fluorine atom and a chlorine atom. As the indolinone compound, compounds described in US5792783 (or Japanese Patent No. 3231044) can be used, and preferred ones include indolinone compounds in which the above R1, R2, and R3 are each independently a hydrogen atom, a methyl group, an ethyl group, a halogen atom, a formyl group, an acetyl group, a carboxyl group, an ethyloxycarbonyl group, a methoxycarbonylmethyl group, an ethoxycarbonylmethyl group, a 2-carboxyethyl group, a 2-methoxycarbonylethyl group, a 2-ethoxycarbonylethyl group, or a 2-diethylaminoethylamide group, and R4, R5, R6, and R7 are each independently a hydrogen atom, a halogen atom, or a nitro group. More preferred examples include 3-[(2,4-dimethylpyrrol-5-yl)methylene]-2-indolinone (Semaxanib), 3-[(4-(2-carboxyethyl)-3-methylpyrrol-5-yl)methylene]-2-indolinone, 3-[(3-ethoxycarbonyl-2,4-dimethylpyrrol-5-yl)methylene]-2-indolinone, 5-chloro-3-[(2,4-dimethylpyrrol-5-yl)methylene]-2-indolinone (Chloro-Semaxanib), and 5-fluoro-3-[(3-(2-diethylaminoethyl)carbamoyl-2,4-dimethylpyrrol-5-yl)methylene]-2-indolinone (Sunitinib).More preferably, the compound has the following chemical formula:

[0050] [ka] Examples include 3-[(2,4-dimethylpyrrol-5-yl)methylene]-2-indolinone (Semaxanib), which has the formula:

[0051] The indolinone compound of the present invention can be synthesized according to the method described in US Pat. No. 5,792,783. Pharmaceutically acceptable salts of indolinone compounds include acid addition salts, such as inorganic acid salts such as hydrochloride, hydrobromide, and sulfate, and organic acid salts such as citrate, oxalate, tartrate, fumarate, and maleate. The "brain-derived neurotrophic factor" of the present invention refers to BDNF (Brain-derived neurotrophic factor). BDNF is widely distributed in the brain and triggers the differentiation of neurons, promoting their maturation. It also increases the expression of neurotransmitter synthesis enzymes, increasing the number of synaptic molecules and enhancing their function, thereby improving the efficiency of synaptic transmission. The term "vascular endothelial growth factor (VEGF)" as used herein generally refers to VEGF-A. Other members of the VEGF family, including placental growth factor (PLGF), VEGF-B, VEGF-C, and VEGF-D, play important roles in angiogenesis and lymphangiogenesis. Named after its discovery as an angiogenic growth factor, VEGF has since been recognized to have important neurotrophic activity and to support neuroplasticity, essential for maintaining neuronal survival, neurogenesis, axonal outgrowth, and brain functions such as normal memory and learning. These VEGF family proteins induce autophosphorylation of the receptor tyrosine kinase VEGF receptor (VEGFR). Intracellular signaling via this VEGF / VEGF receptor system triggers biological functions. The "vascular endothelial growth factor (VEGF) signaling pathway proteins" of the present invention include VEGF-A, VEGF-C, VEGF-D, VEGF receptor-2 (a receptor for VEGF-A, VEGF-C, and VEGF-D), VEGF receptor-3 (a receptor for VEGF-C and VEGF-D), and neuropilin 2 (a coreceptor for VEGF-A, VEGF-C, and VEGF-D). VEGF-A regulates angiogenesis and vascular permeability, while VEGF-C and VEGF-D primarily regulate lymphangiogenesis. VEGF-A plays an important role in maintaining neuronal survival. VEGF-A also plays an important role in neuroplasticity, including the formation of spines and synapses, which are reduced by depression and PTSD, as well as the re-extension and branching of dendrites. VEGF-C and VEGF-D play important roles in promoting the proliferation and differentiation of neural stem cells. VEGF also binds to a receptor complex consisting of both neuropilin and VEGF receptors. This receptor complex is known to have VEGF signaling activity. Therefore, the use of recombinant VEGF-C and VEGF-D protein preparations as therapeutic agents for various neurodegenerative diseases such as Down's syndrome and Alzheimer's disease has been investigated (Patent Documents 4 and 5). However, because these are protein preparations, it has been difficult to control their delivery to target tissues in the brain.

[0052] The indolinone compounds of the present invention are characterized by enhancing or promoting the expression of the above-mentioned BDNF and / or VEGF signaling pathway proteins. Furthermore, some indolinone compounds, such as semaxanib, are characterized by simultaneously enhancing the expression of the above-mentioned BDNF and VEGF signaling pathway proteins. It has been recognized that BDNF and VEGF signaling pathway proteins must act in conjunction to physiologically exert therapeutic effects (Non-Patent Document 7). As a result, it has been newly discovered that indolinone compounds promote neuroplasticity (dendrite re-extension and branching, spine formation, and synapse formation) as well as the proliferation and differentiation of neural stem cells. Therefore, use of the indolinone compounds of the present invention is believed to promote neuroplasticity and neural stem cell proliferation and differentiation, in addition to maintaining neuronal cell survival, thereby improving neuropsychiatric disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, Huntington's disease, age-related neurological disorders (dementia, etc.), depression, schizophrenia, bipolar disorder, post-traumatic stress disorder (PTSD), anxiety disorder, panic disorder, autism spectrum disorder, developmental disorders, Rett syndrome, and Down syndrome. Furthermore, since the indolinone compounds promote the proliferation and differentiation of neural stem cells, they replenish lost neurons, and are therefore believed to be useful for symptom recovery after cerebral ischemia, functional recovery after spinal cord injury, extension of healthy lifespan, and maintenance and establishment of transplanted neural stem cells in regenerative medicine.

[0053] The dissociative anesthetic ketamine was approved by the US FDA in 2019 as a potent, fast-acting antidepressant. It has been reported that administration or amplification of BDNF alone is insufficient for ketamine to exert its antidepressant effects; conjugation with VEGF is required (Non-Patent Document 7). Therefore, to ensure its antidepressant effects, simultaneous promotion of the expression of both BDNF and VEGF signaling proteins is believed to be essential for the expression of its physiological function as an antidepressant. By simultaneously promoting the expression of BDNF and VEGF signaling proteins and physiologically coupling them, ketamine is thought to be more effective in treating and ameliorating the symptoms of a variety of neuropsychiatric disorders, including amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, Huntington's disease, age-related neurological disorders (e.g., dementia), depression, schizophrenia, bipolar disorder, post-traumatic stress disorder (PTSD), anxiety disorder, panic disorder, autism spectrum disorder, developmental disorders, Rett syndrome, and Down syndrome, as well as cerebral ischemia and neuronal damage. Moreover, since the compound of the present invention is a low molecular weight compound, it can be easily transported into the brain and is stable and easy to store. Furthermore, because the compound of the present invention is stable and easy to store, it is possible to develop simple pharmaceuticals such as nasal sprays and patches, which were not possible with protein preparations.

[0054] The "neuron activating agent" of the present invention promotes neuroplasticity (dendrite re-extension and branching, spine formation and synapse formation) in damaged neurons, dysfunctional neurons or transplanted neural stem cells, helps maintain the survival of neurons by avoiding apoptosis (cell death of damaged neurons), or promotes the proliferation and differentiation of neural stem cells, thereby maintaining neurons. Damaged neurons include, for example, neurons damaged in cerebral ischemic injury or spinal cord injury. Damaged neurons also include neurons lost in diseases that result in the loss of damaged neurons, such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, and Huntington's disease. Dysfunctional neurons include, for example, neurons responsible for depression, schizophrenia, bipolar disorder, post-traumatic stress disorder (PTSD), panic / anxiety disorder, Rett syndrome, Down syndrome, autism spectrum disorder, developmental disorders, and age-related neurological disorders, including dementia. Transplanted neural stem cells include, for example, transplanted neural stem cells used to treat spinal cord injury, Alzheimer's disease, or Parkinson's disease, and neurons derived from iPS cells used to treat macular degeneration. Examples of "neuropsychiatric disorders" in the present invention include amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, Huntington's disease, age-related neurological disorders (dementia, etc.), depression, schizophrenia, bipolar disorder, post-traumatic stress disorder (PTSD), anxiety disorder, panic disorder, autism spectrum disorder, developmental disorder, Rett syndrome, and Down syndrome.

[0055] The "expression promoter" of the present invention refers to promoting or enhancing the expression of BDNF and / or VEGF signaling pathway proteins. Co-expression of BDNF and VEGF signaling pathway proteins can more effectively promote neuroplasticity and neural stem cell proliferation and differentiation. The expression promoter of the present invention has an excellent BDNF expression-promoting effect, and increased BDNF expression leads to re-elongation and branching of atrophied dendrites, promoting spine formation and improving neuroplasticity. Furthermore, memory reconstitution in the hippocampus is promoted. Furthermore, the expression promoter of the present invention has excellent expression promotion properties for VEGF and VEGF receptors and their co-receptors, and increased expression of VEGF and VEGF receptors and their co-receptors suppresses motor neuron death in ALS and promotes neural stem cell proliferation and differentiation. The expression-promoting agents of the present invention, when co-expressing BDNF and VEGF signaling proteins, are expected to have a synergistic effect, going beyond the therapeutic effects previously achieved using each substance alone. This synergistic effect approaches the biological dynamics of neuronal maintenance, neuroplasticity improvement, and neural stem cell proliferation and differentiation promotion, thereby enabling an integrated approach to the treatment and prevention of neuropsychiatric disorders, as well as symptom recovery after stroke and functional recovery after spinal cord injury. The synergistic effect of co-promoting BDNF and VEGF signaling proteins enables more effective treatment of neuropsychiatric disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, Huntington's disease, age-related neurological disorders (dementia, etc.), depression, schizophrenia, bipolar disorder, post-traumatic stress disorder (PTSD), anxiety disorder, panic disorder, autism spectrum disorder, developmental disorders, Rett syndrome, Down syndrome, symptom recovery after stroke, and functional recovery after spinal cord injury. Furthermore, coupling between BDNF and VEGF signaling proteins is essential for the improvement of depression. Furthermore, as an excellent adjuvant for regenerative medicine using neural stem cell transplantation, it can be clinically applied to the treatment of Parkinson's disease, Alzheimer's disease, and spinal cord injury, further promoting regenerative medicine.

