JP7602791B2 - Culture medium for direct transdifferentiation of macrophages into neural cells and use thereof - Google Patents

Description

The present invention relates to a medium used for directly transdifferentiating macrophages into neurons and uses thereof. Specifically, the present invention relates to a medium used for directly transdifferentiating macrophages into neurons, a method for producing neurons using the medium, and a method for directly transdifferentiating macrophages into neurons.

The direct conversion of somatic cells into specific differentiated cells without first transforming them into pluripotent stem cells is called "direct reprogramming," and in recent years, research and development into direct reprogramming has been actively conducted as a new trend in basic research, drug discovery, and regenerative medicine.

For example, Non-Patent Documents 1 to 3 report direct transdifferentiation of somatic cells such as astrocytes and fibroblasts into neural cells.

On the other hand, macrophages have a migratory function. Due to this function, macrophages accumulate at the lesion, for example, during the acute phase of cerebral infarction. If it were possible to directly reprogram these accumulated macrophages into nerve cells in vivo, this would open up new possibilities for neuroregenerative medicine.

Hu W et al., “Direct Conversion of Normal and Alzheimer's Disease Human Fibroblasts into Neuronal Cells by Small Molecules.”, Cell stem cell, Vol. 17, Issue 2, pp. 204-212, 2015. Yin JC et al., “Chemical Conversion of Human Fetal Astrocytes into Neurons through Modulation of Multiple Signaling Pathways.”, Stem cell reports, Vol. 12, Issue 3, pp. 488-501, 2019. Mahato B et al., “Pharmacologic fibroblast reprogramming into photoreceptors restores vision.”, Nature, Vol. 581, Issue 7806, pp. 83-88, 2020.

However, there have been no reports of direct transdifferentiation of macrophages into neurons.

The present invention has been made in consideration of the above circumstances, and provides a culture medium for directly transdifferentiating macrophages into neurons, as well as a method for producing neurons using the culture medium and a method for directly transdifferentiating macrophages into neurons.

That is, the present invention includes the following aspects.
(1) A medium used for directly transdifferentiating macrophages into neurons, comprising:
A medium comprising a glycogen synthase kinase 3 inhibitor, an adenylate cyclase activator, a Rho kinase inhibitor, a neuronal differentiation factor, and a PU.1 inhibitor.
(2) The medium according to (1), wherein the PU.1 inhibitor is DB2313.
(3) The medium according to (1) or (2), wherein the content of the PU.1 inhibitor is more than 0.1 μmol/L and less than 1 μmol/L relative to the total volume of the medium.
(4) The medium according to any one of (1) to (3), wherein the neuronal differentiation inducer is ISX-9.
(5) The medium according to any one of (1) to (3), further comprising an AMP-activated protein kinase inhibitor.
(6) A method for producing nerve cells, comprising a transdifferentiation step of culturing macrophages using a medium containing a glycogen synthase kinase 3 inhibitor, an adenylate cyclase activator, a Rho kinase inhibitor, a neuronal differentiation factor, and a PU.1 inhibitor, and directly transdifferentiating the macrophages into nerve cells.
(7) A method for directly transdifferentiating macrophages into neurons, comprising the steps of:
A method comprising a transdifferentiation step of culturing macrophages using a medium containing a glycogen synthase kinase 3 inhibitor, an adenylate cyclase activator, a Rho kinase inhibitor, a neuronal differentiation factor, and a PU.1 inhibitor, and directly transdifferentiating the macrophages into neurons.

The medium of the above aspect can provide a medium for directly transdifferentiating macrophages into nerve cells. The manufacturing method of the above aspect can produce nerve cells that have been directly transdifferentiated from macrophages. The method of the above aspect can directly transdifferentiate macrophages into nerve cells.

1 shows differential interference contrast microscope images of cells cultured in each medium in Example 1. 1 is a graph showing the expression levels of pluripotency marker genes in cells cultured in medium No. 2 in Example 1. 1 is a fluorescent staining image of cells cultured in medium No. 2 in Example 1. 1 is a graph showing the expression levels of each neuronal marker gene and each macrophage marker gene in cells cultured in medium No. 2 in Example 1. 1 shows a differential interference microscope image and a fluorescent stained image of cells cultured in medium No. 2 in Example 1. 1 is a fluorescent staining image of cells cultured in medium No. 2 in Example 2. 1 is a graph showing the relationship between the DB2313 concentration in the medium and the cell survival rate and the rate of transdifferentiation into neural cells in Example 3. 1 is a graph showing the relationship between the ISX-9 concentration in the medium and the cell survival rate and the rate of transdifferentiation into neural cells in Example 4.

<Culture medium used to directly transdifferentiate macrophages into neurons>
The medium used for directly transdifferentiating the macrophages of this embodiment into neurons (hereinafter simply referred to as the "medium of this embodiment") contains a glycogen synthase kinase 3 inhibitor (GSK3 inhibitor), an adenylate cyclase activator, a Rho kinase inhibitor (ROCK inhibitor), a neuronal differentiation inducer, and a PU.1 inhibitor.

The medium of this embodiment has the above-mentioned composition, and thus can directly transdifferentiate macrophages into nerve cells.
As used herein, the term "direct transdifferentiation" refers to directly converting a somatic cell into a specific differentiated cell without passing through a pluripotent stem cell, and is used synonymously with the term "direct reprogramming."

Previous reports have shown that it is possible to chemically reprogram fibroblasts or astrocytes into neurons by culturing them in a medium with a specific composition. However, when the medium used for these cells was directly applied to macrophages, it was not possible to directly reprogram them into neurons. This is thought to be because the components or factors required for chemically direct reprogramming vary greatly depending on the type of somatic cells used as the raw material, and the appropriate components or factors for direct reprogramming from macrophages to neurons have not yet been identified.

