JP2023182520A - Production method for stage 5 neurons - Google Patents

Abstract

To provide a technique for maturing stage 3 neurons with immature dendrites into stage 5 neurons with dendritic spines and postsynapses.SOLUTION: A production method for stage 5 neurons with mature dendrites includes a step for culturing stage 3 neurons with immature dendrites in a culture medium containing B-27 Plus.SELECTED DRAWING: None

Description

The present invention relates to a method for producing stage 5 neurons. More specifically, the present invention relates to a method for producing stage 5 neurons, a method for screening stage 5 neurons, and therapeutic agents for neurological diseases.

There are high hopes for technology that induces differentiation of human nerve cells (neurons) from pluripotent stem cells for regenerative medicine and drug discovery for neurological diseases. In particular, many methods for inducing the differentiation of induced pluripotent stem cells (iPSCs) into neurons have been established, but there are many unknowns regarding the process and conditions by which these neurons acquire functional maturity.

It is sometimes reported that electrophysiological functions spontaneously appear when iPSC-derived neurons are cultured for a long period of time, ranging from tens to hundreds of days. It is known that the formation of postsynapses (postsynapses) and the excitatory responses mediated by postsynaptic NMDA receptors are incomplete in many cases (see, for example, Non-Patent Document 1).

It is known that excitatory postsynapses in the brain are formed in spines, which are cellular microstructures on the dendrites of neurons, and their morphological and functional maturation is important for cognition, learning, etc. It plays an important role in higher brain functions. For example, the actin-binding protein drebrin is used as a marker for spine formation and dynamics. There are two types of drebrin isoforms: the embryonic isoform E is expressed in young nerves and is present in growth cones, and in mature nerves it is converted to the adult isoform A (brain-type drebrin) by splicing. It gathers in granules (clusters) in dendritic spines.

In addition, changes in the localization of drebrin due to excitatory responses mediated by NMDA receptors are an important mechanism of synaptic plasticity, and the expression of drebrin clusters in vivo or in primary cultured rodent cells is associated with neuroexcitotoxicity and neurodegeneration. It is used as an evaluation marker for synaptic disorders in diseases, etc.

In principle, the formation of spines containing drebrin clusters is expected even in sufficiently mature human iPSC-derived neurons. However, in reality, the existence of spines that can show functional maturity due to changes in the localization of drebrin, etc. has not been reported in human iPSC-derived neurons, and human iPSC-derived neurons that exhibit such functional maturity have not been reported. It has been suggested that it is difficult to obtain (for example, see Non-Patent Document 1).

In particular, for the development of animal alternative methods using human iPSC-derived neurons and drug discovery screening techniques related to synaptic disorders, a highly stable method for producing mature neurons suitable for industrial practical use is required.

Generally, neurons that express some level of neuron-specific markers and that fire electrically are often called mature neurons. However, according to this definition, stage 3 neurons whose dendrites are immature and whose postsynapses are not functioning are also included in mature neurons.

Herein, we distinguish between "stage 3 neurons with immature dendrites" and "stage 5 neurons with mature dendrites."

The differentiation and maturation process of neurons in in vitro culture is strictly divided into the following five stages (Dotti C., et al., The establishment of polarity by hippocampal neurons in culture, J. Neurosci., 8 , 1454-1468, 1988., Arimura N. and Kaibuchi K., Neuronal polarity: From extracellular signals to intracellular mechanisms, Nat. Rev. Neurosci., 8, 194-205, 2007.).

Stage 1: Immediately after seeding, there are no neurites yet, and filopodia are emerging.
Stage 2: A state in which immature protrusions (immature neurites) with a growth cone at the tip begin to elongate. It is not yet possible to distinguish between dendrites and axons.
Stage 3: A state in which nerve polarization occurs and a single immature process grows long and becomes an axon. Other immature processes remain short. At this point, axons express, for example, tau (MAPT) and neurofilament (NF), and immature processes that are about to become dendrites express microtubule-associated protein 2 (MAP2). They can be distinguished by their expression.
Stage 4: After axons have grown to a certain extent, dendrites begin to elongate and branch, becoming mature.
Stage 5: A state in which spines are formed in mature dendrites and neurotransmission via chemical synapses and synaptic plasticity are possible.

FIG. 1 is a schematic diagram showing the differentiation/maturation process of iPSC-derived neurons hypothesized by the inventors. It was hypothesized that the differentiation/maturation process of iPSC-derived neurons follows the five stages described above. Figure 1 also shows changes in the localization of drebrin. In this specification, neurons in stages 1 to 5 are distinguished based on the differentiation/maturation process shown in FIG. 1.

As shown in Figure 1, stage 1 neurons are neural progenitor cells that do not yet have processes. In stage 2 neurons, initiation of process extension is observed. Furthermore, drebrin E (details will be described later) is present around the cell body and within the growth cone at the tip of the extending process. Neural polarization is observed in stage 3 neurons. Dendrites and axons can be distinguished, and neurogenesis has been completed, but both morphology and function are immature. In stage 4 neurons, dendrite maturation (elongation/branching) and synapse formation begin. In stage 5 neurons, dendrites mature, drebrin A (described in detail later) aggregates into granules, and spines and stable postsynaptic structures are formed.

It is known that human iPSC-derived neurons relatively easily reach stage 3, where dendrites are still immature. However, as described above, it is difficult to mature human iPSC-derived neurons to stage 5, where dendritic spines and postsynapses have been formed.

Non-Patent Document 1 discloses a post-synaptic maturation evaluation method in which human iPSCs are differentiated into neural progenitor cells and then induced to differentiate into neurons, and brain-type drebrin is used as an index.

Non-Patent Document 1 states that in order to determine that human iPSC-derived neurons are truly mature, it is necessary not only to evaluate the expression of genetic markers and the presynapse, which are also observed at stages 3 and 4, but also to evaluate the spine. It is stated that it is necessary to confirm post-synapse formation.

However, the neurons described in Non-Patent Document 1 express very few brain-type drebrin clusters (4 to 5 per cell), and do not express clusters of postsynaptic density protein 95 (PSD95), which is a postsynaptic marker. Its existence was not recognized, and electrophysiological evaluation using a multipoint electrode array did not show any synaptic transmission activity. As described above, Non-Patent Document 1 does not succeed in producing stage 5 neurons having structurally and functionally mature dendrites.

An object of the present invention is to provide a technique for maturing stage 3 neurons having immature dendrites into stage 5 neurons in which dendritic spines and postsynapses have been formed.

The method for producing stage 5 neurons with mature dendrites according to the present invention includes the step of culturing stage 3 neurons with immature dendrites in a medium containing B-27 Plus.

According to the present invention, it is possible to provide a technique for maturing stage 3 neurons having immature dendrites into stage 5 neurons in which dendritic spines and postsynapses have been formed.