[0056] The "therapeutic agent" of the present invention refers to a pharmaceutical composition containing the compound of the present invention as an active ingredient and used for the prevention or treatment of neuropsychiatric disorders. These pharmaceutical compositions are prepared as general pharmaceutical preparations and administered orally or parenterally. When administered orally, they can be administered in dosage forms commonly used in the art. When administered parenterally, they can be administered in dosage forms such as topical preparations (such as transdermal preparations), injections, and nasal preparations. Oral preparations include, for example, tablets, powders, granules, capsules, and liquid preparations. Injectable solutions include, for example, sterile solutions or suspensions. Topical preparations include, for example, conventional patches and nasal drops. These preparations are prepared using commonly used pharmaceutical carriers. The pharmaceutical carriers used are those commonly used in the pharmaceutical field and which do not react with the compounds of the present invention. Specific examples of pharmaceutical carriers used in the manufacture of tablets, capsules, granules, and powders include excipients such as lactose, corn starch, sucrose, mannitol, calcium sulfate, and crystalline cellulose, disintegrants such as carmellose sodium, modified starch, and carmellose calcium, binders such as methylcellulose, gelatin, gum arabic, ethylcellulose, hydroxypropylcellulose, and polyvinylpyrrolidone, and lubricants such as light anhydrous silicic acid, magnesium stearate, talc, and hardened oil. Tablets may be coated using conventional coating agents by known methods. Specific examples of carriers used in the production of syrups include sweeteners such as sucrose, glucose, and fructose; suspending agents such as gum arabic, tragacanth, carmellose sodium, methylcellulose, sodium alginate, crystalline cellulose, and Veegum; and dispersing agents such as sorbitan fatty acid esters, sodium lauryl sulfate, and polysorbate 80. Injectable preparations are usually prepared by dissolving the above-mentioned active ingredient in distilled water for injection, and if necessary, solubilizers, buffers, pH adjusters, isotonicity agents, soothing agents, preservatives, etc. Furthermore, the compound may be in the form of a suspension injection prepared by suspending it in distilled water for injection or vegetable oil, and if necessary, a base, suspending agent, viscosity modifier, etc. may be added.

[0057] Second Aspect of the Invention In the second aspect of the present invention, the term "indolinone compound" refers to a low molecular weight compound having an indolinone skeleton, which promotes the expression of the survivin gene and also promotes the expression of neurotrophic factors such as BDNF. For example, semaxanib can be mentioned. The term "iPS cells" used in this invention refers to induced pluripotent stem cells (iPSCs) produced by incorporating Yamanaka factors or other factors into human somatic cells. Human somatic cells may be from healthy individuals or patients with neurological disorders. Patients with neuropsychiatric disorders include those with amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, Huntington's disease, age-related neurological disorders (dementia, etc.), depression, schizophrenia, bipolar disorder, post-traumatic stress disorder (PTSD), anxiety disorder, panic disorder, autism spectrum disorder, developmental disorder, Rett syndrome, Down syndrome, cerebral ischemic injury, or spinal cord injury.

[0058] The term "survivin" used in this invention refers to a protein that inhibits caspase activation and suppresses apoptosis. Survivin is often highly expressed in cancer cells, whereas expression is almost nonexistent in many fully differentiated cells with short turnover periods, such as those found in skin tissue. Therefore, survivin has been considered an ideal target for cancer therapy. However, it has also been shown that survivin is essential for the normal proliferation and differentiation of hematopoietic stem cells, T lymphocytes, and erythroblasts, raising concerns about the potential for survivin-targeted cancer therapy (Bird. Nature Reviews Immunology (2004) 4:166.) (Leung et al., J Exp Med (2007) 204:1603-11). Furthermore, survivin expression is maintained in areas such as the cerebral cortex, where mature neurons are maintained for a long period of time, and is thought to play a major role in the long-term maintenance of neurons. Survivin mRNA expression is known to increase with the onset of differentiation induction of neural progenitor cells. Survivin is essential for the proliferation and differentiation of neural progenitor cells in the adult hippocampus, and has been shown to play a major role in neurogenesis and recovery after traumatic brain injury (Zhang et al., Neuroscience (2015) 300:219-228). Aging is associated with decreased hippocampal neural stem cell activity, which is thought to be associated with age-related cognitive decline. A retroviral approach to survivin expression in 13-week-old mice restored the hippocampal neural stem cells to their 3-week-old state (Miranda CJ, et al., Aging Cell (2012) 11;542-552 "Aging brain microenvironment decreases hippocampal neurogenesis through Wnt-mediated survivin signaling"). Enhanced survivin expression may also contribute to extending healthy lifespan.

[0059] The "expression promoter" of the present invention refers to a substance that promotes the expression of the survivin gene, thereby enabling the suppression of apoptosis and cell activation by the expressed survivin protein. The term "xeno-free" in the present invention means "xenogeneic component free," and is used in its shortened form as the word "xeno-free." That is, the term xeno-free means "free of xenogeneic components," "free of animal-derived substances," or "free of non-human-derived components." The "xeno-free culture system" of the present invention refers to a cell culture environment that is xeno-free. For example, a xeno-free culture system is said to be one in which no animal-derived components (such as bovine serum, feeder cells, or mouse sarcoma-derived Matrigel) are used in the culture medium. The "embryoid body (cell aggregate)" of the present invention is also called an embryoid body (EB). When iPS cells are cultured in suspension, ball-shaped cell aggregates are formed, and these cell aggregates are called embryoid bodies. When cultured in the embryoid body state for about two weeks, differentiation into various cell types is observed, making it difficult to isolate single cells.

[0060] The "human neurogenin 2 gene" of the present invention is one of the factors that promote the differentiation of neural stem cells into neurons, and is a bLHL-type transcription factor. The term "iPS cells transfected with the human neurogenin 2 gene" used herein refers to iPS cells transfected with the human neurogenin 2 gene by RNA lipofection, or with a vector containing the neurogenin 2 gene in a plasmid or viral vector, enabling transient expression. RNA lipofection is a method in which a negatively charged DNA is bound to a positively charged cationic liposome to form a complex, and the DNA is taken up into the cell through the cell surface by endocytosis (Chesnoy S. et al. Annu. Rev. Biophys. Biomol. Struct. (2000) 29:27-47). Alternatively, a vector containing the neurogenin 2 gene in a plasmid or viral vector, enabling transient expression, can be prepared according to the method of Kim et al. (Method in Molecular Biology (2016) 1357:111-131). For example, according to the method of Kim et al., a vector that transiently expresses neurogenin 2 in the presence of doxycycline can be prepared and then transfected into iPS cells.

[0061] "Laminin coating" in the present invention refers to coating a culture vessel (such as a culture dish) with a human recombinant laminin fragment. Preferably, coating is performed with iMatrix-511 (Nippi). Differentiation of iPS cells and iPS cell-derived neurons, as well as the maintenance of differentiated neurons, are preferably achieved by two-dimensional culture on a laminin-coated culture dish. For differentiation of iPS cells into neurons and maintenance of neurons, Neurobasal Medium or a 1:1 mixture of Neurobasal Medium and DMEM / F12 can be used. In the present invention, "transient induction of expression" refers to the introduction of the human neurogenin 2 gene into iPS cells to transiently express the transcription factor neurogenin 2 protein. Expression induction is achieved by lipofection in the RNA lipofection method, but when a vector is used, for example, a vector that transiently expresses neurogenin 2 in the presence of doxycycline is used, allowing neurogenin 2 to be transiently expressed only in the presence of doxycycline.

[0062] The term "small molecule TGFβ family inhibitor" as used herein refers to a small molecule compound that inhibits signal transduction by the TGFβ family, which has a similar structure to TGF-β and transmits signals via a similar pathway. The TGFβ family is involved in a wide variety of cellular functions, including cell proliferation, differentiation, development, and apoptosis control. By suppressing or inhibiting this action, the proliferation and function of, for example, blood cells and lymphocytes can be promoted. Examples of small molecule inhibitors of the TGFβ family include SB431542, LY364947 (4-[3-(2-pyridinyl)-1H-pyrazol-4-yl)-quinoline), LDN-193189, Galunisertib (LY2157299), LY2109761, SB525334, SB505124, and GW788388.

[0063] The term "adenylate cyclase activator" used in the present invention refers to a compound that activates adenylate cyclase, an enzyme that converts ATP into cAMP, a second messenger for many neurotransmitters and hormones, to synthesize it. Examples of adenylate cyclase activators include F0855 (forskolin) and DG02877 (colforsin hydrochloride daropate). The term "GSK3 inhibitor" used in the present invention refers to an inhibitor of glycogen synthase kinase 3, which is involved in cell division, cell proliferation, cell motility, and cell survival. Examples of such inhibitors include CHIR-99021 (6-[[2-[[4-(2,4-Dichlorophenyl)-5-(4-methyl-1H-imidazole-2-yl)-2-pyrimidinyl]amino]ethyl]amino]nicotinonitrile), A1070722 (1-(7-Methoxyquinolin-4-yl)-3-[6-(trifluoromethyl)pyridin-2-yl]urea), BIO((2'Z,3'E)-6-Bromoindirubin-3'-oxime), and SB2 Examples include 16763 (3-(2,4-Dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione), SB415286 (3-[(3-Chloro-4-hydroxyphenyl)amino]-4-(2-nitrophenyl)-1h-pyrrole-2,5-dione), TC-G24 (N-(3-Chloro-4-methylphenyl)-5-(4-nitrophenyl)-1,3,4-oxadiazol-2-amine), and TCS2002 (2-Methyl-5-[3-[4-(methylsulfinyl)phenyl]-5-benzofuranyl]-1,3,4-oxadiazole).