In response to this, the inventors focused on the transcription factor PU.1 from among the many characteristics and marker genes of macrophages, and discovered that the use of a PU.1 inhibitor can suppress the expression of the characteristics inherent to macrophages, and that suppressing the expression of these characteristics is effective for efficiently and directly transdifferentiating macrophages into neurons. Furthermore, they discovered that the minimum effective components for directly transdifferentiating macrophages into neurons are a combination of a glycogen synthase kinase 3 inhibitor, an adenylate cyclase activator, a Rho kinase inhibitor, and a neuronal differentiation inducer, in addition to a PU.1 inhibitor, and thus completed the present invention.

Below, we will explain in detail each component contained in the medium of this embodiment.

[PU.1 Inhibitors]
PU.1 is expressed in granulocytes, monocytes and B lymphocytes, and is known to be an essential transcription factor for the differentiation of these cell lineages. Therefore, by suppressing the expression or function of PU.1 in macrophages, it is possible to suppress the exertion of the inherent properties of macrophages. This allows for efficient direct transdifferentiation of macrophages into nerve cells.

That is, the PU.1 inhibitor is not particularly limited as long as it can inhibit the expression or function of PU.1. Specific examples of PU.1 inhibitors include low molecular weight compounds, PU.1 expression inhibitors, and PU.1 specific binding substances. Among these, low molecular weight compounds are preferred as PU.1 inhibitors.

Examples of low molecular weight compounds that are PU.1 inhibitors include DB1976 (CAS number: 2369663-93-2), DB2115 (CAS number: 1366231-70-0), and DB2313 (CAS number: 2170606-74-1). Among these, DB2313 is preferred.

Examples of PU.1 expression inhibitors include siRNA, shRNA, miRNA, ribozymes, and antisense nucleic acids. By administering these PU.1 expression inhibitors, the expression level of PU.1 can be reduced, and the inherent properties of macrophages can be inhibited from being exerted. As a result, macrophages can be directly and efficiently transdifferentiated into nerve cells.

siRNA (small interfering RNA) is a small double-stranded RNA with 21 to 23 base pairs that is used for gene silencing by RNA interference. When siRNA is introduced into cells, it binds to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNA that has a sequence complementary to the siRNA, thereby suppressing gene expression in a sequence-specific manner.

The siRNA can be prepared by synthesizing sense and antisense oligonucleotides using an automatic DNA/RNA synthesizer, denaturing them in an appropriate annealing buffer at 90°C to 95°C for about 1 minute, and then annealing them at 30°C to 70°C for about 1 hour to 8 hours.

The siRNA, shRNA, miRNA, ribozyme, and antisense nucleic acid may contain various chemical modifications to improve stability and activity. For example, to prevent degradation by hydrolases such as nucleases, phosphate residues may be replaced with chemically modified phosphate residues such as phosphorothioate (PS), methylphosphonate, and phosphorodithioate. In addition, at least a portion of the nucleic acid may be composed of a nucleic acid analog such as peptide nucleic acid (PNA).

The PU.1 specific binding substance includes substances that specifically bind to PU.1 and inhibit the function of PU.1, such as antibodies, antibody fragments, aptamers, etc. Antibodies can be produced, for example, by immunizing an animal such as a mouse with the PU.1 protein or a fragment thereof as an antigen. Alternatively, they can be produced, for example, by screening a phage library. Antibody fragments include Fv, Fab, scFv, etc. The above-mentioned antibodies are preferably monoclonal antibodies. They may also be commercially available antibodies.

An aptamer is a substance that has a specific binding ability to a target substance. Examples of aptamers include nucleic acid aptamers and peptide aptamers. Nucleic acid aptamers that have a specific binding ability to a target peptide can be selected, for example, by the systematic evolution of ligand by exponential enrichment (SELEX) method. Peptide aptamers that have a specific binding ability to a target peptide can be selected, for example, by the two-hybrid method using yeast.

The concentration of the PU.1 inhibitor in the medium is preferably more than 0.1 μmol/L and less than 1 μmol/L (hereinafter, "mol/L" is abbreviated as M) relative to the total volume of the medium, more preferably 0.15 μM or more and 0.95 μM or less, even more preferably 0.2 μM or more and 0.9 μM or less, particularly preferably 0.3 μM or more and 0.7 μM or less, and most preferably 0.35 μM or more and 0.65 μM or less.
By setting the concentration of the PU.1 inhibitor in the medium to the above lower limit or higher, the expression or function of PU.1 can be more effectively suppressed. On the other hand, by setting the concentration to the above upper limit or lower, the toxicity of the PU.1 inhibitor to cells can be further suppressed, and the cell viability can be further improved.

[GSK3 inhibitors]
By using a GSK3 inhibitor, it is possible to promote Wnt signaling, and as a result, it is possible to promote transdifferentiation into neural cells.

Examples of GSK3 inhibitors include CHIR99021 (CAS number: 252917-06-9), Kenpaullone (CAS number: 142273-20-9), 6-Bromoindirubin-3'-oxime (BIO, CAS number: 667463-62-9), etc. Among these, CHIR99021 is preferred.

The concentration of the GSK3 inhibitor in the medium is typically 0.1 μM or more and 10 μM or less, preferably 0.5 μM or more and 7 μM or less, more preferably 1 μM or more and 5 μM or less, and even more preferably 2 μM or more and 4 μM or less, relative to the total volume of the medium.
By setting the concentration of the GSK3 inhibitor in the medium to be equal to or higher than the lower limit, the effect of the GSK3 inhibitor can be more effectively exhibited, whereas by setting the concentration to be equal to or lower than the upper limit, the toxicity of the GSK3 inhibitor to cells can be further suppressed, and the cell survival rate can be further improved.