FIG. 1 is a schematic diagram showing the differentiation/maturation process of iPSC-derived neurons. FIG. 2 is a phase contrast microscope image showing the results of Experimental Example 1. FIG. 3 is a representative fluorescence microscope image showing the results of immunostaining in Experimental Example 1. FIG. 4 is a graph showing the results of principal component analysis in Experimental Example 2. FIG. 5 is a graph showing the average vector of each cell type created in Experimental Example 2. FIG. 6 is a heat map showing the results of analyzing the correlation with the developmental process of the human brain in Example 2. FIG. 7 is a heat map of the RNA-Seq expression profile in Experimental Example 3. FIG. 8 is a graph showing the results of classifying gene expression profiles into six patterns in Experimental Example 4. FIG. 9A is a graph showing the top 20 significantly enriched gene ontology terms in cluster DOWN1 in Experimental Example 4. FIG. 9B is a graph showing the top 20 significantly enriched gene ontology terms in cluster DOWN2 in Experimental Example 4. FIG. 9C is a graph showing the top 20 significantly enriched gene ontology terms in cluster DOWN3 in Experimental Example 4. FIG. 10A is a graph showing the top 20 significantly enriched gene ontology terms in cluster UP1 in Experimental Example 4. FIG. 10B is a graph showing the top 20 significantly enriched gene ontology terms in cluster UP2 in Experimental Example 4. FIG. 10C is a graph showing the top 20 significantly enriched gene ontology terms in cluster UP3 in Experimental Example 4. FIG. 11 is a representative fluorescence microscope image showing the results of immunostaining in Experimental Example 5. FIG. 12 is a representative fluorescence microscope image showing the results of immunostaining in Experimental Example 6. FIG. 13 is a representative fluorescence microscopy image showing the results of immunostaining in Example 7. FIG. 14 is a diagram comparing the base sequences of two drebrin isoforms identified by mapping RNA-Seq data to the DBN1 locus of the human reference genome in Experimental Example 8. FIG. 15 is a graph showing the expression levels of drebrin isoforms detected by analysis of RNA-Seq data in Experimental Example 8. FIG. 16 is a graph showing the results of quantitative real-time RT-PCR analysis of the expression levels of drebrin (total) and drebrin isoform A in Experimental Example 8. FIG. 17 is a graph showing the results of measuring the relative expression levels of GRIN1, GRIN2A, and GRIN2B, which are subunits of the NMDA receptor, by quantitative real-time RT-PCR in Experimental Example 9. FIG. 18 is a graph showing the results of measuring the relative expression levels of tau (total), 3R tau isoform, 4R tau isoform, and drebrin isoform A by quantitative real-time RT-PCR in Experimental Example 10. be. FIG. 19 is a graph showing the number of electrical activity detection electrodes of iPSC-derived neurons and the synchronized network burst frequency measured in Experimental Example 11. FIG. 20 is a graph showing a typical collective firing frequency histogram measured in Experimental Example 11. FIG. 21 is a dose-response curve of 4-AP and D-AP5 created based on FIG. 20. FIG. 22 is a raster plot of firing frequency and a typical collective firing frequency histogram showing network bursts on Day 63 and Day 84 measured in Example 11. FIG. 23 is a graph showing a typical nerve transmission map in which the action potential propagation path was reconstructed on the electrode surface by the axon tracing assay on Day 63 and Day 84 in Example 11. FIG. 24 is a representative fluorescence microscope image showing the results of immunostaining in Experimental Example 12. FIG. 25 is a representative fluorescence microscope image showing the results of immunostaining in Experimental Example 13. FIG. 26 is a representative fluorescence microscope image showing the results of immunostaining in Experimental Example 14. FIG. 27 is a representative fluorescence microscope image showing the results of immunostaining in Experimental Example 15.

[Method for manufacturing stage 5 neurons]
In one embodiment, the present invention provides a method for producing stage 5 neurons with mature dendrites, comprising culturing stage 3 neurons with mature dendrites in a medium containing B-27 Plus. .

As described later in Examples, according to the manufacturing method of this embodiment, stage 3 neurons with immature dendrites can be matured into stage 5 neurons with dendritic spines and postsynapses formed. Can be done. Therefore, the manufacturing method of this embodiment can also be said to be a method for inducing maturation of stage 3 neurons into stage 5 neurons.

B-27 Plus is a supplement used for neuronal cell culture, and is added to the basic medium. B-27 Plus is sold by Thermo Fisher Scientific. The main components of B-27 Plus are shown in Table 1 below.

In the production method of this embodiment, a basic medium supplemented with B-27 Plus can be used as the medium.

The basic medium is not particularly limited, and any serum-free basic medium for cell culture can be used. For example, a synthetic medium buffered to a pH of 7.2 or more and 7.6 or less can be used. More specifically, for example, NeuroBasal medium (Thermo Fisher Scientific), NeuroBasal Plus medium (Thermo Fisher Scientific), BrainPhys medium (Stem Cell Technologies), neuron medium containing rat glial cell culture supernatant ( Fujifilm Wako Pure Chemical Industries, Ltd.), Advanced Dulbecco's Modified Eagle Medium/Ham's F-12 Mixed Medium (DMEM/F12), RPMI1640 Medium, Advanced RPMI Medium, and the like.

B-27 Plus is preferably added to the basal medium in an amount of, for example, 0.1% by volume or more, 0.5% by volume or more, 1% by volume or more, or 1.5% by volume or more. The amount of B-27 Plus added to the basic medium may be, for example, 10% by volume or less, 8% by volume or less, 6% by volume or less, 4% by volume or less, 3% by volume or less, or 2% by volume or less. These upper and lower limits can be arbitrarily combined. For example, B-27 Plus may be added to the basal medium at 0.1% to 10% by volume, 0.5% to 5% by volume, and 1% to 3% by volume. % or less, or 2% by volume.

Additives other than B-27 Plus may be added to the medium. Such additives include, for example, CultureOne (Thermo Fisher Scientific), GlutaMAX (Thermo Fisher Scientific), ascorbic acid, antibiotics, N-2 supplements, and cell growth agents such as DAPT and Ara-C. Examples include inhibitors, growth factors such as BDNF, GDNF, and IGF-1.

As described later in the Examples, stage 5 neurons are produced by culturing stage 3 neurons in a medium containing B-27 Plus for, for example, 50 days or more, 60 days or more, 70 days or more, or 80 days or more. can do. Although there is no upper limit to the culture time, it is realistic to set it to about 12 months or less, 10 months or less, 8 months or less, or 6 months or less, for example.

In the production method of this embodiment, stage 3 neurons preferably express neural markers including MAP2 and neurofilament heavy chain (NF-H) and are polarized. Furthermore, it is preferable that the stage 3 neurons are obtained by inducing differentiation of pluripotent stem cells.