[0064] The "Smoothened agonist" of the present invention refers to an agonist (promoter) of Smoothened, a seven-transmembrane protein that enhances the proliferation and survival of nerve cells. Examples include palmorfamine (2-(1-naphthoxy)-6-(4-morpholinoanilino)-9-cyclohexylpurine) and SAG (3-chloro-N-[trans-4-(methylamino)cyclohexyl]-N-[3-(4-pyridinyl)benzyl]-1-benzothiophene-2-carboxamide). The term "ROCK inhibitor" as used herein refers to an inhibitor of ROCK (p160-Rho-associated coiled-coil kinase), a phosphorylating enzyme involved in basic cellular functions such as cell proliferation and gene expression. ROCK inhibitors are substances that exhibit very strong cell death-suppressing effects, and examples of such inhibitors include Y-27632, thiazovivin, RKI-1447, GSK-429286A, and Fasudil Hydrochloride.

[0065] The "neurotrophic factors" of the present invention are cell growth and proliferation factors and cytokines that have an effect on nerves, and examples thereof include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 / 5 (NT-4 / 5), glial cell line-derived neurotrophic factor (GDNF), insulin-like growth factor-1 (IGF-1), and vascular endothelial growth factor (VEGF). As used herein, "low cell activity" refers to inactive cells that stagnate or die during differentiation. For example, when iPS cells derived from a patient with an organic disease resulting from a genetic mutation or epigenetic abnormality are induced to differentiate, the stress placed on the iPS cells weakens their cell activity, causing differentiation to stagnate or the cells to die during the differentiation process.

[0066] The term "neuropsychiatric disorder patient" as used herein refers to a patient suffering from a disease that results in motor or cognitive impairment due to a lesion in the nerves themselves, such as the brain, spinal cord, or peripheral nerves, or a lesion in the muscles themselves. Examples of such disorders include Parkinson's disease, spinocerebellar degeneration, amyotrophic lateral sclerosis (ALS), Alzheimer's disease, viral or bacterial neuritis or myelitis, myasthenia gravis, muscular dystrophy, and polymyositis. Further examples include dementia, cerebrovascular disorders with a wide range of pathologies, from acute stroke to gradually progressive vascular dementia, Huntington's disease, and aging. Other examples include depression, schizophrenia, bipolar disorder, panic disorder, PTSD, anxiety disorder, and other conditions associated with neurite retraction, decreased spine density, and attenuated hippocampal neurogenesis. Furthermore, examples include developmental disorders such as autism spectrum disorder and attention deficit hyperactivity disorder, which are caused by abnormal synaptic pruning during brain development.

[0067] The "method for screening therapeutic agents for neurological diseases" of the present invention involves inducing differentiation of iPS cells derived from a patient with a neurological disease into neurons, particularly motor neurons, to produce disease-specific neurons that reflect the patient's neurons (particularly motor neurons), and then using these cells to screen for therapeutic agents optimal for the disease. The disease-specific neurons differentiated by the present invention can be used to screen for compounds (e.g., pharmaceutical compounds, solvents, small molecules, peptides, or polynucleotides) effective in treating neurological diseases. For example, screening can be performed using drugs alone or in combination with other compounds, and the screened compounds can be evaluated by analyzing morphological or functional changes in the differentiated disease-specific neurons. Examples of morphological changes that can be evaluated include dendrite extension, increased dendritic branching, increased spines on dendrites, and axonal elongation. Examples of functional changes that can be evaluated include changes in gene expression or changes in the type or expression level of produced proteins. Furthermore, the differentiation-induced disease-specific nerve cells of the present invention can be used to analyze the specific dynamics of nerve disease-specific nerve cells.

[0068] The "cell activator" of the present invention can simultaneously promote the activity of multiple factors that are required to work together under physiological conditions. This significantly differs from previous drug discovery efforts that target single molecules and serves as the basis for an entirely new type of therapeutic agent with physiological functionality. It will play an important role in the development of future therapeutic agents for neuropsychiatric disorders for which no effective treatments have been established to date, including amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, Huntington's disease, age-related neurological disorders (e.g., dementia), depression, schizophrenia, bipolar disorder, post-traumatic stress disorder (PTSD), anxiety disorders, panic disorders, autism spectrum disorders, developmental disorders, Rett syndrome, Down syndrome, and spinal cord injury. Furthermore, the cell activator is a low-molecular-weight compound, inexpensive, easily stored, and likely to cross the blood-brain barrier, making it distinct from neurotrophic factor replacement therapies that have been explored to date. In the present invention, "capable of maintaining culture" means that terminally differentiated neurons can be stably cultured and maintained for at least four months using a minimal number of steps and reagents under xeno-free culture conditions that do not contain unidentified contaminants. In the case of iPS cells derived from patients with neurological disorders, even if the cells have low activity, adding an indolinone compound, for example, can produce terminally differentiated neurons without causing cell death during the culture and enable their maintenance in culture. [Example]

[0069] The present invention will be explained in more detail below based on examples and test examples, but the present invention is not limited thereto in any way.

[0070] First aspect of the present invention (Example 1-1) Establishment of a feeder-free iPS cell culture system (1) Materials and reagents 1) iPS cells derived from healthy individuals (HSP0003, 0009, 0029) and ALS patient-derived iPS cells (HSP0290 (familial), 0292 (familial), 0134 (sporadic), 0140 (familial)) (RIKEN BioResource Center (hereafter referred to as RIKEN BRC); 3) iPS cell culture medium StemFit AK02N Ajinomoto (Takara Bio); 4) iMatrix-511 Nippi (Takara Bio); 5) Y-27632 10 mM solution (Fujifilm Wako Pure Chemical Industries); 6) TrypLE Select (Thermo Fisher); 7) STEM-CELLBANKER (Takara Bio); 8) D-PBS(-) (Nacalai Tesque); 10) Bicelle Freezing Container (Funakoshi).

[0071] (2) Method 20 μl of iMatrix-511 was added to a 60 mm culture dish containing 1 mL of D-PBS(-). The dish was incubated for at least 60 minutes in a 37°C, 5% CO2 incubator to coat the dish with laminin. The solution was then removed and replaced with StemFit medium. The dish was then placed in a 37°C, 5% CO2 incubator to allow for incubation. The required amount (4 mL per dish) of StemFit medium was supplemented with Y-27632 to a final concentration of 10 μM (hereafter referred to as StemFit+Y medium). The StemFit medium was removed from the laminin-coated culture dish, and the iPS cells were suspended in StemFit+Y medium and seeded onto the laminin-coated dish. The next day, the medium was replaced with StemFit medium without Y-27632. The medium was changed every other day or daily. The cells were passaged when they had proliferated to approximately 60–70% of the culture area. Passage was performed as follows. First, StemFit medium was removed from the culture dish, and the dish was washed once with 4 mL of D-PBS(-). 1 mL of TrypLE Select was added and the dish was incubated for 9 minutes in a 37°C, 5% CO2 incubator. 4 mL of StemFit medium was then added, and the cells were dispersed well by pipetting. The cells were then transferred to a centrifuge tube and collected by centrifugation. The collected iPS cells were dispersed in 1 mL of StemFit+Y medium and counted, resulting in approximately 4 x 10 cells per culture dish. 4iPS cells were dispersed in 4 ml of StemFit+Y medium and seeded onto a laminin-coated culture dish. After several passages, the cells were adapted to the feeder-free culture system. For cell freezing, collect approximately 1x10 cells. 6 The cells were suspended in STEM-CELLBANKER to a final concentration of 1 / ml, dispensed into cryotubes, placed in bicells, frozen at -80°C for at least 3 hours, and then transferred to liquid nitrogen and stored within a few days.

[0072] (3) Results A microscopic image of a culture of iPS cells from a healthy individual is shown in Figure 1A. The fact that these iPS cells cultured in a feeder-free environment proliferated while maintaining their iPS cell characteristics can be seen in Figure 1B, a microscopic photograph of an iPS cell cluster cultured using feeder cells (from the iPS Research Institute website https: / / www.jiji.com / jc / d4?p=yns002-05728491&d=d4_news). All iPS cell lines were able to be transferred to a feeder-free culture system.

[0073] (Example 1-2) Differentiation of iPS cells into motor neurons (1) Materials and reagents 1) iPS cells derived from healthy individuals (HSP0003, 0009, 0029) and ALS patient-derived iPS cells (HSP0290 (familial), 0292 (familial), 0134 (sporadic), 0140 (familial)) (RIKEN BRC); 2) Human iPS cell transposon vector piggyBac, All-in-One PB-TAG-ERN (RIKEN BRC) (Kim et al., 2016, Methods in Molecular Biology. 1357: 111-131); 3) pCAG-PBase (RIKEN BRC); 4)Gateway LR Clonase II (Invitrogen); 5) Gataway pENTR1A (Thermo Fisher); 6) Human neurogenin-2 gene (Kazusa Genome Technologies); 7) TransIT-LT1 (Takara Bio); 8) 50 mg / mL G418 (Nacalai Tesque); 9) doxycycline 1 mg / mL (LAT Laboratories); 10) DMEM / HAM's F12 medium (Fujifilm Wako Pure Chemical Industries); 11) Gibco Neurobasal Medium (Thermo Fisher); 12) L-Glutamine (x100) (Fujifilm Wako Pure Chemical Industries); 13) N2 Supplement (x100) (Fujifilm Wako Pure Chemical Industries); 14) B27 Supplement (x50) (Thermo Fisher); 15) L-Ascorbic acid 10mg / mL (x1000) (Sigma-Aldrich); 16) SB431542 (Fujifilm Wako Pure Chemical Industries, Ltd. 033-2431) 5 mM / DMSO; 17) CHIA99021 (Fujifilm Wako Pure Chemical Industries) 10 mM / DMSO; 18) Forskolin (Fujifilm Wako Pure Chemical) 10 mM / DMSO; 19) Parmorphamine (Fujifilm Wako Pure Chemical Industries) 10 mM / DMSO; 20) Recombinant BDNF 10 μg / mL D-PBS(-) (R&D System, Cosmo Bio); 21) Recombinant GDNF 10 μg / mL D-PBS(-) (R&D System, Cosmo Bio); 22) Recombinant IGF-1 10 μg / mL D-PBS(-) (Fujifilm Wako Pure Chemical Industries); 23) Neuronal differentiation medium (NDM) is a 1:1 mixture of DMEM / HAM's F12 medium and Neurobasal Medium, containing N2 supplement, B27 supplement, L-glutamine, and 10 μg / mL L-ascorbic acid; 24) Isogen (Nippon Gene); 27) PrimeScript RT Reagent Kit (Takara Bio); 28) TB Green Premix EX taq II (Takara Bio); 29)SmartCycler(Cepheid SC2500N5-1); 30) Real-time PCR primers (β-actin, PAX6, HB9, glutamate receptor 1) (Table 1)

[0074] (2) Method A. Establishment of iPS cell lines capable of transiently inducing neurogenin 2 expression by doxycycline The human neurogenin 2 gene obtained from Kazusa Genome Technologies lacked a stop codon, so a stop codon was inserted by base insertion according to standard procedures and the gene was cloned into pENTR1A. An iPS cell line in which neurogenin 2 expression was transiently induced by doxycycline was created according to the method described by Kim et al. (Method in Molecular Biology. (2016) 1357:111-131). However, gene introduction was performed by reverse transfection using TransIT-LT1 instead of electroporation. iPS cells carrying the vector were selected using G418 (gradually increasing the concentration from 50 μg / mL to 100 μg / mL), and five or more cell lines capable of transiently inducing neurogenin 2 expression with doxycycline were established by limiting dilution. (Hereinafter, iPS cells transiently expressing neurogenin 2 with doxycycline are collectively referred to as iPS-PBN2.)