[Adenylate cyclase activators]
Adenylate cyclase activators are also called MAP kinase inhibitors.
By using an adenylate cyclase activator, the intracellular concentration of AMP can be increased, thereby activating adenylate cyclase, and as a result, transdifferentiation into nerve cells can be promoted.

Examples of adenylate cyclase activators include forskolin (CAS number: 66575-29-9).

The concentration of the adenylate cyclase activator in the medium is usually 0.1 μM or more and 100 μM or less, preferably 1 μM or more and 50 μM or less, more preferably 5 μM or more and 15 μM or less, and even more preferably 8 μM or more and 12 μM or less, relative to the total volume of the medium.
When the concentration of the adenylate cyclase activator in the medium is equal to or higher than the lower limit, the effect of the adenylate cyclase activator can be more effectively exhibited, whereas when the concentration is equal to or lower than the upper limit, the toxicity of the adenylate cyclase activator to cells can be further suppressed, and the cell viability can be further improved.

[ROCK Inhibitor]
By using a ROCK inhibitor, it is possible to suppress cell apoptosis and maintain high cell survival rates.

Examples of ROCK inhibitors include Y-27632 (CAS number: 146986-50-7), Fasudil (CAS number: 105628-07-7), Y39983 (CAS number: 203911-26-6), Wf-536 (CAS number: 539857-64-2), SLx-2119 (CAS number: 911417-87-3), azabenzimidazole-aminofurazans (CAS number: 850664-21-0), DE-104, H-1152P (CAS number: 872543-07-6), and Rho kinase α inhibitors (ROKα inhibitor), XD-4000, HMN-1152, 4-(1-aminoalkyl)-N-(4-pyridyl)cyclohexane-carboxamides, Rhostatin, BA-210, BA-207, Ki-23095, VAS-012, etc. Among these, Y-27632 is preferred.

The concentration of the ROCK inhibitor in the medium is usually 0.1 μM or more and 100 μM or less, preferably 1 μM or more and 50 μM or less, more preferably 5 μM or more and 15 μM or less, and even more preferably 8 μM or more and 12 μM or less, relative to the total volume of the medium.
When the concentration of the ROCK inhibitor in the medium is equal to or higher than the lower limit, the effect of the ROCK inhibitor can be more effectively exhibited, whereas when the concentration is equal to or lower than the upper limit, the toxicity of the ROCK inhibitor to cells can be further suppressed, and the cell survival rate can be further improved.

[Neurodifferentiation Inducing Factor]
By using a neuronal differentiation inducer, neuronal differentiation can be promoted by acting on voltage-dependent calcium channels and NMDA receptors to promote calcium influx and enhance the expression of the neuroD gene.

Examples of neuronal differentiation inducers include isoxazole 9 (ISX-9) (CAS number: 832115-62-5).

The concentration of the neuronal differentiation induction factor in the culture medium is typically 0.1 μM or more and less than 10 μM, preferably 1 μM or more and less than 9 μM, more preferably 2.5 μM or more and less than 7.5 μM, and even more preferably 2.7 μM or more and less than 5 μM, relative to the total volume of the culture medium.
When the concentration of the neuronal differentiation inducer in the medium is equal to or higher than the lower limit, the effect of the neuronal differentiation inducer can be more effectively exerted, whereas when the concentration is equal to or lower than the upper limit, the toxicity of the neuronal differentiation inducer to cells can be further suppressed, and the cell survival rate can be further improved.

[AMP-activated protein kinase inhibitors]
In addition to the above components, the medium of the present embodiment preferably further contains an AMP-activated protein kinase inhibitor, which allows the medium of the present embodiment to more efficiently transdifferentiate macrophages directly into neurons.
AMP-activated protein kinase inhibitors are also called BMP pathway inhibitors.

Examples of AMP-activated protein kinase inhibitors include dorsomorphin (CAS number: 866405-64-3), chordin (human Genbank accession numbers: NP_001291401.1, NP_001291402.1, NP_001291403.1, NP_003732.2, etc.), noggin (human Genbank accession number: NP_005441.1, etc.), follistatin (human Genbank accession numbers: NP_006341.1, NP_037541.1, etc.), etc.), (6-[4-(2-piperidin-1-yl-ethoxy)phenyl]-3-pyridin-4-yl-pyrazolo[1,5-a]pyrimidine), DMH1 (4-[6-(4-isopropoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline, 4-[6-[4-(1-methylethoxy)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline), LDN193189, (4-(6-(4-(piperidin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline). Among these, dorsomorphin is preferred.

The concentration of the AMP-activated protein kinase inhibitor in the medium is usually 0.5 μM or more and 10 μM or less, preferably 0.75 μM or more and 5 μM or less, and more preferably 1 μM or more and 3 μM or less, relative to the total volume of the medium.

[Other ingredients]
The medium of this embodiment may further contain growth factors, neurobiological supplements, medium supplements, serum, serum substitutes, antibacterial agents, and serum-derived proteins such as insulin and albumin, etc. The concentrations of these components in the medium may be appropriately determined by those skilled in the art with reference to known literature, etc.

Examples of growth factors include the neurotrophin family, such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT3), and neurotrophin 4/5; glial cell line-derived neurotrophic factor (GDNF); acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF); insulin-like growth factor (IGF), and the like.

Neurobiological supplements include B27 supplement (Thermo Fisher brand name) which contains biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinol, retinyl acetate, sodium selenite, triiodothyronine (T3), DL-α-tocopherol (vitamin E), albumin, insulin, and transferrin; and N2 supplement which contains human transferrin, bovine insulin, progesterone, putrescine, and sodium selenite.

Examples of medium supplements include glutamic acid-containing supplements such as the "GlutaMax series" (Thermo Fisher product name) which contain L-glutamic acid and dipeptides derived from L-glutamic acid, aqueous amino acid solutions such as "MEM Non-Essential Amino Acids Solution" (Thermo Fisher product name), and 2-mercaptoethanol.