As described later in Examples, the production method of this embodiment uses a transcription factor induction method that allows stage 3 neurons to be produced with high efficiency directly from pluripotent stem cells without going through neurospheres. It is particularly effective for That is, stage 3 neurons are preferably those induced to differentiate by inducing the expression of transcription factors in pluripotent stem cells.

The expression of transcription factors in pluripotent stem cells can be induced by transfecting the transcription factor mRNA into the cells, by using RNA viruses such as Sendai virus vectors, or by using lentivirus vectors etc. A gene construct whose expression is induced using a DNA virus may be introduced, or a gene construct whose expression is induced using genetic modification such as genome editing may be introduced. In particular, mRNA transfection or expression induction using an RNA virus is preferred, since there is no risk of a foreign gene being integrated into the genome and remaining over a long period of time.

Examples of pluripotent stem cells include embryonic stem cells (ESC), induced pluripotent stem cells (iPSC), and the like. Preferably, the pluripotent stem cells are cells of mammalian origin. Examples of mammals include rodents such as mice, rats, hamsters, and guinea pigs; ungulates such as pigs, cows, goats, horses, and sheep; carnivores such as dogs and cats; humans, rhesus monkeys, cynomolgus monkeys, and marmosets. , orangutans, chimpanzees, and other primates.

The pluripotent stem cells are preferably human cells. Furthermore, the pluripotent stem cells may be wild-type cells, cells derived from a neurological disease patient, or genetically modified wild-type cells.

In the manufacturing method of this embodiment, dendritic spines are formed in stage 5 neurons. Moreover, it is preferable that a post-synaptic structure containing drebrin and PSD95 is formed in the stage 5 neuron.

[Stage 5 Neuron]
In one embodiment, the invention provides human iPS cell-derived stage 5 neurons with mature dendrites that are isolated and cultured in a container. "Isolated and cultured" means that it is not, for example, a mature neuron present in a tissue section.

To date, there has been no report of successful production of functional stage 5 neurons having dendritic spines and postsynapses in vitro in human iPSC-derived cells.

On the other hand, as described later in Examples, the inventors succeeded for the first time in vitro in producing stage 5 neurons derived from human iPSCs in which dendritic spines and postsynapses were formed.

Stage 5 neurons of this embodiment can be produced in vitro from stage 3 neurons by the method described above. Therefore, the stage 5 neurons of this embodiment are isolated and cultured in a container.

The container is not particularly limited, and containers commonly used for cell culture can be used. More specifically, examples include dishes, multiwell plates, glass slides, planar microelectrode arrays (MEA), and the like.

The stage 5 neurons of this embodiment are preferably cultured in a plane (2D culture) or in a spheroid culture (3D culture). Furthermore, it is preferable that the stage 5 neurons of this embodiment are composed of only nerve cells and do not contain other cells.

That is, the stage 5 neurons of this embodiment are distinguished from brain organoids that contain a plurality of cells and are cultured in 3D.

[Screening method for therapeutic drugs for neurological diseases]
In one embodiment, the present invention provides drug screening comprising the step of evaluating the function or phenotype of stage 5 neurons induced to differentiate from iPSCs derived from neurological disease patients in the presence and/or absence of a test substance. provide a method. Stage 5 neurons can typically be obtained by the methods described above. Evaluation of functions or expression systems in the presence and/or absence of a test substance includes, for example, exposure to the test substance during the maturation stage from stage 3 neurons to stage 5 neurons, or after maturation to stage 5 neurons. The function or phenotype is evaluated by comparing the case with exposure and the case without exposure.

The test substance is not particularly limited, and for example, a natural compound library, a synthetic compound library, an existing drug library, etc. can be used.

The function or phenotype of stage 5 neurons is not particularly limited, and includes the degree of dendritic maturity, electrophysiological properties, and the like. Examples of the degree of maturity of dendrites include the shape of dendritic spines, the presence or absence of formation of postsynaptic structures, the shape of postsynaptic structures, the localization of drebrin, and the localization of PSD95. Electrophysiological properties include, for example, the number of electrodes detecting electrical activity, spontaneous firing, synchronized network burst frequency, electrically dependent calcium influx, and the effects of drugs on these. The drugs include potassium channel inhibitors, N-methyl-D-aspartate (NMDA) receptor inhibitors, α-amino-3-hydroxy-5-methoxazole-4-propionic acid (AMPA) receptor inhibitors, Examples include agonists and antagonists of various neurotransmitters (eg, glutamic acid, etc.).

As iPSCs derived from neurological disease patients, iPSCs produced from cells derived from neurological disease patients, iPSCs into which the same genetic mutations as those of neurological disease patients have been introduced, etc. can be used.

If the function or phenotype of stage 5 neurons in the presence of the test substance is closer to the wild type than in the absence of the test substance, the test substance is considered a candidate for a therapeutic agent for neurological diseases. It can be determined that Conversely, if the function or phenotype of stage 5 neurons in the presence of the test substance is more characteristic of the disease than in the absence of the test substance, then the test substance is a causative agent of the neurological disease. Alternatively, it can be determined that the substance is a candidate for a substance that promotes a disease.

(First embodiment)
In the screening method of the first embodiment, exposure to the test substance is carried out at the stage of maturation from stage 3 neurons to stage 5 neurons. When a therapeutic drug candidate for a neurological disease is obtained by the screening method of this embodiment, the therapeutic drug can be administered before stage 3 neurons mature into stage 5 neurons, thereby causing the expression of the neurological disease phenotype. It is hoped that this can be prevented. That is, the candidate compound obtained by the screening method of this embodiment may be used as a preventive drug for neurological diseases.

(Second embodiment)
In the screening method of the second embodiment, exposure to the test substance is performed after maturation into stage 5 neurons. Therefore, the screening method of the second embodiment differs from the screening method of the first embodiment mainly in the timing at which the test substance is applied.

In the screening method of the first embodiment, the test substance is applied in the process of obtaining stage 5 neurons from stage 3 neurons, whereas in the screening method of the second embodiment, the test substance is applied after obtaining stage 5 neurons. let

In the screening method of the second embodiment, the test substance, the phenotype of stage 5 neurons, iPSCs derived from neurological disease patients, etc. are the same as the screening method of the first embodiment.