[0075] Neurogenin is a transcription factor that acts as a switch when pluripotent stem cells differentiate into neurons. There are three types of neurogenin: neurogenin 1, neurogenin 2, and neuron 3. We performed experiments to differentiate iPS cells into neurons by transiently expressing all combinations of neurogenins and each alone. We found that differentiation into neurons was possible only when neurogenin 2 was added, and that differentiation into neurons was also possible with neurogenin 2 alone. Therefore, we decided to use neurogenin 2 alone. A classic method is to differentiate stem cells into neurons through cell division (forming embryoid somatic cells (cell aggregates)). However, it is difficult to form embryoid somatic cells (cell aggregates) when culturing without using feeder cells. This method requires a complex process and the addition of many drugs, many of which are cytotoxic, and differentiation takes a long time, more than one month. Therefore, a simpler method was developed.

[0076] B. Differentiation of iPS cells into motor neurons 5x10 per 60mm laminin-coated culture dish 4 iPS-PBN2 cells were suspended and seeded in 4 mL of (StemFit+Y) + 1 μg / mL doxycycline medium. After 16 hours, the medium was replaced with NDM + 10 μM Y-27632 + 3 μM SB431542 + 3 μM CHIA99021 + 3 μM forskolin (D1). By day 4, neural progenitor cells proliferated vigorously, occupying approximately 70% of the culture area. Cells were collected as described in Example 1-1. First, remove the NDM + 10 μM Y-27632 + 3 μM SB431542 + 3 μM CHIA99021 + 3 μM forskolin medium from the culture dish, wash once with 4 mL of D-PBS(-), add 1 mL of TrypLE Select, and incubate at 37°C for 9 minutes in a 5% CO2 incubator. Then, add 4 mL of NDM medium, disperse the cells well by pipetting, transfer to a centrifuge tube, and collect the cells by centrifugation. Disperse the cells in 1 mL of NDM medium, count the number of cells, and collect 2 x 10 cells per culture dish. 5Cells were suspended in 4 mL of NDM + 10 μM Y-27632 + 3 μM forskolin + 3 μM parmorphamine medium and seeded onto laminin-coated 60 mm culture dishes (D4). On day 6, the medium was replaced with NDM + Y-27632 + neurotrophic factors (10 ng / mL recombinant BDNF + 10 ng / mL recombinant GDNF + 10 ng / mL recombinant IGF-1) (D6). The medium was replaced with fresh NDM + Y-27632 + neurotrophic factors medium every three days. RNA was extracted over time using Isogen's standard method and reverse-transcribed using the PrimeScript RT reagent kit. The cDNA products obtained from the reverse transcription and TB Green Premix EX taq II reagent were used to measure the time-dependent changes in mRNA expression of β-actin, PAX6, HB9, and GluA1 using a SmartCycler.

[0077] HB9 is widely used as the most reliable motor neuron marker. Because mature neurons are required to transmit neurotransmitter signals functionally, the neurotransmitter receptor GluA1 was used as a mature neuron marker. PAX6 was used because primer design was easy.

[0078] (3) Results Figures 2A and 2B show the morphological changes associated with differentiation of the iPS cell line HSP0003-PBN2#2 derived from a healthy individual into motor neurons following neurogenin 2 induction. On day 33 of differentiation induction, axons and dendrites extended from the cell body, confirming morphological characteristics of neurons (Figure 2B). Differentiation into motor neurons was also observed in other iPS-PBN2 lines (iPS0009-PBN2#2, iPS0029-PBN2#2). Figure 2C is a schematic diagram of the expression patterns of a neural progenitor cell marker (PAX6), a motor neuron marker (HB9), and a mature neuron marker (neurotransmitter glutamate receptor 1 (GluA1)) that accompany the differentiation of iPS cells into motor neurons. The diagram shows that the changes in expression of each marker gene follow the differentiation of iPS cells into motor neurons. Figure 3 shows an example of changes in marker gene mRNA expression accompanying the differentiation of HSP0003-PBN2#2 derived from a healthy individual into motor neurons. Expression of the neural progenitor cell marker (PAX6) transiently increased significantly after differentiation induction, and then decreased as the motor neurons matured. Following the expression of PAX6, mRNA expression of the motor neuron marker (HB9) increased. As the neurons matured, mRNA of the mature neuron marker (neurotransmitter receptor GluA1) significantly increased, following the expression of HB9. The results shown in Figures 2 and 3 were used as guidelines for subsequent experiments on differentiating iPS cells into motor neurons.

[0079] (Example 1-3) Effect of adding semaxanib during differentiation of iPS cells into motor neurons (1) Materials and reagents 1) Neurogenin 2 expression can be transiently induced by doxycycline in the iPS cell line HSP0003-PBN2#2 derived from a healthy individual and the iPS cell line HSP0134-PBN2#1 derived from a sporadic ALS patient; 3) iPS cell culture medium StemFit AK02N Ajinomoto (Takara Bio); 4) iMatrix-511 Nippi (Takara Bio); 5) Y-27632 10 mM solution (Fujifilm Wako Pure Chemical Industries); 6) doxycycline 1 mg / mL (LAT Laboratories); 7) 10 mM SU5416 (Semaxanib) (Selleck S2845); 8) TrypLE Select (Thermo Fisher); 9) DMEM / HAM's F12 medium (Fujifilm Wako Pure Chemical Industries); 10) Gibco Neurobasal Medium (Thermo Fisher); 11) L-Glutamine (x100) (Fujifilm Wako Pure Chemical Industries); 12) N2 Supplement (x100) (Fujifilm Wako Pure Chemical Industries); 13) B27 Supplement (x50) (Thermo Fisher); 14) L-Ascorbic acid 10mg / mL (x1000) (Sigma-Aldrich); 15) Neuronal differentiation medium (NDM) is a 1:1 mixture of DMEM / HAM's F12 medium and Neurobasal Medium, containing N2 supplement, B27 supplement, L-glutamine, and 10 μg / mL L-ascorbic acid; 16) SB431542 (Fujifilm Wako Pure Chemical Industries) 5 mM / DMSO; 17) CHIA99021 (Fujifilm Wako Pure Chemical Industries) 10 mM / DMSO; 18) Forskolin (Fujifilm Wako Pure Chemical Industries) 10 mM / DMSO (x3000); 19) Parmorphamine (Fujifilm Wako Pure Chemical Industries) 10 mM / DMSO; 20) Recombinant BDNF 10 μg / mL D-PBS(-) (R&D System, Cosmo Bio); 21) Recombinant GDNF 10 μg / mL D-PBS(-) (R&D System, Cosmo Bio); 22) Recombinant IGF-1 10 μg / mL D-PBS(-) (Fujifilm Wako Pure Chemical Industries); 23) Isogen (Nippon Gene); 24) PrimeScript RT reagent Kit (Takara Bio); 25) TB Green Premix EX taq II (Takara Bio); 26)SmartCycler(Cepheid SC2500N5-1); 27) Primers for real-time PCR (β-actin, brain-derived neurotrophic factor (BDNF)) were selected from Table 1 below.

[0080] [Table 1]

[0081] (2) Method The iPS cell line HSP0003-PBN2#2 derived from a healthy individual and the iPS cell line HSP0134-PBN2#1 derived from a sporadic ALS patient, in which neurogenin 2 expression can be transiently induced by doxycycline, were cultured at 5x10 cells per laminin-coated 60mm culture dish. 4 The cells were suspended in 4 mL of (StemFit+Y) + 1 μg / mL doxycycline medium and seeded. An indolinone compound (semaxanib) was added to the culture medium to a final concentration of 2.5 μM. DMSO, a solvent, was added to the experimental control group (control). After 16 hours, the medium was changed to NDM + 10 μM Y-27632 + 3 μM SB431542 + 3 μM CHIA99021 + 3 μM forskolin (D1). Semaxanib was added to the culture medium to a final concentration of 2.5 μM. By day 4, differentiated neural progenitor cells proliferated vigorously and formed colonies. In order to space the cells apart so that they are suitable for subsequent observation of neurons, the cells were collected by the method shown in (Example 1-1), that is, first, NDM + 10 μM Y-27632 + 3 μM SB431542 + 3 μM CHIA99021 + 3 μM forskolin medium was removed from the culture dish, and then washed once with 4 mL of D-PBS (-), 1 mL of TrypLE Select was added, and the mixture was incubated at 37 ° C. in a 5% CO2 incubator for 9 minutes, after which 4 mL of StemFit medium was added, and the cells were dispersed well by pipetting, transferred to a centrifuge tube, and collected by centrifugation. After that, the procedure was followed as in (Example 1-2) except for the addition of semaxanib. That is, the collected cells were dispersed in 1 mL of NDM medium, and the cell number was counted, and 2 x 10 per culture dish was obtained. 5Cells were suspended in 4 mL of NDM + 10 μM Y-27632 + 3 μM forskolin + 3 μM parmorphamine medium and seeded onto laminin-coated 60 mm culture dishes (D4). On day 6, the medium was replaced with NDM + Y-27632 + neurotrophic factors (10 ng / mL recombinant BDNF + 10 ng / mL recombinant GDNF + 10 ng / mL recombinant IGF-1) medium (D6). Every three days, the medium was replaced with fresh NDM + neurotrophic factors + 2.5 μM semaxanib medium or NDM + neurotrophic factors + DMSO medium. On day 9 of differentiation induction, the cells were divided into two groups: one group in which the medium was replaced with fresh NDM + Y-27632 + neurotrophic factors + 2.5 μM semaxanib medium every three days, and one group in which the medium was not renewed. RNA was extracted over time using Isogen by standard methods, and reverse transcription was performed using the PrimeScript RT reagent Kit. The cDNA products obtained by reverse transcription and TB Green Premix EX taq II reagent were used to measure the time-dependent changes in mRNA expression of β-actin, brain-derived neurotrophic factor (BDNF), VEGF-A, C, D, VEGF receptor-1, 2, 3, and neuropilin 2 using a SmartCycler.