Examples of antibacterial agents include penicillin antibiotics, cephalosporin antibiotics, macrolide antibiotics, tetracycline antibiotics, fosfomycin antibiotics, aminoglycoside antibiotics, and new quinolone antibiotics.

It is preferable that the medium of this embodiment does not contain a γ-secretase inhibitor such as N-[N-(3,5-difluorophenylacetyl-L-alanyl)]-S-phenylglycine tert-butyl ester (DAPT) (CAS number: 208255-80-5) and a TGF-β receptor inhibitor such as RepSox (CAS number: 446859-33-2).
These components have been reported to be effective in promoting the differentiation of various stem cells, such as ES cells and iPS cells, into neural cells. However, the medium of the present embodiment does not contain these components, and therefore can more efficiently transdifferentiate macrophages directly into neural cells.

[Basal medium]
The medium of this embodiment can be prepared by adding the above-mentioned components to a basal medium.

Examples of basal media include BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM (GMEM) medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle MEM medium, αMEM medium, DMEM medium, F-12 medium, DMEM/F12 medium, IMDM/F12 medium, Ham's medium, RPMI 1640 medium, Fischer's medium, X-VIVO ^™ 15 serum-free lymphocyte medium (manufactured by Lonza), and NeuroBasal Medium (manufactured by Gibco); mixed media thereof; and media obtained by reducing components related to neural differentiation from these media.

<Method of producing nerve cells>
The method for producing nerve cells according to the present embodiment (hereinafter simply referred to as the "production method according to the present embodiment") includes a transdifferentiation step of culturing macrophages using a medium containing a GRK3 inhibitor, an adenylate cyclase activator, a ROCK inhibitor, a neuronal differentiation factor, and a PU.1 inhibitor, and directly transdifferentiating them into nerve cells.

According to the manufacturing method of this embodiment, it is possible to produce nerve cells that have been directly transdifferentiated from macrophages. In other words, the manufacturing method of this embodiment can also be said to be a method of directly transdifferentiating (directly reprogramming) macrophages into nerve cells.

The steps that make up the manufacturing method of this embodiment are described in detail below.

[Transdifferentiation process]
In the transdifferentiation step, macrophages are cultured using a medium containing the above-mentioned components and directly transdifferentiated into nerve cells.

The composition of the medium is as described above in "Culture medium used for direct transdifferentiation of macrophages into neurons."

The culture period is usually at least one day, preferably between three and fourteen days, and more preferably between five and nine days.

The culture temperature is usually from 30°C to 50°C, preferably from 32°C to 45°C, and more preferably from 34°C to 40°C.

The carbon dioxide content in the culture vessel is usually 1% to 15% by volume, preferably 2% to 10% by volume, and more preferably 3% to 7% by volume.

The form of the culture vessel is not particularly limited, but examples include known forms such as flasks, tissue culture flasks, culture dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, multi-plates, multi-well plates, chamber slides, petri dishes, tubes, trays, culture bags, microcarriers, stack plates, spinner flasks, and cover glasses.

The animal species from which the macrophages used in the transdifferentiation process are derived is not particularly limited, but examples include mammals such as rodents, ungulates, felines, and primates. Rodents include mice, rats, hamsters, and guinea pigs. Ungulates include pigs, cows, goats, horses, and sheep. Felidae include dogs and cats. Primates refer to mammals belonging to the order Primates, and examples of primates include the prosimian suborder, such as lemurs, lorises, and tree shrews, and the anthropoid suborder, such as monkeys, apes, and humans. Of these, humans are preferred.

The macrophages used in the transdifferentiation process may be a known cell line, or may be cells induced to differentiate from monocytes. The phenotype of the macrophages may be either M1 or M2.

[Other steps]
When monocyte-derived macrophages are used, prior to the transdifferentiation step, the monocytes are cultured in a medium containing GM-CSF (Granulocyte-Macrophage Colony Stimulating Factor) or M-CSF (Macrophage Colony Stimulating Factor) to induce differentiation of the monocytes into macrophages of an M1 or M2 phenotype (hereinafter also referred to as a "differentiation induction step" or a "macrophage production step").

The inventors also attempted to directly transdifferentiate monocytes into nerve cells using a medium of the above composition, but confirmed that the above composition, particularly the PU.1 inhibitor, is highly toxic to monocytes, and therefore it is not possible to directly transdifferentiate monocytes into nerve cells using a medium of the above composition. Therefore, after inducing differentiation of monocytes into macrophages, the macrophages are directly transdifferentiated into nerve cells using a medium of the above composition.

The culture period, culture temperature, carbon dioxide content in the culture vessel, and shape of the culture vessel in the differentiation induction process are the same as those in the "transdifferentiation process" described above.

The monocytes may be an established cell line or may be isolated from a biological sample such as animal blood.

The medium used for culturing monocytes can be prepared by adding GM-CSF or M-CSF to a basal medium. Examples of the basal medium include those exemplified in the "transdifferentiation process" above.

The medium used for culturing monocytes may further contain, in addition to GM-CSF or M-CSF, medium supplements, serum, serum substitutes, antibacterial agents, and serum-derived proteins such as insulin and albumin. Examples of medium supplements and antibacterial agents include those similar to those exemplified in the "transdifferentiation process" above.

Furthermore, after the transdifferentiation process, the obtained cells may be evaluated to confirm whether or not they are neural cells (hereinafter, also referred to as the "evaluation process").

Evaluation methods include, for example, observing cell morphology, confirming the expression of marker genes or marker proteins in nerve cells, and measuring the action potential of cells.

The cell morphology may be observed with the naked eye or by a method using an optical microscope or the like. If the obtained cells have a dendrite-like structure, it can be evaluated that nerve cells have been obtained.