In the screening method of the second embodiment, the predetermined period for culturing stage 5 neurons in the presence of the test substance is not particularly limited, and may be, for example, 1 minute or more, 3 minutes or more, 5 minutes or more, 10 minutes or more, 30 minutes or more. The duration may be longer than one minute, longer than 1 hour, longer than 1 day, longer than 3 days, longer than 1 week, longer than 2 weeks, longer than 1 month, longer than 3 months, etc. The predetermined period for culturing stage 5 neurons in the presence of the test substance is, for example, 3 months or less, 1 month or less, 1 week or less, 3 days or less, 1 day or less, 12 hours or less, 6 hours or less, 1 hour. Hereinafter, the duration may be 30 minutes or less, 10 minutes or less, etc. These upper and lower limits can be arbitrarily combined. The predetermined period for culturing stage 5 neurons in the presence of the test substance may be, for example, 1 minute, 3 minutes, 5 minutes, or 10 minutes. It may be for 30 minutes, it may be for 1 hour, it may be for 6 hours, it may be for 12 hours, it may be for about 1 day, It may be about one week or about one month.

If a candidate for a therapeutic drug for a neurological disease is obtained by the screening method of the second embodiment, the therapeutic drug can be administered to stage 5 neurons that have already exhibited a phenotype of a neurological disease, thereby changing the phenotype to a more normal one. There is hope that it can be treated.

[Experiment example 1]
(Examination of neurons induced to differentiate from iPSCs by inducing expression of transcription factors)
Human iPSC-derived neurons (product name: "Quick-Neuron ^™ Excitatory-Human iPSC-derived Neurons", Elixagen Scientific) were cultured, and their shapes were observed. These neurons were obtained by introducing mRNA of a transcription factor that promotes induction of differentiation into neurons into iPSCs, and cryopreserved 3 days later.

Human iPSC-derived neurons were thawed, suspended in the manufacturer's recommended medium, and placed in a 96-well culture plate previously coated with 0.05% polyethyleneimine (PEI, Sigma) and 20 μg/mL laminin solution (Sigma). Cells were seeded at a density of 50,000 cells/ ^cm2 .

The day when transcription factors were introduced into iPSCs and differentiation induction was started was defined as Day 0. Since the cells were frozen and preserved on Day 3, the day after thawing and seeding was designated as Day 4. Half of the medium was replaced every 3 to 4 days starting from Day 4. On any day from Day 4 to Day 10, add 2% B-27 Plus (Thermo Fisher Scientific) to Neurobasal Plus basal medium (Thermo Fisher Scientific), and neuronal medium (Fujifilm Wako Pure Chemical Industries, Ltd.). company), 1% CultureOne (Thermo Fisher Scientific), 1% GlutaMAX (Thermo Fisher Scientific), 0.1% 200mM ascorbic acid, and 1% antibiotic (Penicillin Streptomycin). I replaced half of it.

FIG. 2 is phase-contrast microscopic images of cells on day 1 (Day 4) and Day 8 after seeding. Scale bar is 100 μm. As a result, it was confirmed that protrusions began to grow on Day 4, and long axons were formed on Day 8.

On Days 10, 37, and 71, cells were fixed with paraformaldehyde, and the expression of MAP2, a dendrite marker, and neurofilament heavy chain (NF-H), an axon marker, was examined by immunostaining. In addition, the nucleus was stained with DAPI.

Figure 3 is a representative fluorescence microscopy image showing the results of immunostaining. Scale bar is 100 μm. The results showed that on Day 10, the early stage of culture, the cell body expressed MAP2, but dendrites were hardly developed at this stage. In addition, it was observed that each cell extended one long process, and an NF-H-positive axon was formed in the extension. From the above results, it was suggested that at Day 10, the neuron was in a stage 3 neuron state with neural polarity formed. On Day 37, the late stage of culture, it was shown that neurofilament heavy chain (NF-H) positive axons were developing and MAP2 positive dendrites were elongating and branching. On Day 71, more MAP2-positive dendrites were shown to be elongating and branching. The above results suggested that at the time of Day 71, the neuron had reached a stage state later than that of the stage 3 neuron on Day 10.

[Experiment example 2]
(RNA-seq analysis 1)
Human iPSC-derived neurons (product name: "Quick-Neuron ^TM Excitatory-Human iPSC-derived Neurons", Elixagen Scientific) were cultured in the same manner as in Experimental Example 1, and RNA-Seq analysis was performed. However, in order to easily handle a large amount of cells, they were seeded in a non-adhesive U-bottom 96-well culture plate at a density of 10,000 cells/well to form spheroids.

RNA-Seq analysis was performed on Day 0, Day 10, Day 32, Day 57, and Day 88. For Day 0, which is a control, two independently cultured undifferentiated iPSCs (iPSC-1 and iPSC-2) were analyzed.

FIG. 4 is a graph showing the results of principal component analysis. The results showed two types of trajectories: rapid differentiation from immature iPS cells and maturation requiring long-term culture.

The direction of differentiation into each cell type was estimated by plotting the average vector of factor loadings of cell type-specific marker genes. Cell type-specific marker gene sets were obtained from Polioudakis D., et al., A Single-Cell Transcriptomic Atlas of Human Neocortical Development during Mid-gestation, Neuron 103, 785-801, 2019. For a given cell type, the average of the corresponding factor loadings was defined as the direction of differentiation V (PC1, PC2), calculated based on the following formula (1).

［式（１）中、Ｖは分化の方向のベクトルを示し、ＧはＰＣ１／ＰＣ２因子負荷ベクトルを示し、ｉは細胞型のマーカー遺伝子を示し、ｎは細胞型のマーカー遺伝子の総数を示す。］

[In formula (1), V indicates a vector in the direction of differentiation, G indicates a PC1/PC2 factor loading vector, i indicates a cell type marker gene, and n indicates the total number of cell type marker genes. ]

FIG. 5 is a graph showing the average vector for each cell type. In Figure 5, "Pg" indicates a proliferating immature cell, "IP" indicates an intermediate progenitor cell, "ExN" indicates a migrating excitatory neuron, and "ExM" indicates an excitatory neuron in the middle of maturation. "ExM-U" indicates supracortical condensed excitatory neurons, "ExDp1" indicates deep cortical layer 1, "ExDp2" indicates deep cortical layer 2, "RG" indicates radial glia, and "Mic" indicates microglia and “OPC” indicates oligodendrocyte precursor cells.

As a result, cells at Day 0 are close to proliferating immature cells (Cycling Progenitors), at Day 10 they are in an intermediate state between intermediate progenitor cells and migrating excitatory neurons/excitatory neurons in the process of maturing, and at Day 32 It was shown that the state was close to that of the upper cortical layer condensed excitatory neurons, and on Days 57 to 88, it was close to that of mature excitatory deep layer neurons.

This result indicates that although cell cycle termination and neurogenesis are completed on Day 10, neurons change to a more mature state over time. We also show that the state changes further stepwise after Day 32, including switching of drebrin isoforms and switching of NMDA receptor subunits, which are considered to be characteristic molecular events of spine formation and postnatal brain development, which will be described later.