[0082] (3) Results As shown in Figure 4A, the iPS cell line HSP0003-PBN2#2 derived from a healthy donor differentiated into motor neurons regardless of whether semaxanib was added. Microscopic images of motor neurons on day 1 (Figure 4A, left column) and day 33 (Figure 4A, right column) of differentiation induction are shown. Meanwhile, Figure 4B (left column) shows microscopic images on day 1 of differentiation induction. In the iPS cell line HSP0134-PBN2#1 derived from a sporadic ALS patient, both the control and semaxanib-treated groups differentiated into neural progenitor cells and proliferated until day 4. However, from day 8 of differentiation induction, cells in the control group ceased to elongate neurites and completely died by day 15 of differentiation induction (Figure 4B, middle column, top panel; microscope image on day 15, bold red frame). Cells in the semaxanib-treated group maintained survival (Figure 4B, middle column, bottom panel). The control group and semaxanib-treated group were observed in five independent culture dishes each, and similar results were observed in all five. The lower right image of Figure 4B shows a microscopic image of semaxanib-treated motor neurons on day 27 of differentiation induction. These results suggest that semaxanib has a survival-maintaining effect on ALS patient-derived motor neurons. Furthermore, in this ALS patient-derived neuronal cell culture, the neurons in the three culture dishes in which the renewal of neurotrophic factors, which are thought to be essential for neuronal culture, had stopped on the ninth day after differentiation induction, also survived well, and under a microscope they were judged to be in better condition than the neurons in the two culture dishes in which new neurotrophic factors had been added every three days. This led us to suspect an autocrine neurotrophic factor. Using real-time PCR, we examined changes in mRNA expression of brain-derived neurotrophic factor (BDNF), one of the three neurotrophic factors added. As shown in Figure 5, in the iPS cell line HSP0003PBN2#2 derived from a healthy individual, semaxanib treatment increased BDNF expression by approximately 5-fold compared to the control group and approximately 14-fold compared to iPS cells. In the iPS cell line HSP0134PBN2#1 derived from a sporadic ALS patient, semaxanib treatment resulted in an approximately 3.5-fold increase in BDNF expression compared to iPS cells, although the control group died on day 15, meaning that comparison was only possible with iPS cells. These results demonstrate that semaxanib promotes BDNF expression in motor neurons differentiated from iPS cells derived from healthy individuals and ALS patients. Furthermore, the microscopic images in Figure 4 show that nearly 100% of the motor neurons have neurite extensions, indicating that these motor neurons are autocrine. While healthy individuals transition to autocrine expression of BDNF, ALS patients are unable to achieve this transition, presumably resulting in decreased BDNF expression and death. Thus, semaxanib acts on motor neurons to promote autocrine BDNF expression. It is believed that semaxanib can efficiently activate motor neurons by promoting autocrine BDNF expression in motor neurons.

[0083] In recent years, exercise has been recommended to improve depression and cognitive function and prevent dementia. This is believed to be due to the increased production of BDNF and VEGF, which improves neuroplasticity and promotes neurogenesis in the hippocampus (Kumagai, A. et al., Research in Exercise Epidemiology (2006) 9: 1-45 "Exercise Epidemiology in Relation to Cognitive Function and Brain-Derived Neurotrophic Factor"); Cotman, C.W. and Berchtold, N.C., Trends in Neurosciences (2002) 25(6); 295-301 "Exercise: a behavioral intervention to enhance brain health and plasticity"; Fabel, K., et al., Eur J Neurosci. (2003) 18: 2803-2812 "VEGF is necessary for excised-induced adult hippocampal neurogenesis"); Duman, R.S., Neurobiol Aging (2005)26: 88-93 "Neurotrophic factors and regulation of mood: role of exercise, diet and metabolism”).

[0084] (Example 1-4) Evaluation of the expression of protein genes related to the maintenance of survival and neuroplasticity of motor neurons differentiated from iPS cells, and the promotion of neuronal proliferation and differentiation (1) Materials and reagents 1) Neurogenin 2 expression can be transiently induced by doxycycline in the iPS cell line HSP0003-PBN2#2 derived from a normal individual and the iPS cell line HSP0134-PBN2#1 derived from a sporadic ALS patient; 3) iPS cell culture medium StemFit AK02N Ajinomoto (Takara Bio); 4) iMatrix-511 Nippi (Takara Bio); 5) Y-27632 10 mM solution (Fujifilm Wako Pure Chemical Industries); 6) doxycycline 1 mg / mL (LAT Laboratories); 7) 10 mM SU5416 (Semaxanib) (Selleck S2845); 8) TrypLE Select (Thermo Fisher); 9) DMEM / HAM's F12 medium (Fujifilm Wako Pure Chemical Industries); 10) Gibco Neurobasal Medium (Thermo Fisher); 11) L-Glutamine (x100) (Fujifilm Wako Pure Chemical Industries); 12) N2 Supplement (x100) (Fujifilm Wako Pure Chemical Industries); 13) B27 Supplement (x50) (Thermo Fisher); 14) L-Ascorbic acid 10mg / mL (x1000) (Sigma-Aldrich); 15) Neuronal differentiation medium (NDM) is a 1:1 mixture of DMEM / HAM's F12 medium and Neurobasal Medium, containing N2 supplement, B27 supplement, L-glutamine, and 10 μg / mL L-ascorbic acid; 16) SB431542 (Fujifilm Wako Pure Chemical Industries) 5 mM / DMSO; 17) CHIA99021 (Fujifilm Wako Pure Chemical Industries) 10 mM / DMSO; 18) Forskolin (Fujifilm Wako Pure Chemical Industries) 10 mM / DMSO (x3000); 19) Parmorphamine (Fujifilm Wako Pure Chemical Industries) 10 mM / DMSO; 20) Recombinant BDNF 10 μg / mL D-PBS(-) (R&D System, Cosmo Bio); 21) Recombinant GDNF 10 μg / mL D-PBS(-) (R&D System, Cosmo Bio); 22) Recombinant IGF-1 10 μg / mL D-PBS(-) (Fujifilm Wako Pure Chemical Industries); 23) Isogen (Nippon Gene); 24) PrimeScript RT reagent Kit (Takara Bio); 25) TB Green Premix EX taq II (Takara Bio); 26)SmartCycler(Cepheid SC2500N5-1); 27) Primers for real-time PCR (β-actin, vascular endothelial growth factor (VEGF)-A, VEGF-C, VEGF-D, VEGF receptor-1, VEGF receptor-2, VEGF receptor-3, and neuropilin 2) were selected from Table 1.

[0085] (2) Method The iPS cell line HSP0003-PBN2#2 derived from a healthy individual and the iPS cell line HSP0134-PBN2#1 derived from a sporadic ALS patient, in which neurogenin 2 expression can be transiently induced by doxycycline, were cultured at 5x10 cells per laminin-coated 60mm culture dish. 4The cells were suspended in 4 mL of (StemFit+Y) + 1 μg / mL doxycycline and seeded. The indolinone compound (semaxanib) was added to the culture medium to a final concentration of 2.5 μM. DMSO, a solvent, was added to the experimental control group (control). After 16 hours, the medium was replaced with NDM + 10 μM Y-27632 + 3 μM SB431542 + 3 μM CHIA99021 + 3 μM forskolin (D1). Semaxanib was added to the culture medium to a final concentration of 2.5 μM. DMSO, a solvent, was added to the experimental control group (control). By day 4, differentiated neural progenitor cells proliferated vigorously and formed colonies. To ensure adequate spacing between cells for subsequent neuronal observation, the cells were collected using the method described in Example 1-1. That is, first, remove NDM + 10 μM Y-27632 + 3 μM SB431542 + 3 μM CHIA99021 + 3 μM forskolin medium from the culture dish, wash once with 4 mL of D-PBS (-), add 1 mL of TrypLE Select, react for 9 minutes in a 37 ° C, 5% CO2 incubator, add 4 mL of NDM medium, disperse well by pipetting, transfer to a centrifuge tube, and collect the cells by centrifugation. After that, follow the procedure (Example 1-2) except for the addition of semaxanib. That is, the collected cells were dispersed in 1 mL of NDM medium and counted, and 2 x 10 per culture dish were collected. 5The cells were suspended in 4 mL of NDM + 10 μM Y-27632 + 3 μM forskolin + 3 μM parmorphamine + 2.5 μM semaxanib medium or 4 mL of NDM + 10 μM Y-27632 + 3 μM forskolin + 3 μM parmorphamine + DMSO medium and seeded onto laminin-coated 60 mm culture dishes (D4). On day 6, the medium was replaced with NDM + Y-27632 + neurotrophic factors (10 ng / mL recombinant BDNF + 10 ng / mL recombinant GDNF + 10 ng / mL recombinant IGF-1) + 2.5 μM semaxanib or NDM + Y-27632 + neurotrophic factors (10 ng / mL recombinant BDNF + 10 ng / mL recombinant GDNF + 10 ng / mL recombinant IGF-1) + DMSO (D6). Every 3 days, the medium was replaced with fresh NDM + neurotrophic factors + 2.5 μM semaxanib or NDM + Y-27632 + neurotrophic factors (10 ng / mL recombinant BDNF + 10 ng / mL recombinant GDNF + 10 ng / mL recombinant IGF-1) + DMSO. RNA was extracted over time using Isogen, followed by reverse transcription using the PrimeScript RT reagent Kit. The cDNA products obtained by reverse transcription and TB Green Premix EX taq II reagent were used to measure the time-dependent changes in mRNA expression of β-actin, VEGF-A, C, D, VEGF receptors-1, 2, 3, and neuropilin 2 using a SmartCycler. Real-time PCR reactions were performed using the primer pairs listed in Table 1 for β-actin, vascular endothelial growth factor (VEGF)-A, VEGF-C, VEGF-D, VEGF receptor-1, VEGF receptor-2, VEGF receptor-3, and neuropilin 2, and TB Green Premix EX taq II reagent, respectively, and detection was performed using a SmartCycler.