Examples of marker genes or marker proteins for nerve cells include β-Tubulin 3 (TUJ1), microtubule-associated protein 2 (MAP2), doublecortin (DCX), Myelin Transcription Factor 1-Like (MYT1L), Achaete-scute homolog 1 (ASCL1), Neurogenic Differentiation 1 (NeuroD1), neuronal specific POU transcription factor (BRN), Neurogenin-2 (NGN2), Neuronal nuclei (NEUN), etc. Expression of the marker gene or marker protein can be evaluated by, for example, RT-PCR, Western blotting, ELISA, immunostaining, etc.
When the expression of these marker genes or marker proteins for nerve cells is higher than the expression level in macrophages or is at the same level as the expression level in nerve cells, it can be evaluated that nerve cells have been obtained.

In addition, the expression of a marker gene or a marker protein of a macrophage may be further confirmed. Examples of the marker gene or the marker protein of a macrophage include integrin αM (CD11b), ionized calcium binding adapter protein 1 (Iba1), etc. The expression of these can be evaluated by the same method as that for evaluating the marker gene or the marker protein of a nerve cell.
When the expression of these macrophage marker genes or marker proteins is reduced compared to the expression level in macrophages, the characteristics of macrophages are completely lost, and when combined with the result of the expression of the above-mentioned neuronal marker genes or marker proteins, it can be evaluated that macrophages have been transdifferentiated into neurons.

<Other embodiments>
The medium in the above embodiment and the nerve cells obtained by the method for producing nerve cells in the above embodiment are expected to be applied to regenerative medicine for brain and nerve diseases such as cerebral infarction and neurodegenerative diseases.
That is, in one embodiment, the present invention provides a method for treating a neurological disease, comprising administering an effective amount of the culture medium in the above embodiment or the nerve cells obtained by the production method in the above embodiment to a patient or animal suffering from the neurological disease.

Examples of the target cranial nerve disease include cerebral infarction, and neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Parkinson's disease, and Alzheimer's disease. Examples of the patient or animal patient include the animal species exemplified in the above "culture medium".
The effective amount of nerve cells is an amount effective for treating the target neurological disease, and can be appropriately set depending on the type and progression of the disease, the weight and age of the patient or animal, the symptoms of the patient or animal, the size of the lesion, etc.

In addition, by directly administering the components contained in the medium in the above embodiment, specifically, a GSK3 inhibitor, an adenylate cyclase activator, a ROCK inhibitor, a neurodifferentiation inducer, and a PU.1 inhibitor (preferably, a GSK3 inhibitor, an adenylate cyclase activator, a ROCK inhibitor, a neurodifferentiation inducer, a PU.1 inhibitor, and an AMP-activated protein kinase inhibitor), to endogenous macrophages accumulated in the lesion of a brain and nerve disease, a novel regenerative medicine method can be provided in which differentiation of macrophages into nerve cells is induced in vivo.
That is, in one embodiment, the present invention provides a method for treating a cranial nerve disease, comprising administering an effective amount of each of a GSK3 inhibitor, an adenylate cyclase activator, a ROCK inhibitor, a neurodifferentiation inducer, and a PU.1 inhibitor to a patient or animal suffering from a cranial nerve disease.
Preferably, in one embodiment, the present invention provides a method for treating a cranial nerve disease, comprising administering an effective amount of each of a GSK3 inhibitor, an adenylate cyclase activator, a ROCK inhibitor, a neurodifferentiation factor, a PU.1 inhibitor, and an AMP-activated protein kinase inhibitor to a patient or animal suffering from the cranial nerve disease.
More preferably, in one embodiment, the present invention provides a method for treating a cranial nerve disease, comprising administering an effective amount of each of CHIR99021, forskolin, Y-27632, ISX-9, ISX-9, and dorsomorphin to a patient or animal suffering from a cranial nerve disease.

The set of the above-mentioned compounds can be called a set of therapeutic agents for brain and nerve diseases.
Alternatively, the composition containing the above-mentioned five (preferably six) types of compounds alone or a mixture thereof can be called a pharmaceutical composition for treating cranial nerve diseases. Hereinafter, the above-mentioned five (preferably six) types of compounds alone or a mixture thereof may be collectively referred to as a "therapeutic agent for cranial nerve diseases."

The pharmaceutical composition may be in a dosage form for oral use or a dosage form for parenteral use, but a dosage form for parenteral use is preferred. Dosage forms for oral use include, for example, tablets, capsules, elixirs, microcapsules, etc. Dosage forms for parenteral use include, for example, injections, ointments, patches, etc.

As the pharma- ceutically acceptable carrier, those usually used in the preparation of pharmaceutical compositions can be used without any particular restrictions. More specifically, examples include binders such as gelatin, corn starch, gum tragacanth, and gum arabic; excipients such as starch and crystalline cellulose; swelling agents such as alginic acid; solvents for injections such as water, ethanol, and glycerin; and adhesives such as rubber-based adhesives and silicone-based adhesives.

The pharmaceutical composition may contain additives. Examples of additives include lubricants such as calcium stearate and magnesium stearate; sweeteners such as sucrose, lactose, saccharin, and maltitol; flavorings such as peppermint and saffron oil; stabilizers such as benzyl alcohol and phenol; buffers such as phosphates and sodium acetate; solubilizers such as benzyl benzoate and benzyl alcohol; antioxidants; and preservatives.

The pharmaceutical composition can be formulated by mixing the above-mentioned five (preferably six) compounds alone or in a mixture thereof with the above-mentioned pharma- ceutically acceptable carriers and additives in a unit dose form required for generally accepted pharmaceutical practice.