Similar experiments were performed using planar cultured human iPSC-derived neurons. The results showed that even when human iPSC-derived neurons were cultured in a plane, they followed the same trajectory as in spheroid culture and reached the lower right position on Day 88. These results showed that neurons mature equally in both spheroid culture and flat culture.

Next, using the RNA-Seq data obtained in this experimental example, the correlation with Brainspan data was determined on the time axis. Brainspan is a public database that compiles comprehensive analyzes of gene expression during the development and development of the brain from the human fetus to postnatal birth (http://www.brainspan.org, Li M., et al., Integrative functional genomic analysis of human brain development and neuropsychiatric risks, Science 362, 139-148, 2018). FIG. 6 is a heat map showing the Pearson correlation coefficient between the global gene expression of human iPSC-derived neurons from Days 10 to 88 and the global gene expression of the human prefrontal cortex at each time period. As a result, it was found that up to Day 32, there is a high correlation with the prenatal brain condition (8 to 24 weeks after conception), and from Day 57 onward, there is a high correlation with the postnatal brain condition (after birth). 4 months to 40 years). On Day 88, the correlation with the prenatal period further decreased, and it was found that the correlation with the prenatal period was transferred to the cranial nerves after birth.

Therefore, cells up to Day 32 contain many stage 3 neurons in an immature state similar to that of the fetal brain, transition to more mature stage 5 neurons between Day 32 and Day 57, and by Day 88 they are in a state similar to cranial nerves after birth. It was suggested that many stage 5 neurons were included. This transferability of gene expression in human iPSC-derived neurons from the fetal brain to the postnatal brain state has so far been reported only in brain organoids cultured in long-term 3D for more than 250 days (Gordon A., et al., Long-term maturation of human cortical organoids matches key early postnatal transitions, Nat. Neurosci. 24, 331-342, 2021). However, there have been no reports of successful cases in which most of the cells transition to the postnatal brain state in a relatively short period of time (57 to 88 days) using flat cultures or simple spheroid cultures composed only of neurons. , it has been suggested that it is difficult. In addition, brain organoids have complex culture conditions, contain multiple types of cells, and aggregate cells, making it impossible to visualize and quantify spines and postsynaptic structures, which will be described later. Therefore, a method for producing mature nerves that is more suitable for practical use is required.

[Experiment example 3]
(RNA-seq analysis 2)
Based on the RNA-Seq data obtained in Experimental Example 2, the expression profile of known neuron-specific markers was examined.

Figure 7 is a heat map of RNA-Seq expression profiles. Expression of major undifferentiated and neuron-specific markers is shown. The results confirmed time-dependent regulation of the transcriptional program. Specifically, stem cell markers and proliferation markers were downregulated on Day 10, suggesting that most cells were efficiently differentiated.

Day 10 neurons expressed neural progenitor cell markers (PAX6, NES, etc.) and several neuronal markers (NEUROD1, CDH2, TUBB3, MAP2, MAPT, etc.). This result indicated that they were at an immature neuron stage.

Afterwards, the expression of most neural progenitor cell markers and immature neuron markers decreased. In contrast, increases in mature neuron markers (NEFH, BDNF), presynaptic markers (SYN1, SYP, SNAP25), and postsynaptic markers (DLG4, CAMK2) were observed after Day 32.

In addition, upregulation of glutamate vesicle transporters (SLC17A6, SLC17A7) and AMPA receptors (GRIA1-4), followed by upregulation of NMDA receptors (GRIN1, -2A, -2B) was observed, resulting in excitatory transmission. The presence of possible glutamatergic neurons was demonstrated.

[Experiment example 4]
(RNA-seq analysis 3)
Based on the RNA-Seq data obtained in Experimental Example 2, genes were grouped by hierarchical clustering analysis of expression profiles, and gene ontology (GO) enrichment analysis was performed.

FIG. 8 is a graph showing the results of classifying gene expression profiles into six patterns based on the average expression level of each gene on Day0, Day10, Day32, Day57, and Day88.

FIGS. 9A, 9B, and 9C are graphs showing the top 20 significantly enriched gene ontology terms in clusters DOWN1, DOWN2, and DOWN3, respectively.

FIGS. 10A, 10B, and 10C are graphs showing the top 20 significantly enriched gene ontology terms in clusters UP1, UP2, and UP3, respectively.

The results showed that genes that were immediately upregulated on Day 10 (cluster UP1) were mainly associated with axon formation, neuronal projection, and presynaptic vesicle formation and transport, indicating active neuronal development. .

In contrast, genes strongly upregulated after Day 32 (cluster UP2) are associated with functions that require more mature postsynaptic structures (learning or memory, plasticity, long-term potentiation), transmembrane ion transport, and regulation of synaptic function. was involved. This suggested increased synapse formation and neurotransmission activity at later time points.

Cluster UP3 is rich in purine metabolism, oxidative phosphorylation, and respiratory functions, which may be due to meeting the high demand for ATP synthesis in neurogenesis and neurotransmission.

Downregulated clusters were mostly enriched in RNA processing and splicing functions, reflecting the dramatic translational changes required to achieve complex cell differentiation. Cluster DOWN2 contained genes related to nuclear division and chromosome/chromatid segregation, possibly related to cell cycle exit in differentiated neurons.

The above RNA-Seq analysis results suggested that following the initial induction of differentiation, specific long-term transcriptional programs regulate neuronal maturation and synapse formation.

[Experiment example 5]
(Immunostaining study 1)
Human iPSC-derived neurons (product name: "Quick-Neuron ^TM Excitatory-Human iPSC-derived Neurons", Elixagen Scientific) cultured in the same manner as in Experimental Example 1 were immunostained to determine neurite growth and synaptic structure. The formation of was observed.

FIG. 11 is a fluorescence microscopy image showing typical results of immunostaining of each neuron on Day 37 and Day 71. The lower rows each show an enlarged image of the area surrounded by a square in the image of Day 71 in the middle row. The upper scale bar is 100 μm, and the lower scale bar is 20 μm. In Figure 11, the staining results for neurofilament heavy chain (NF-H), an axon marker, and synapsin I, a presynaptic marker, are shown on the left, and the staining results for dendritic marker MAP2 and postsynaptic marker are shown on the left. The staining results for a certain drebrin are shown in the center, and a schematic diagram for interpreting the state of each neuron is shown on the right.

iPSC-derived neurons developed short MAP2-positive dendrites and rapidly elongating neurofilament heavy chain (NF-H)-positive axons one month after culture initiation. Similar to Example 1, MAP2-positive dendrites continued to grow and branch over time.

Concomitantly, expression of the presynaptic marker synapsin I was increased. Synapsin I diffused around axons and cell bodies in early cultures, and aggregated along mature dendrites in late cultures on Day 71. This indicates that synapsin I becomes concentrated at synaptic sites as neuron development progresses from stage 4 onwards.