[0086] (3) Results Figure 6A shows the results of evaluating the promotion of protein gene expression related to the maintenance of survival and neuroplasticity in motor neurons differentiated from the iPS cell line HSP0003-PBN2#2 derived from a healthy individual, as well as the promotion of neuronal proliferation and differentiation. Compared to the control group, the indolinone compound (semaxanib)-added group exhibited the following gene expression promoting effects. 1) VEGF-A expression increased approximately three-fold (upper left image). 2) The expression of VEGF-C and VEGF-D increased approximately 9.5-fold (upper middle panel) and approximately 18-fold (upper right panel), respectively. 3) It has previously been observed that the expression of VEGF receptor-2 decreases with differentiation into neurons, but the addition of semaxanib maintained expression (lower left figure). 4) Expression of VEGF receptor-3 increased approximately fourfold (bottom center figure). 5) The expression of neuropilin 2, a co-receptor for VEGF receptor-2 and receptor-3, increased approximately fourfold (bottom right). Figure 6B shows the results of evaluating the promotion of protein gene expression related to maintaining survival, neuroplasticity, and promoting neuronal proliferation and differentiation in motor neurons differentiated from the iPS cell line HSP0134-PBN2#1 derived from a sporadic ALS patient. In the control group, cells died within 15 days of differentiation, but in the semaxanib-added group, the expression of each gene was promoted as follows. 1) VEGF-A expression increased approximately 2-fold compared to iPS cells (top left). 2) The expression of VEGF-C and VEGF-D increased approximately 5.5-fold (upper middle panel) and approximately 4-fold (upper right panel), respectively, compared to iPS cells. 3) Expression of VEGF receptor-2, which decreases with differentiation into neurons, was maintained at approximately 30% of that in iPS cells by adding semaxanib (lower left figure). 4) VEGF receptor-3 expression was maintained at approximately 80% compared to iPS cells (bottom center figure). 5) Expression of neuropilin 2, a co-receptor for VEGF receptor-2 and receptor-3, increased approximately five-fold compared to iPS cells (bottom right figure). These results demonstrate that semaxanib promotes the expression of VEGF signaling pathway proteins in motor neurons differentiated from iPS cells. Thus, it has become clear that semaxanib acts on motor neurons and promotes the expression of VEGF signaling pathway proteins as an autocrine.

[0087] Second Aspect of the Invention In the examples, the following materials were used to induce differentiation of iPS cells into motor neurons. (Materials and Reagents) 1) iPS cells derived from healthy individuals (HSP0003, 0009, 0029) and ALS patient-derived iPS cells (HSP0290 (familial), 0292 (familial), 0134 (sporadic), 0140 (familial)) (RIKEN BRC); 2) iPS cell culture medium StemFit AK02N Ajinomoto (Takara Bio); 3) iMatrix-511 Nippi (Takara Bio); 4) ROCK inhibitor (Y-27632) 10 mM solution (Fujifilm Wako Pure Chemical Industries); 5) TrypLE Select (Thermo Fisher); 6) Human iPS cell transposon vector piggyBac, All-in-One PB-TAG-ERN (RIKEN BRC) (Kim et al., Methods in Molecular Biology. 1357: 111-131, 2016); 7) pCAG-PBase (RIKEN BRC); 8)Gateway LR Clonase II (Invitrogen); 9) Gataway pENTR1A (Thermo Fisher); 10) Neurogenin-2 gene (Kazusa Genome Technologies); 11) TransIT-LT1 (Takara Bio); 12) 50 mg / mL G418 (Nacalai Tesque); 13) doxycycline 1 mg / mL (LKT Laboratories); 14) DMEM / HAM's F12 medium (Fujifilm Wako Pure Chemical Industries); 15) Gibco Neurobasal Medium (NB) (Thermo Fisher); 16) L-Glutamine (x100) (Fujifilm Wako Pure Chemical Industries); 17) N2 Supplement (x100) (Fujifilm Wako Pure Chemical Industries); 18) B27 Supplement (x50) (Thermo Fisher); 19) L-Ascorbic acid 10mg / mL (x1000) (Sigma-Aldrich); 20) TGFβ family inhibitor (SB431542, Fujifilm Wako Pure Chemical Industries, Ltd. 033-2431) 5mM / DMSO; 21) GSK3 inhibitor (CHIR-99021, Fujifilm Wako Pure Chemical Industries) 10 mM / DMSO; 22) Adenylate cyclase activator (forskolin, Fujifilm Wako Pure Chemical Industries) 10 mM / DMSO (x3000); 23) Smoothened agonist (palmorphamine, Fujifilm Wako Pure Chemical) 10 mM / DMSO; 24) Recombinant BDNF 10 μg / mL D-PBS(-) (R&D System, Cosmo Bio); 25) Recombinant GDNF 10 μg / mL D-PBS(-) (R&D System, Cosmo Bio); 26) Recombinant IGF-1 10 μg / mL D-PBS(-) (Fujifilm Wako Pure Chemical Industries); 27) Semaxanib 10mM (Selleck); 28) Neuronal differentiation medium (NDM) is a 1:1 mixture of DMEM / HAM's F12 medium and Neurobasal Medium, containing N2 supplement, B27 supplement, L-glutamine, and 10 μg / mL L-ascorbic acid; 29) Isogen (Nippon Gene); 30) PrimeScript RT Reagent Kit (Takara Bio); 31) TB Green Premix EX taq II (Takara Bio); 32)SmartCycler(Cepheid SC2500N5-1); 33) Real-time PCR primers (β-actin, PAX6, HB9, glutamate receptor 1, survivin) (Table 2)

[0088] [Table 2]

[0089] (Example 2-1) Preparation of iPS cells derived from healthy individuals and iPS cells derived from ALS patients transfected with the human neurogenin 2 gene (1) Method A stop codon was inserted into the human neurogenin 2 gene purchased from Kazusa Technologies and cloned into pENTR1A. iPS cells transiently expressing neurogenin 2 in response to doxycycline were generated according to Kim et al. (2016, Method in Molecular Biology. 1357:111-131). However, gene transfer was performed using reverse transfection with TransIT-LT1 instead of electroporation.

[0090] (2) Results iPS cells carrying the vector were selected using G418 (gradually increasing from 50 μg / mL to 100 μg / mL), and from each iPS cell line, iPS0003-PBN2#1,2,3,4,5, iPS0009-PBN2#1,2,3,4,5, and iPS0029-PBN2#1,2,3,4,5 were established from iPS cells derived from normal individuals as cell lines in which neurogenin 2 expression could be transiently induced by doxycycline. Similarly, we established iPS0290-PBN2#1,2,3,4,5, iPS0292-PBN2#1,2,3,4,5, iPS0134-PBN2#1,2,3,4,5, and iPS0140-PBN2#1,2,3,4,5 from iPS cells derived from ALS patients (hereafter, iPS cells in which neurogenin 2 is transiently expressed by doxycycline are collectively referred to as iPS-PBN2).

[0091] (Example 2-2) Initiation of differentiation of iPS cells derived from healthy individuals transfected with human neurogenin 2 gene (1) Method 20 μl of iMatrix-511 was added to a 60 mm culture dish containing 1 mL of D-PBS(-), and the dish was incubated in a 37°C, 5% CO2 incubator for 60 minutes or more to coat the dish with laminin. The solution was then removed and replaced with StemFit medium, which was then placed in the incubator to allow for incubation. A ROCK inhibitor (Y-27632) was added to the required amount of StemFit medium (4 mL culture dish) to a final concentration of 10 μM, and the dish was placed in the incubator. Each iPS-PBN2 cell obtained in Example 1 was suspended and seeded onto the laminin-coated culture dish from which the StemFit medium had been removed. For differentiation induction, 5x10 cells were cultured in a laminin-coated 60 mm culture dish as described above. 4 iPS-PBN2 cells were suspended in 4 mL of (StemFit+Y)+1 μg / mL doxycycline (DOX), seeded, and cultured for 16 hours.

[0092] (2) Results After 16 hours of culture of iPS cells derived from a healthy individual, iPS0003-PBN2#2, most of the iPS cells extended processes and underwent a neural progenitor cell-like morphology, as shown in the right panel of the phase-contrast micrograph in Figure 8. Even after 24 hours of culture in the presence of DOX, the neural progenitor cell-like morphology was maintained, as shown in the right panel of Figure 9. Furthermore, after 48 hours with DOX added, large, round cells with a blurred periphery began to appear. After 5 days with DOX added, the cells changed into colonies with a changed morphology. Therefore, it was revealed that 16 to 20 hours of induction of Neurogenin 2 by DOX is sufficient, and that prolonged induction of Neurogenin 2 has a negative effect on differentiation into neurons.