The pharmaceutical composition may be used in combination with a therapeutic agent for other diseases. For example, the therapeutic agent for the cranial nerve disease may be used in combination with other therapeutic agents for the cranial nerve disease, etc., to treat the cranial nerve disease while improving the symptoms of the specific cranial nerve disease.
The therapeutic agent for cranial nerve disease and the other drug may be in the same preparation or in separate preparations. Moreover, each preparation may be administered by the same administration route or by separate administration routes. Furthermore, each preparation may be administered simultaneously, sequentially, or separately at a certain time or period. In one embodiment, the therapeutic agent for cranial nerve disease and the other drug may be in a set (therapeutic agent set for cranial nerve disease) including them.

Administration to a patient can be by methods known to those skilled in the art, for example, intrathecal injection, intraarterial injection, intravenous injection, subcutaneous injection, intranasal injection, transbronchial injection, intramuscular injection, transdermal injection, or oral injection.

The effective amount of each compound is an amount effective for treating the target neurological disease, and can be set appropriately depending on the type and progression of the disease, the weight and age of the patient or animal, the symptoms of the patient or animal, the method of administration, etc.

In one embodiment, the present invention provides a method for evaluating the efficacy of a test substance, comprising a step of contacting the medium in the above embodiment or the nerve cells obtained by the production method in the above embodiment with a test substance (hereinafter also referred to as "step X"), and a step of examining the effect of the test substance on the nerve cells (hereinafter also referred to as "step Y").

In step X, examples of test substances include natural compound libraries, synthetic compound libraries, existing drug libraries, metabolite libraries, etc. New drugs can also be used as test substances.

In step Y, the effect of the test substance on nerve cells can be assayed, for example, by Western blotting, ELISA, immunostaining, etc.

Furthermore, before step X, a step (hereinafter also referred to as "step x") of transplanting neural cells into the brain of a mammal can be included. When neural cells exhibit a pathological condition similar to that of a specific disease, such as Alzheimer's disease, including step x makes it possible to evaluate the efficacy of the test substance in an environment closer to that of a living body suffering from a specific disease.

In one embodiment, the present invention provides a medium supplement for direct reprogramming of macrophages into neurons (or a medium supplement for producing neurons), comprising each of the components contained in the medium in the above embodiment, i.e., a GSK3 inhibitor, an adenylate cyclase activator, a ROCK inhibitor, a neuronal differentiation inducer, and a PU.1 inhibitor (preferably, a GSK3 inhibitor, an adenylate cyclase activator, a ROCK inhibitor, a neuronal differentiation inducer, a PU.1 inhibitor, and an AMP-activated protein kinase inhibitor).
The above supplement, the above-mentioned basal medium, and, if necessary, the above-mentioned other components can also be made into a medium kit for directly transdifferentiating (direct reprogramming) macrophages into neurons (or a medium kit for producing neurons).

The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[Example 1]
(Investigation of medium components that directly transdifferentiate monocyte-derived macrophages into neurons)
Human mononuclear cells isolated from blood samples collected with consent from healthy subjects were seeded onto a cover glass (Matsunami Glass Industry Co., Ltd., round cover glass 18 round, product code: C018001) to a density of 5.0 x ¹⁰⁶ cells/ ^cm2 or less, and cultured for 1 hour at 37°C in a 5% _CO2 environment using Monocyte Attachment Medium (PromoCell, Inc., C-28051). Next, the floating cells in the medium were removed together with the medium, and the adherent cells (monocytes) on the cover glass were cultured in a medium (composition: 10 w/v% fetal bovine serum (FBS), X-VIVO ^™ 15 serum-free lymphocyte medium (Lonza, 04-418Q)) containing 10 ng/mL GM-CSF (Granulocyte-Macrophage Colony Stimulating Factor) relative to the total volume of the medium for 7 days in a 37°C, 5% by volume _CO2 environment to obtain macrophages (hereinafter sometimes abbreviated as "Mφ") having an M1 phenotype. The fact that Mφ was obtained was confirmed by detecting enhanced expression of the CD11b gene and Iba1 gene, which are marker genes for Mφ, by quantitative PCR.

Next, the obtained human monocyte-derived M1 type Mφ was further cultured for 7 days at 37° C. in a 5% by volume CO ₂ environment using a medium having the composition shown below.

Figure 1 shows differential interference microscope images (scale bar 50 μm) of cells cultured for 7 days using each of the above No. 1 to No. 3 media, taken with a differential interference microscope (magnification: 400x (upper image) and 200x (lower image), Olympus, IX83).

As can be seen from Figure 1, the cells cultured using medium No. 1 and No. 2 were observed to have a morphology with neuron-like dendrites. On the other hand, the cells cultured using medium No. 3 did not show any change to neuron-like cells.
From these findings, it was revealed that the minimum essential components of the medium for directly transdifferentiating Mφ into neurons are a glycogen synthase kinase 3 inhibitor, an adenylate cyclase activator, a Rho kinase inhibitor, a neuronal differentiation inducer, and a PU.1 inhibitor, which are used for directly transdifferentiating macrophages into neurons.

Next, total RNA was collected from cells cultured for 0, 1, and 7 days using medium No. 2 using NucleoSpin (registered trademark) RNA Plus (740984-50) manufactured by Takara, and the expression of the pluripotency marker genes PAX6 gene and SOX2 gene was confirmed by RT-PCR using Thermal Cycler Dice Real Time System manufactured by Takara. The results are shown in Figure 2. In Figure 2, "NS" stands for "not significant" and means that it is not significant.

As shown in Figure 2, the expression of the pluripotency marker genes PAX6 gene and SOX2 gene was negative in cells cultured for 7 days using medium No. 2. In other words, it was confirmed that the transdifferentiation of Mφ into neural cells using medium No. 2 was a direct reprogramming that did not go through pluripotent stem cells.