Next, we evaluated the intracellular localization of drebrin, an actin-binding protein. During early culture from the start of culture to Day 37, drebrin was distributed around the cell body and in the growth cone at the end of the developing dendrite. On Day 71, drebrin formed clusters along mature MAP2-positive dendrites.

[Experiment example 6]
(Study by immunostaining 2)
Using a neuron differentiation kit (product name "Quick-Neuron ^TM Excitatory-SeV Kit", Elixagen Scientific), stage 3 neurons were produced from iPSCs derived from healthy individuals according to the manufacturer's protocol. The 1231A3 strain was used as the iPSC derived from a healthy individual. By using a different strain from Experimental Example 1, we confirmed the reproducibility of maturation and obtained higher resolution immunostaining images.

FIG. 12 is a fluorescence microscopy image showing representative results of immunostaining of iPSC-derived neurons on Day 73. The lower rows are enlarged images of the areas surrounded by squares in the upper row. The upper scale bar is 100 μm, and the lower scale bar is 10 μm.

As a result, expression of Postsynaptic density protein 95 (PSD95) was also observed. It was also shown that the presynaptic marker synapsin I and the postsynaptic markers drebrin and PSD95 form clusters on or near dendrites.

In addition, in the center diagram of Figure 12, clusters of synapsin I and drebrin are adjacent or partially colocalized (white arrows), and in the dendritic spine, presynapses and postsynapses are connected to form synaptic connections. It was shown that it was established.

The above results indicate that iPSC-derived neurons matured into stage 5 neurons with mature dendrites. That is, it was shown that stage 5 neurons can be produced by culturing stage 3 neurons in a medium containing B-27 Plus.

[Experiment Example 7]
(Immunostaining study 3)
Human iPSC-derived neurons (product name: "Quick-Neuron ^TM Excitatory-Human iPSC-derived Neurons", Elixagen Scientific, Inc.), cultured in the same manner as in Experimental Example 1, were immunostained and subjected to immunostaining for known nerve-specific markers. Expression was observed. In addition, the nucleus was stained with DAPI.

FIG. 13 is a fluorescence microscopy image showing representative results of immunostaining of iPSC-derived neurons. Scale bar is 100 μm. As a result, it was confirmed that most (more than 95%) of the cells expressed vGLUT1, a glutamatergic nerve marker, at Day 28, and NeuN, a mature nerve marker, at Day 85. Furthermore, it was confirmed that some cells (5% or less) expressed GAD64, a GABAergic nerve marker, and TH, a dopaminergic nerve marker, on Day 85.

[Experiment example 8]
(Study of drebrin isoforms)
Changes in the subcellular localization of drebrin during neuronal development are known to be caused by alternative splicing of drebrin. The embryonic isoform drebrin E plays a role in immature neuron migration and neurite outgrowth, and is a mature brain-specific regulator that regulates dendritic spine formation and plasticity as neurons mature. It replaces the body isoform drebrin A.

FIG. 14 is a diagram comparing the base sequences of two drebrin isoforms identified by mapping RNA-Seq data to the DBN1 locus of the human reference genome.

The transcript containing additional exon 11 was assumed to be the mature brain-specific adult isoform A, and the shorter transcript was assumed to be the embryonic isoform E.

Based on the RNA-Seq data obtained in Experimental Example 2, the expression levels of drebrin isoforms were analyzed. FIG. 15 is a graph showing the expression level (transcripts per million, TPM) of all drebrin transcripts and each isoform detected by analysis of RNA-Seq data.

The results showed that drebrin (total) and embryonic isoform E are expressed in undifferentiated iPSCs, upregulated upon induction of differentiation, and downregulated again at later time points. It was shown that adult isoform A is almost absent in the early stages of differentiation, and its expression is induced in the later stages.

FIG. 16 is a graph showing the results of quantitative real-time RT-PCR analysis of the expression levels of drebrin (total) and drebrin isoform A in human iPSC-derived neurons cultured in the same manner as in Experimental Example 1. In Figure 16, "DBN1" indicates the expression level of drebrin (total), "DBN1 isoform A" indicates the expression level of drebrin isoform A, and "*" indicates p<0.05. Indicates that there is a significant difference, and "**" indicates that there is a significant difference at p<0.001. As a result, it was confirmed that isoform A was particularly upregulated after Day 57.

These results indicate that the maturation of iPSC-derived neurons appears to recapitulate the molecular event of drebrin isoform switching previously known in rodent hippocampal neurons.

[Experiment example 9]
(Study of NMDA receptor subunits)
It is known that the properties of the NMDA receptor change during maturation, with a developmental switch from the subunit GluN2B (GRIN2B) to GluN2A (GRIN2A).

FIG. 17 shows the relative expression levels of GRIN1, GRIN2A, and GRIN2B, which are subunits of the NMDA receptor, measured by quantitative real-time RT-PCR for human iPSC-derived neurons cultured in the same manner as in Experimental Example 1. It is a graph showing the results. In Figure 17, "***" indicates a significant difference with p<0.001 from the Day 10 result, and "###" indicates a significant difference with p<0.001 from the Day 57 result of GRIN2B. Indicates that there is a significant difference.

The results showed that GRIN1 and GRIN2B were significantly up-regulated on Day 32, and GRIN2A was significantly up-regulated on Day 57. Furthermore, GRIN2B was again significantly downregulated on Day 88, suggesting induction of a developmental switch from GluN2B to GluN2A.

[Experiment example 10]
(Study of tau isoforms)
Tau (gene name: MAPT), which stabilizes microtubules in nerve cells, expresses six isoforms through alternative splicing, three of which have three repeats (3R tau isoform) and three others. It is known that species can be distinguished by having four repeat sequences (4R tau isoforms). It is known that only 3R tau is expressed during the embryonic period, but the number of isoforms expressed increases with maturation, and 4R tau is expressed in the brain of living organisms.

Figure 18 shows the relative expression levels of tau (total), 3R tau, 4R tau, and drebrin isoform A measured by quantitative real-time RT-PCR on human iPSC-derived neurons cultured in the same manner as in Experimental Example 1. This is a graph showing the results. The results showed that tau (total) and 3R tau were highly expressed from Day 38, whereas 4R tau was upregulated on Day 66 and Day 87, similar to drebrin isoform A.

[Experiment example 11]
(Study of functional activity of excitatory postsynaptic proteins 1)
Electrophysiological responses of human iPSC-derived neurons were analyzed using a high-density microelectrode array system (HD-MEA, Maxwell Biosystems).

Human iPSC-derived neurons (product name: "Quick-Neuron ^TM Excitatory-Human iPSC-derived Neurons", Elixagen Scientific) 100,000 cells/well on the electrodes of a high-density microelectrode array (HD-MEA) Culture was carried out in the same manner as in Experimental Example 1 except that the cells were seeded at different densities, and the local electric field potential was measured over time.