[0093] (Example 2-2) Induction of differentiation of neural stem cell-like cells derived from iPS cells derived from healthy individuals into neurons (1) Method The culture medium was replaced in the culture dish that had been cultured in the presence of DOX for 16 hours as in Example 2, and differentiation into neurons was induced as follows. a) Primary culture The culture medium for neural progenitor-like cells was replaced with NDM medium containing a ROCK inhibitor (10 μM Y-27632), a TGFβ family inhibitor (3 μM SB431542), a GSK3 inhibitor (3 μM CHIR99021), and an adenylate cyclase activator (3 μM forskolin). By day 4, neural progenitor cells had proliferated vigorously, occupying approximately 70% of the culture area. After removing the NDM + 10 μM Y-27632 + 3 μM SB431542 + 3 μM CHIA99021 + 3 μM forskolin medium from the culture dish, the cells were washed once with 4 mL of D-PBS(-), and incubated with 1 mL of TrypLE Select in a 37°C, 5% CO2 incubator for 9 minutes. After adding 4 mL of NDM medium and dispersing well by pipetting, the cells were transferred to a centrifuge tube and collected by centrifugation. The collected cells were dispersed in 1 ml of NDM medium and the number of cells was counted. b) Secondary culture The collected cells were cultured at 2 x 10 per 60 mm culture dish. 5The cells were suspended in 4 mL of NDM medium containing a ROCK inhibitor (10 μM Y-27632), an adenylate cyclase activator (3 μM forskolin), and a Smoothened agonist (3 μM palmorphamin) to obtain a total of 10 cells per 1000 cells. These cells were then seeded onto a laminin-coated 60 mm culture dish. The cells were then cultured until day 6 of differentiation induction. c) Tertiary culture (final differentiation culture) After the 6th day of differentiation induction, the medium in the culture dish was replaced with NDM medium containing a ROCK inhibitor (Y-27632) and neurotrophic factors (10 ng / mL recombinant BDNF, 10 ng / mL recombinant GDNF, and 10 ng / mL recombinant IGF-1). Thereafter, the medium was replaced with fresh NDM medium containing neurotrophic factors every 3 days. RNA was extracted over time (typically, day 1 without doxycycline was used as a control, and days 1, 4, 15, 18, 22, and 33 after doxycycline addition) using standard methods using Isogen, and reverse transcription was performed using the PrimeScript RT reagent Kit. Using the cDNA products obtained from the reverse transcription reaction and TB Green Premix EX taq II reagent, changes in mRNA expression of β-actin, PAX6, HB9, and GluA1 were measured using a SmartCycler.

[0094] (2) Results As shown in the phase-contrast micrographs in Figure 10, neurite extension began from all cells on day 1 of differentiation induction (upper right panel, b). On day 3, cells were proliferating and further neurite development was observed (lower left panel, c). On day 32 of differentiation induction, neurons with well-developed neurites were observed (lower right panel, d). Figure 11 shows the dynamics of neuronal differentiation marker mRNA expression. The expression of the neural stem cell marker PAX6 first increased rapidly and then decreased as differentiation progressed (top panel a). Next, the expression of the motor neuron marker HB9 increased rapidly and maintained a high level of expression while gradually increasing as differentiation progressed (middle panel b). When the expression of GluA1 was examined as a marker for mature neurons, it increased rapidly on day 18, when the expression of PAX6 began to decrease, and remained at a high level (bottom panel c). Differentiated neurons could be observed for up to four months. Thus, it is thought that differentiated neurons will become the majority from 18 days onwards after the start of differentiation induction, and that after about one month they will have become mature neurons and differentiation induction will be complete.

[0095] (Example 2-3) Initiation of differentiation of ALS patient-derived iPS cells transfected with human neurogenin 2 gene (1) Method 20 μL of iMatrix-511 was added to a 60 mm culture dish containing 1 mL of D-PBS(-), and the dish was incubated in a 5% CO2 incubator at 37°C for 60 minutes or more to perform laminin coating. The solution was then removed and replaced with StemFit medium, which was then placed in the incubator to allow for incubation. A ROCK inhibitor (Y-27632) was added to the required amount (4 mL / dish) of StemFit medium to a final concentration of 10 μM, and the dish was placed in the incubator. The sporadic ALS patient-derived iPS0134-PBN2#1 obtained in Example 1 was suspended in the StemFit+Y-27632 medium that had been placed in the incubator, and the suspension was seeded onto the laminin-coated culture dish. For differentiation induction, 5x10 cells were cultured in a laminin-coated 60 mm culture dish as described above. 4 iPS0134-PBN2#1 cells derived from an ALS patient were suspended in 4 mL of (StemFit+Y) + 1 μg / mL doxycycline (DOX), seeded, and cultured for 16 hours. A group was treated with an indolinone compound (semaxanib) (2.5 μM) and a group was not treated with an indolinone compound (DMSO).

[0096] (2) Results As shown in the phase-contrast micrograph b) in Figure 12, after differentiation induction, iPS cells derived from ALS patients were cultured for 16 hours, and most of the iPS cells in both the group without indolinone compound addition (left) and the group with indolinone compound (semaxanib) addition (right) underwent morphological changes resembling neural progenitor cells.

[0097] (Example 2-4) Induction of differentiation of neural progenitor-like cells derived from ALS patient-derived iPS cells into neurons (1) Method iPS0134-PBN2#1 cells derived from a sporadic ALS patient were used. Differentiation induction was initiated as described in Example 2, with the exception of an indolinone compound (semaxanib)-treated group and a non-treated group. In the indolinone compound (semaxanib)-treated group, doxycycline (DOX) was added to a final concentration of 2.5 μM at the same time as differentiation initiation. After 16 hours of culture in the presence of DOX, the medium was replaced in the culture dish as follows, and differentiation into neurons was induced. a) Primary culture The medium for neural stem cell-like cells was replaced with NDM medium containing a ROCK inhibitor (10 μM Y-27632), a TGFβ family inhibitor (3 μM SB431542), a GSK3 inhibitor (3 μM CHIR99021), and an adenylate cyclase activator (3 μM forskolin). In the indolinone compound (semaxanib) group, a final concentration of 2.5 μM was added. By day 4 of differentiation with DOX addition, neural progenitor cells proliferated vigorously, reaching approximately 70% of the culture area. After removing the NDM + 10 μM Y-27632 + 3 μM SB431542 + 3 μM CHIA99021 + 3 μM forskolin medium from the culture dish, the dish was washed once with 4 mL of D-PBS(-), and 1 mL of TrypLE Select was added. The dish was incubated at 37°C in a 5% CO2 incubator for 9 minutes. 4 mL of NDM medium was then added and the cells were dispersed well by pipetting. The cells were then transferred to a centrifuge tube and centrifuged to collect the cells. The collected cells were dispersed in 1 mL of NDM medium and counted. b) Secondary culture The collected cells were cultured at 2 x 10 per 60 mm culture dish. 5The cells were suspended in 4 mL of NDM medium containing a ROCK inhibitor (1 μM Y-27632), an adenylate cyclase activator (3 μM forskolin), and a Smoothened agonist (3 μM palmorfamine) to a final concentration of 100 μM, and seeded onto laminin-coated 60 mm culture dishes. For the indolinone compound (semaxanib)-treated group, a final concentration of 2.5 μM was added. Culture was continued until day 6 of differentiation induction. c) Tertiary culture (final differentiation culture) After the 6th day of secondary culture, the culture medium was replaced with NDM medium containing a ROCK inhibitor (Y-27632) and neurotrophic factors (10 ng / mL recombinant BDNF, 10 ng / mL recombinant GDNF, and 10 ng / mL recombinant IGF-1). For the indolinone compound (semaxanib) treatment group, a final concentration of 2.5 μM was added. The medium was replaced with fresh NDM medium containing neurotrophic factors every 3 days. Observations were continued from the start of differentiation induction until day 28. RNA was extracted over time (day 1 for the control without Dox, days 1, 4, and 21 after Dox addition) using standard methods using Isogen, and reverse transcription was performed using the PrimeScript RT reagent kit. Using the cDNA products obtained by the reverse transcription reaction and TB Green Premix EX taq II reagent, changes in mRNA expression of β-actin, PAX6, HB9, and GluR1 were measured using a SmartCycler.

[0098] (2) Results As shown in the phase-contrast micrograph c) in Figure 12, on day 15 of differentiation induction, the group to which the indolinone compound was not added (left image) had died, but the group to which the indolinone compound (semaxanib) was added (right image) survived and further extended neurites, and as shown in d), on day 26 of differentiation induction, neurons with developed neurites were observed in the indolinone compound-added group. Note that the group to which the indolinone compound was not added had died on day 15 of differentiation induction, and therefore was not shown in the left image of d).

[0099] (Example 2-5) Effect of indolinone compound (semaxanib) on promoting survivin gene expression (1) Method Similar to Example 5, an indolinone compound (semaxanib)-treated group and a control group were created, and iPS cells iPS0003-PBN2#2 derived from a healthy donor and iPS cells iPS0134-PBN2#1 derived from a sporadic ALS patient were differentiated into neurons. RNA was extracted over time using standard methods using Isogen, and reverse transcription was performed using the PrimeScript RT reagent Kit. Changes in survivin mRNA expression were measured using a SmartCycler with the cDNA product obtained by reverse transcription and TB Green Premix EX taq II reagent.

[0100] (2) Results No significant differences were observed during the subculture process between iPS cells derived from healthy individuals and those derived from ALS patients. However, when the expression of the apoptosis inhibitor protein survivin was evaluated, the expression of survivin was significantly reduced in iPS cells derived from ALS patients (approximately 18% of that in iPS cells derived from healthy individuals) (Figure 13). Therefore, we added the indolinone compound (semaxanib) to iPS cells derived from healthy volunteers and iPS cells derived from ALS patients, and differentiated them into neurons. Without the addition of the indolinone compound (semaxanib), neurons induced to differentiate from ALS patient-derived iPS cells died on day 15 of differentiation induction, as described in Example 2-5. However, with the addition of the indolinone compound (semaxanib), neurons induced to differentiate from ALS patient-derived iPS cells remained viable even on day 21 of differentiation induction, as shown in Figure 12d). As shown in Figure 13, survivin gene expression was increased (approximately 3-fold compared to ALS patient-derived iPS cells, and approximately 80% of that in neurons derived from healthy volunteers without the indolinone compound). Similarly, survivin gene expression was increased 2.7-fold on day 22 of differentiation induction in neurons induced to differentiate from healthy volunteer-derived neurons. Thus, it was found that the indolinone compound (semaxanib) promotes the expression of the cybabin gene in neurons induced from both iPS cells derived from healthy donors and iPS cells derived from ALS patients when added from the start of differentiation. Furthermore, due to its effect of promoting cybabin gene expression during differentiation induction, it was shown to be able to suppress the death of ALS patient-derived neurons, which are vulnerable and susceptible to death due to the stress of neuronal differentiation induction. [Industrial Applicability]

[0101] First aspect of the present invention The indolinone compounds of the present invention act on motor neurons and have the effect of promoting the expression of BDNF and / or VEGF signaling pathway proteins as an autocrine. Therefore, it has been revealed that the indolinone compounds of the present invention are extremely useful therapeutic agents for a wide range of neuropsychiatric disorders. Furthermore, as shown by semaxanib, indolinone compounds that promote the simultaneous expression of BDNF and VEGF signaling pathway proteins can further improve neuronal maintenance and neuroplasticity by conjugating BDNF and VEGF signaling pathway proteins to perform physiological functions (e.g., depression, Non-Patent Document 7) and / or by synergistic effects, thereby promoting the proliferation and differentiation of neural stem cells and activating the biological dynamics of motor neurons. The indolinone compounds of the present invention, by promoting the expression of BDNF and / or VEGF signaling proteins as autocrine pathways, are extremely useful therapeutic agents for a wide range of neuropsychiatric disorders, including amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, Huntington's disease, age-related neurological disorders (dementia, etc.), depression, schizophrenia, bipolar disorder, post-traumatic stress disorder (PTSD), anxiety disorder, panic disorder, autism spectrum disorder, developmental disorders, Rett syndrome, Down syndrome, symptom recovery after stroke, and functional recovery after spinal cord injury. Furthermore, they can be clinically applied as an excellent adjuvant to regenerative medicine using neural stem cell transplantation for the treatment of Parkinson's disease, Alzheimer's disease, and spinal cord injury, thereby promoting regenerative medicine and contributing to the extension of healthy lifespan, which is an urgent issue.