Cells cultured for 7 days in medium No. 2 were stained with a specific antibody against β-Tubulin 3 (TUJ1), a neuronal marker (Biolegend, 801201), as well as a specific antibody against PAX6 (Covance, PRB-278P) and a specific antibody against SOX2 (R&D, AF2018), which are pluripotency markers. The cells were stained with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) (Vector Laboratories, H-2000-10). The cells were observed under a fluorescent microscope (400x magnification, Carl Zeiss, LSM710). The results are shown in Figure 3 (top: TUJ1/PAX6/DAPI, bottom: TUJ1/SOX2/DAPI). In Figure 3, the scale bar is 50 μm. In the upper and lower fluorescent staining images in Figure 3, the image on the left is a merged image of detection images using three types of antibodies: anti-TUJ1 antibody, anti-PAX6 antibody or anti-SOX2 antibody, and DAPI. The image in the middle is a fluorescent staining image using anti-TUJ1 antibody, and the image on the right is a fluorescent staining image using anti-PAX6 antibody or anti-SOX2 antibody.

As shown in Figure 3, in cells cultured for 7 days using medium No. 2, the expression of TUJ1, a marker for neural cells, was confirmed, but the expression of PAX6 and SOX2, pluripotency markers, was not confirmed.
From this, similar to the results in Figure 2, it was confirmed that transdifferentiation from Mφ to neural cells using medium No. 2 was a direct reprogramming that did not involve pluripotent stem cells.

Next, total RNA was extracted from cells cultured for 0, 3, and 7 days using medium No. 2 using NucleoSpin (registered trademark) RNA Plus (740984-50) manufactured by Takara Corporation, and seven types of neuronal marker genes (TUJ1 gene, microtubule-associated protein 2 (MAP2) gene, doublecortin (DCX) gene, Myelin Transcription Factor 1-Like (MYT1L) gene, Achaete-scute homolog 1 (ASCL1) gene, Neurogenic Differentiation The expression of the 1 (NeuroD1) gene, the neuron-specific POU transcription factor (BRN) gene, and the Neurogenin-2 (NGN2) gene, as well as two macrophage marker genes (integrin αM (CD11b) gene and ionized calcium binding adapter protein 1 (Iba1 gene)) was confirmed by RT-PCR using a Thermal Cycler Dice Real Time System manufactured by Takara. The results are shown in FIG. 4. In FIG. 4, "NS" stands for "not significant" and means non-significant. In addition, "*" means P<0.05, "**" means P<0.01, and "***" means P<0.001.

As shown in Figure 4, it was confirmed that as the number of days of culture increased, the expression of neuronal marker genes increased, while the expression of Mφ marker genes decreased.

Next, the cells were cultured for 7 days using medium No. 2 and then fluorescently stained using a specific antibody against TUJ1 (Biolegend, 801201) and a specific antibody against Neuronal Nuclei (NEUN) (Millipore, ABN78). The cells were also stained nuclear with DAPI. The cells were observed using a fluorescent microscope (400x magnification, Carl Zeiss, LSM710). The results are shown in Figure 5 (left: differential interference contrast image, right: fluorescent stained image). In Figure 5, the scale bar is 50 μm. In addition, in the fluorescent staining images on the right side of Figure 5, the upper left image is an image obtained by merging detection images using three types of antibodies: anti-NEUN antibody, anti-TUJ1 antibody, and DAPI, the upper right image is a fluorescent staining image using anti-TUJ1 antibody, and the lower left image is a fluorescent staining image using anti-NEUN antibody.

As shown in Figure 5, expression of TUJ1 and NEUN, which are markers for neural cells, was confirmed at the protein level in cells cultured for 7 days using medium No. 2.

[Example 2]
(Investigation of direct reprogramming to neural cells using monocyte-derived M2 type Mφ)
Next, using medium No. 2, we examined whether direct reprogramming to neurons could be achieved similarly for Mφ with a different phenotype (monocyte-derived M2 type Mφ).

Human mononuclear cells isolated from blood samples collected with consent from healthy subjects were seeded onto a cover glass (Matsunami Glass Industry Co., Ltd., round cover glass 18 round, product code: C018001) to a density of 5.0 x ¹⁰⁶ cells/ ^cm2 or less, and cultured for 1 hour at 37°C in a 5% _CO2 environment using Monocyte Attachment Medium (PromoCell, Inc., C-28051). Next, the floating cells in the medium were removed together with the medium, and the adherent cells (monocytes) on the cover glass were cultured in a medium (composition: 10 w/v% fetal bovine serum (FBS), X-VIVO ^™ 15 serum-free lymphocyte medium (Lonza, 04-418Q)) containing 50 ng/mL M-CSF (Macrophage Colony Stimulating Factor) relative to the total volume of the medium for 7 days at 37°C in a 5% by volume _CO2 environment to obtain Mφ having an M2 phenotype. The fact that Mφ was obtained was confirmed by detecting enhanced expression of the CD11b gene and Iba1 gene, which are marker genes for Mφ, using a quantitative PCR method.

Next, the obtained human monocyte-derived M2 type Mφ was further cultured for 7 days at 37° C. in a 5% by volume CO ₂ environment using the above-mentioned medium No. 2 (for composition, see Table 1 above).

Then, the cells were cultured for 7 days using medium No. 2 and fluorescently stained using a specific antibody against TUJ1, a neuronal marker (Biolegend, 801201). The cells were observed using a fluorescent microscope (400x, Carl Zeiss, LSM710). The results are shown in Figure 6. Note that the scale bar in Figure 6 is 50 μm.

As shown in Fig. 6, expression of TUJ1, a marker for nerve cells, at the protein level was confirmed in cells cultured for 7 days using medium No. 2.
From the above, it was confirmed that direct reprogramming of monocyte-derived M2 Mφ into neural cells can be achieved by using a medium containing a combination of specific components, similar to that for monocyte-derived M1 Mφ.

[Example 3]
(Investigation of optimal concentration of PU.1 inhibitor in culture medium)
Next, the optimal concentration of the PU.1 inhibitor contained in the medium was examined.