FIG. 19 is a graph showing the number of active electrodes and synchronized network burst rate measured over time in four independent wells of cultured iPSC-derived neurons. The number of electrically active detection electrodes is proportional to the presence of cells that spontaneously fire, and is an indicator of firing activity. Synchronized network bursts are indicative of the establishment of connections between cultured neurons.

The results showed that after Day 31, firing activity increased and reproducible network bursts appeared. Network burst was measured stably until about 8 weeks after the start of culture, and then decreased again with maturation. Since no decrease in firing activity was observed, this does not indicate a decrease in the electrical activity or survival rate of individual cells, but rather due to maturation of inhibitory neurons or electrical synapses via gap junctions that do not require postsynaptic function. This was thought to be due to the replacement of chemical synapses that allow for more selective control.

4-aminopyridine (4-AP), a potassium channel inhibitor, and D-2-Amino-5-phosphonopentanoic acid (D-AP5), an NMDA receptor inhibitor, were added to the medium of iPSC-derived neurons 8 weeks after the start of culture. ) were added in increasing concentrations, and the population firing frequency histogram was recorded with 1,024 electrodes per well. Each concentration was stabilized for 1 minute and recorded for 10 minutes. Finally, iPSC-derived neurons were washed with medium exchange three times and left for one day to confirm that the effects of each drug had disappeared.

FIG. 20 is a graph showing a typical collective firing frequency histogram. FIG. 21 is a dose-response curve of 4-AP and D-AP5 created based on FIG. 20. As a result, a dose-dependent response of iPSC-derived neurons to 4-AP and D-AP5 was observed. This result indicates that neurotransmission in iPSC-derived neurons is functional. It was suggested that synaptic transmission via NMDA receptors, which is particularly important in the mature central nervous system, is functioning.

FIG. 22 is a representative collective firing frequency histogram showing a raster plot of firing frequency and network bursts measured for 600 seconds using 1,024 electrodes on Day 63 and Day 84. It was shown that the frequency of network bursts decreased on Day 84 compared to Day 63, and at the same time, the burst duration, average firing frequency within a burst, and burst interval increased.

FIG. 23 is a graph showing a typical nerve transmission map in which action potential propagation paths were reconstructed in a 4 mm x 2 mm area including 26,400 electrodes by performing axon tracing assays on Day 63 and Day 84. . Scale bar is 100 μm. The results showed that the propagation distance of action potentials and the complexity of the network increased on Day 84 compared to Day 63. These results suggest that mature stage 5 neurons exhibit increased neural network connectivity and stability without a decline in electrical activity.

[Experiment example 12]
(Study of functional activity of excitatory postsynaptic proteins 2)
Mature stage 5 neurons derived from human iPSCs were treated with 100 μM glutamic acid for 10 minutes and then immunostained to examine changes in the localization of drebrin. For comparison, a sample immunostained without glutamic acid treatment was also prepared.

FIG. 24 is a fluorescence microscopy image showing representative immunostaining results. In FIG. 24, the left column shows the results of immunostaining without glutamic acid treatment, and the right column shows the results of immunostaining after treatment with 100 μM glutamic acid for 10 minutes.

In FIG. 24, the upper left row is an image obtained by merging the staining results of MAP2, drebrin, and nucleus, and the upper and lower left rows are enlarged images of the area surrounded by the square in the upper left row. The scale bar in the upper left row is 100 μm, and the scale bar in the upper left row is 20 μm.

The lower left upper row is an image obtained by merging the staining results of MAP2, drebrin, and nucleus for another cell, and the lower left row is an enlarged image of the area surrounded by the square in the lower left upper row. The scale bar in the lower left upper row is 100 μm, and the scale bar in the lower left row is 20 μm.

The upper right row is an image obtained by merging the staining results of MAP2, drebrin, and nucleus, and the upper right row and upper right row are enlarged images of the area surrounded by the square in the upper right row. The scale bar in the upper right row is 100 μm, and the scale bar in the upper right row is 20 μm.

The lower right upper row is an image obtained by merging the staining results of MAP2, drebrin, and nucleus for another cell, and the lower right lower row is an enlarged image of the area surrounded by the square in the lower right upper row. The scale bar in the lower right upper row is 100 μm, and the scale bar in the lower right row is 20 μm.

As a result, when mature stage 5 neurons derived from human iPSCs were treated with 100 μM glutamate for 10 minutes, drebrin flowed out from the clusters within the spine and free drebrin moved to the shaft of the dendrite (drebrin exodus). A phenomenon called.

In rodents, it is known that when glutamate is added at a level that does not kill neurons, "drebrin exodus" is observed and "synaptic toxicity" is induced. This is the first time synaptic toxicity has been confirmed in human neurons. Furthermore, this phenomenon is known to be an important mechanism of synaptic plasticity mediated by NMDA receptors, and it has been suggested that synaptic plasticity may also occur in human iPSC-derived neurons.

This result indicates that human iPSC-derived stage 5 neurons have normally functioning postsynapses.

[Experiment example 13]
(Examination of the influence of medium composition of human iPSC-derived neurons)
Human iPSC-derived neurons were cultured in the same manner as in Experimental Example 1, except that various media whose compositions are shown in Table 2 below were used. Subsequently, on Day 77, cells were fixed and immunostained to evaluate whether stage 5 neurons were obtained. In Table 2 below, "NB Plus" indicates NeuroBasal Plus (Thermo Fisher Scientific), "BrainPhys" indicates BrainPhys (Stem Cell Technologies), and "B27 Plus" indicates B-27 Plus (Thermo Fisher Scientific). "CDI" stands for iCell Nervous System Supplement and iCell Nervous Supplement B (Fujifilm Cellular Dynamics), "CultureOne" stands for CultureOne (Thermo Fisher Scientific), "DAPT ” indicates DAPT (CAS number: 208255-80-5), “-” indicates not added, and “+” indicates added. Medium 2 in Table 2 below is the medium used in Experimental Example 1. In addition, "spine OK" means that, as a result of immunostaining described later, mature dendrites were formed in which drebrin aggregated in the spines to form clusters, and stage 5 neurons were obtained. shows. Moreover, "no spine" indicates that no spine was formed and stage 5 neurons were not obtained.

FIG. 25 is a representative fluorescence microscopy image showing the results of immunostaining of human iPSC-derived neurons cultured in medium 2, medium 5, and medium 7, respectively, shown in Table 2 above.

As a result, dendrites were similarly matured in medium 2 and medium 7, and drebrin clusters were observed. On the other hand, in medium 5, the growth of dendrites was poor, and although many drebrin filopodia were generated, it was observed that they had not matured into clustered spines. Table 2 above also shows the results of immunostaining using each medium.