[0102] Second Aspect of the Invention It has been found that the survivin expression promoter of the present invention can enable terminal differentiation of iPS cells that have low cellular activity, reduced survivin gene expression, and are prone to die during differentiation induction. For example, in iPS cells derived from ALS patients, survivin gene expression is significantly reduced, but by adding an indolinone compound during differentiation induction, survivin expression is improved, preventing premature cell death and enabling the production of differentiated neurons. Furthermore, the differentiation induction method of the present invention enables efficient differentiation of neurons from induced pluripotent stem cells and long-term culture of differentiated neurons. Furthermore, differentiation induction is possible in a xeno-free culture system that does not contain unidentified components derived from non-human organisms, such as feeder cells, mouse sarcoma-derived Matrigel, and bovine serum, which vary depending on the lot. The differentiation induction method of the present invention allows for easy generation of ALS-specific neurons from iPS cells derived from ALS patients, enabling the screening of compounds for ALS treatment (e.g., pharmaceutical compounds, solvents, small molecules, peptides, or polynucleotides). Furthermore, the xeno-free culture system eliminates the influence of unidentified components on screening results, allowing for more accurate screening. Furthermore, by inducing differentiation of neurons from iPS cells derived from individual patients, it is possible to preliminarily evaluate the therapeutic effects of drugs on neurological diseases in individual patients. Furthermore, the ability to maintain neuronal cultures for long periods in a xeno-free culture system enables the evaluation of changes over time due to neurological diseases and the screening of therapeutic drugs in more detail. Additionally, the cybavin expression promoters of the present invention, which promote the expression of survivin, which plays an important role in maintaining healthy brain activity, activate neurogenesis in the adult hippocampus, particularly after traumatic brain injury (TBI), and are expected to play a role in therapeutic drugs because survivin plays a major role in neurogenesis in the adult hippocampus and recovery from TBI (Zhang et al. Neuroscience, 300:219-228, 2015). Furthermore, findings that neurogenesis in the adult hippocampus, which plays an important role in learning and memory, declines with age were published in 2018 (Sorrells et al. Nature (2018) 555:377). Animal experiments have shown that enhanced survivin expression restores hippocampal neurogenesis (Miranda CJ et al. Aging Cell (2012) 11;542-552). Therefore, the cybavin expression promoters of the present invention are expected to serve as a starting point for the development of drugs that contribute to extending healthy lifespan.

Claims

1. General formula (1) 【Chemical 1】 [In the formula, R 1 , R ２ and R ３ are each independently a hydrogen atom, a halogen atom, a substituted or unsubstituted lower alkyl group having 1 to 4 carbon atoms, a formyl group, an acetyl group, a carboxyl group, a substituted or unsubstituted lower alkoxycarbonyl group having 1 to 4 carbon atoms, or a substituted or unsubstituted lower alkylamido group having 1 to 4 carbon atoms, and the substituents are a halogen atom, a carboxyl group, a lower alkoxycarbonyl group having 1 to 4 carbon atoms, and a di-lower alkylamino group having 1 to 4 carbon atoms. R ４ , R ５ , R ６ and R ７ are each independently selected from the group consisting of a hydrogen atom, a halogen atom, a nitro group, a substituted or unsubstituted lower alkyl group having 1 to 4 carbon atoms, a lower alkoxy group having 1 to 4 carbon atoms, a lower alkylcarbonyl group having 1 to 4 carbon atoms, a lower alkoxycarbonyl group having 1 to 4 carbon atoms, a carboxyl group, and a hydroxyl group, and the substituents are a halogen atom, a carboxyl group, or a lower alkoxycarbonyl group having 1 to 4 carbon atoms.

2. R 1 , R ２ and R ３ are each independently a hydrogen atom, a methyl group, an ethyl group, a halogen atom, a formyl group, an acetyl group, a carboxyl group, an ethyloxycarbonyl group, a methoxycarbonylmethyl group, an ethoxycarbonylmethyl group, a 2-carboxyethyl group, a 2-methoxycarbonylethyl group, a 2-ethoxycarbonylethyl group, or a 2-diethylaminoethylamide group, and R ４ , R ５ , R ６ and R ７ The expression promoter according to claim 1 , wherein each of the groups independently represents a hydrogen atom, a halogen atom, or a nitro group.

3. R 1 , R ２ and R ３ The halogen atom in R is a chlorine atom, a bromine atom, or an iodine atom. ４ , R ５ , R ６ and R ７ 3. The expression promoter according to claim 1, wherein the halogen atom is a fluorine atom or a chlorine atom.

4. R 1 and R ３ is a methyl group, R ２ is a hydrogen atom, and R ４ , R ５ , R ６ and R ７ The expression promoter according to claim 1 or 2, wherein is a hydrogen atom.

5. The expression promoter according to any one of claims 1 to 4, wherein the expression promotion is promotion of the expression of a BDNF or VEGF signal transduction pathway protein.

6. The expression promoter according to any one of claims 1 to 4, wherein the expression promotion is simultaneous promotion of the expression of BDNF and VEGF signaling pathway proteins.

7. The expression promoter according to any one of claims 1 to 6, wherein the vascular endothelial growth factor (VEGF) signal transduction system protein is one or more of VEGF-A, VEGF-C, VEGF-D, VEGF receptor-2, VEGF receptor-3, and the co-receptor neuropilin 2.

8. General formula (1) 【Chemistry 2】 [In the formula, R 1 , R ２ and R ３ are each independently a hydrogen atom, a halogen atom, a substituted or unsubstituted lower alkyl group having 1 to 4 carbon atoms, a formyl group, an acetyl group, a carboxyl group, a substituted or unsubstituted lower alkoxycarbonyl group having 1 to 4 carbon atoms, or a substituted or unsubstituted lower alkylamido group having 1 to 4 carbon atoms, and the substituents are a halogen atom, a carboxyl group, a lower alkoxycarbonyl group having 1 to 4 carbon atoms, and a di-lower alkylamino group having 1 to 4 carbon atoms. R ４ , R ５ , R ６ and R ７ are each independently selected from the group consisting of a hydrogen atom, a halogen atom, a nitro group, a substituted or unsubstituted lower alkyl group of 1 to 4 carbon atoms, a lower alkoxy group of 1 to 4 carbon atoms, a lower alkylcarbonyl group of 1 to 4 carbon atoms, a lower alkoxycarbonyl group of 1 to 4 carbon atoms, a carboxyl group, and a hydroxyl group, and the substituents are a halogen atom, a carboxyl group, or a lower alkoxycarbonyl group of 1 to 4 carbon atoms.

9. The survivin expression promoter according to claim 8, wherein the iPS cells are derived from an ALS patient.

10. The survivin expression promoter according to claim 8 or 9, wherein the iPS cells are iPS cells induced to differentiate.

11. The survivin expression promoter according to any one of claims 8 to 10, wherein the indolinone compound is semaxanib.

12. The survivin expression promoter according to any one of claims 8 to 11, wherein the iPS cells are capable of inducing transient expression of neurogenin 2.

13. General formula (1) 【Chemistry 3】 [In the formula, R 1 , R ２ and R ３ are each independently a hydrogen atom, a halogen atom, a substituted or unsubstituted lower alkyl group having 1 to 4 carbon atoms, a formyl group, an acetyl group, a carboxyl group, a substituted or unsubstituted lower alkoxycarbonyl group having 1 to 4 carbon atoms, or a substituted or unsubstituted lower alkylamido group having 1 to 4 carbon atoms, and the substituents are a halogen atom, a carboxyl group, a lower alkoxycarbonyl group having 1 to 4 carbon atoms, and a di-lower alkylamino group having 1 to 4 carbon atoms. R ４ , R ５ , R ６ and R ７ are each independently selected from the group consisting of a hydrogen atom, a halogen atom, a nitro group, a substituted or unsubstituted lower alkyl group of 1 to 4 carbon atoms, a lower alkoxy group of 1 to 4 carbon atoms, a lower alkylcarbonyl group of 1 to 4 carbon atoms, a lower alkoxycarbonyl group of 1 to 4 carbon atoms, a carboxyl group, and a hydroxyl group, and the substituents are a halogen atom, a carboxyl group, or a lower alkoxycarbonyl group of 1 to 4 carbon atoms.

14. The survivin expression promoter according to claim 13, wherein the neuron is a motor neuron.

15. The survivin expression promoter according to claim 13 or 14, wherein the nerve cells are nerve cells induced to differentiate from iPS cells derived from an ALS patient.

16. The survivin expression promoter according to any one of claims 13 to 15, wherein the indolinone compound is semaxanib.

17. The survivin expression promoter according to any one of claims 13 to 16, wherein the iPS cells are capable of inducing transient expression of neurogenin 2.

Images