The above results showed that in order to obtain stage 5 neurons, it is important to add B-27 Plus to the medium, and the basal medium may be Neurobasal Plus or BrainPhys.

[Experiment example 14]
(Examination of the effect of the amount of B-27 Plus added to the culture medium of human iPSC-derived neurons)
Human iPSC-derived neurons were cultured in the same manner as in Experimental Example 1. In Experimental Example 1, 2% B-27 Plus (Thermo Fisher Scientific) was added to the culture medium, but in this Experimental Example, it was replaced with 4% B-27 Plus or 10% B-27 Plus. Subsequently, cells were fixed and immunostained on Day 63 to evaluate whether stage 5 neurons were obtained.

Figure 26 is a representative fluorescence microscopy image showing the results of immunostaining of human iPSC-derived neurons cultured in medium supplemented with 2% B-27 Plus, 4% B-27 Plus, and 10% B-27 Plus. be.

As a result, it was observed that dendrites matured and drebrin clusters began to accumulate in all medium compositions. However, because the culture period was set one week shorter than the expected completion time of maturation, there were still many filopodia remaining in B-27 Plus 2%, indicating that drebrin was diffused around the protrusions and accumulation was not completed. observed. Even with B-27 Plus 4%, many filopodia remained, but drebrin expression around the protrusions seemed to be slightly increased. On the other hand, in B-27 Plus 10%, many small granular drebrin clusters were observed, suggesting that the spine was stabilized more quickly.

From the above results, in order to obtain stage 5 neurons, the amount of B-27 Plus added to the basic medium may be as low as 2%, but increasing the amount to 4% or 10% increases the maturation efficiency to stage 5 neurons. It was suggested that it would rise.

[Experiment example 15]
(Creation of stage 5 neurons from iPSCs derived from neurological disease patients)
Stage 3 neurons were generated from iPSCs derived from healthy individuals and iPSCs derived from neurological disease patients using a neuronal differentiation kit (product name: "Quick-Neuron ^TM Excitatory-SeV Kit", Elixagen Scientific) according to the manufacturer's protocol. Created. As iPSCs derived from healthy subjects, 1383D2 strain and 1231A3 strain were used. As iPSCs derived from neurological disease patients, HPS3036 strain derived from Rett syndrome patients was used.

Thereafter, iPSC-derived neurons were cultured in the same manner as in Experimental Example 1. Subsequently, immunostaining was performed on Day 90, Day 91, and Day 112 to evaluate whether stage 5 neurons were obtained.

FIG. 27 is a fluorescence microscopy image showing representative immunostaining results. This is an image obtained by merging staining results of MAP2, drebrin, and nucleus. As a result, the formation of drebrin clusters on or near dendrites was observed, indicating that stage 5 neurons could be obtained using both iPSCs derived from healthy individuals and iPSCs derived from neurological disease patients.

The present invention includes the following aspects.
[1] A method for producing stage 5 neurons with mature dendrites, which includes the step of culturing stage 3 neurons with immature dendrites in a medium containing B-27 Plus.
[2] The manufacturing method according to [1], wherein a dendritic spine is formed in the stage 5 neuron.
[3] The manufacturing method according to [1] or [2], wherein a post-synaptic structure containing drebrin and PSD95 is formed in the stage 5 neuron.
[4] The production method according to any one of [1] to [3], wherein the stage 3 neurons express neural markers including MAP2 and neurofilament heavy chain (NF-H) and are polarized.
[5] The production method according to any one of [1] to [4], wherein the stage 3 neurons are obtained by inducing differentiation of induced pluripotent stem cells (iPSCs).
[6] The production method according to [5], wherein the stage 3 neurons are induced to differentiate by inducing expression of a transcription factor in the iPSC.
[7] Stage 5 neurons with mature dendrites, isolated and cultured in containers.
[8] A step of culturing stage 3 neurons induced to differentiate from iPSCs derived from neurological disease patients in a medium containing B-27 Plus in the presence of a test substance to obtain stage 5 neurons; and evaluating the phenotype of the stage 5 neuron in the presence of the test substance as compared to the absence of the test substance, the stage 5 neuron exhibiting a phenotype closer to the wild type. is a method for screening a therapeutic drug for a neurological disease, the test substance being a candidate for a therapeutic drug for the neurological disease.
[9] The step of culturing stage 5 neurons induced to differentiate from iPSCs derived from neurological disease patients in the presence of a test substance for a predetermined period of time, and the step of evaluating the phenotype of the stage 5 neurons, The fact that the phenotype of the stage 5 neurons in the presence of the test substance is closer to the wild type than in the absence of the substance indicates that the test substance is a candidate for a therapeutic agent for the neurological disease. A screening method for therapeutic drugs for neurological diseases.

JP2020-156463

Togo K., et al., Postsynaptic structure formation of human iPS cell-derived neurons takes longer than presynaptic formation during neural differentiation in vitro, Mol Brain 14:149, 2021.

Claims (9)

culturing stage 3 neurons with immature dendrites in a medium containing B-27 Plus;
Method for producing stage 5 neurons with mature dendrites. The manufacturing method according to claim 1, wherein a dendritic spine is formed in the stage 5 neuron. 3. The manufacturing method according to claim 1, wherein a post-synaptic structure containing drebrin and PSD95 is formed in the stage 5 neuron. The method according to claim 1 or 2, wherein the stage 3 neurons express neural markers including MAP2 and neurofilament heavy chain (NF-H) and are polarized. The manufacturing method according to claim 1 or 2, wherein the stage 3 neurons are obtained by inducing differentiation of induced pluripotent stem cells (iPSCs). 6. The manufacturing method according to claim 5, wherein the stage 3 neurons are induced to differentiate by inducing expression of a transcription factor in the iPSC. Stage 5 neurons with mature dendrites, isolated and cultured in containers. culturing stage 3 neurons induced to differentiate from iPSCs derived from neurological disease patients in a medium containing B-27 Plus in the presence of a test substance to obtain stage 5 neurons;
assessing the phenotype of the stage 5 neurons;
The fact that the phenotype of the stage 5 neurons in the presence of the test substance is closer to the wild type than in the absence of the test substance is that the test substance is a therapeutic agent for the neurological disease. A screening method for therapeutic drugs for neurological diseases that are shown to be candidates for. a step of culturing stage 5 neurons induced to differentiate from iPSCs derived from neurological disease patients for a predetermined period of time in the presence of a test substance;
assessing the phenotype of the stage 5 neurons;
The fact that the phenotype of the stage 5 neurons in the presence of the test substance is closer to the wild type than in the absence of the test substance is that the test substance is a therapeutic agent for the neurological disease. A screening method for therapeutic drugs for neurological diseases that are shown to be candidates for.

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