Human mononuclear cells isolated from blood samples collected with consent from healthy subjects were seeded onto a cover glass (Matsunami Glass Industry Co., Ltd., round cover glass 18 round, product code: C018001) to a density of 5.0 x ¹⁰⁶ cells/ ^cm2 or less, and cultured for 1 hour at 37°C in a 5% _CO2 environment using Monocyte Attachment Medium (PromoCell, Inc., C-28051). Next, the floating cells in the medium were removed together with the medium, and the adherent cells (monocytes) on the cover glass were then cultured in medium containing 10 ng/mL GM-CSF relative to the total volume of the medium (composition: 10 w/v% fetal bovine serum (FBS), X-VIVO ^™ 15 serum-free lymphocyte medium (Lonza, 04-418Q)) at 37°C and 5% by volume _CO2 for 7 days to obtain Mφ having an M1 phenotype. The fact that Mφ were obtained was confirmed by detecting enhanced expression of the CD11b gene and Iba1 gene, which are marker genes for Mφ, by quantitative PCR.

Next, for the obtained human monocyte-derived M1 type Mφ, the DB2313 concentration in medium No. 2 shown in Table 1 above was varied to 0.1, 0.3, 0.5, or 0.9 μM, and four types of medium were prepared in which the other components were the same as those of medium No. 2, and the four types of medium were used to further culture the cells for 7 days in an environment of 37° C. and 5% by volume _CO2 .

Next, for cells cultured for 7 days using each medium, the percentage of the number of cells after culture relative to the number of cells at the start of culture in that medium was calculated as the cell survival rate. In addition, the percentage of the number of cells converted into neurons relative to the number of cells at the start of culture in that medium was calculated as the transdifferentiation efficiency. The conversion to neurons was confirmed by visual morphological changes (expression of dendrite-like structures) and by immunofluorescent staining to be positive for the neuronal marker TUJ1. Figure 7 is a graph showing the relationship between the DB2313 concentration in the medium and the cell survival rate and transdifferentiation efficiency.

As shown in Figure 7, the transdifferentiation efficiency tended to increase as the DB2313 concentration in the medium increased, whereas the cell survival rate tended to increase as the DB2313 concentration in the medium decreased.
It was revealed that when the DB2313 concentration in the medium was 0.3 μM or more and 0.7 μM or less, both the cell survival rate and the transdifferentiation efficiency exceeded 50%, which was particularly good.

[Example 4]
(Investigation of optimal concentration of neuronal differentiation inducer in culture medium)
Next, the optimal concentration of the neuronal differentiation-inducing factor contained in the medium was examined.

Human mononuclear cells isolated from blood samples collected with consent from healthy subjects were seeded onto a cover glass (Matsunami Glass Industry Co., Ltd., round cover glass 18 round, product code: C018001) to a density of 5.0 x ¹⁰⁶ cells/ ^cm2 or less, and cultured for 1 hour at 37°C in a 5% _CO2 environment using Monocyte Attachment Medium (PromoCell, Inc., C-28051). Next, the floating cells in the medium were removed together with the medium, and the adherent cells (monocytes) on the cover glass were then cultured in a medium containing 10 ng/mL GM-CSF relative to the total volume of the medium (composition: 10 w/v% fetal bovine serum (FBS), X-VIVO ^™ 15 serum-free lymphocyte medium (Lonza, 04-418Q) at 37°C and 5% by volume _CO2 for 7 days to obtain Mφ having an M1 phenotype. The fact that Mφ were obtained was confirmed by detecting increased expression of the CD11b gene and Iba1 gene, which are marker genes for Mφ, by quantitative PCR.

Next, for the obtained human monocyte-derived M1 type Mφ, the concentration of ISX-9 in medium No. 2 shown in Table 1 above was varied to 1, 3, 5, or 10 μM, and four types of medium were prepared in which the other components were the same as those of medium No. 2, and the cells were further cultured at 37° C. in a 5% by volume _CO2 environment for 7 days using these four types of medium.

Next, for cells cultured for 7 days using each medium, the percentage of the number of cells after culture relative to the number of cells at the start of culture in that medium was calculated as the cell survival rate. In addition, the percentage of the number of cells converted into neurons relative to the number of cells at the start of culture in that medium was calculated as the transdifferentiation efficiency. The conversion into neurons was confirmed by visual morphological changes (expression of dendrite-like structures) and by immunofluorescent staining to be positive for the neuronal marker TUJ1. Figure 8 is a graph showing the relationship between the ISX-9 concentration in the medium and the cell survival rate and transdifferentiation efficiency.

As shown in Figure 7, the higher the ISX-9 concentration in the culture medium, the higher the transdifferentiation efficiency tended to be, while the lower the ISX-9 concentration in the culture medium, the higher the cell viability tended to be.
It was revealed that when the ISX-9 concentration in the medium was 2.5 μM or more and 7.5 μM or less, both the cell survival rate and the transdifferentiation efficiency exceeded 50%, which was particularly good.

From the above, it has become clear that by using a medium containing a combination of specific components, it is possible to chemically reprogram macrophages into neurons directly.

The medium of this embodiment can provide a medium for directly transdifferentiating macrophages into nerve cells. The production method of this embodiment can produce nerve cells that have been directly transdifferentiated from macrophages. The method of this embodiment can directly transdifferentiate macrophages into nerve cells.

Claims (7)

A medium for use in direct transdifferentiation of macrophages into neural cells, comprising:
A medium comprising a glycogen synthase kinase 3 inhibitor, an adenylate cyclase activator, a Rho kinase inhibitor, a neuronal differentiation factor, and a PU.1 inhibitor. The medium of claim 1, wherein the PU.1 inhibitor is DB2313. The medium according to claim 1 or 2, wherein the content of the PU.1 inhibitor is more than 0.1 μmol/L and less than 1 μmol/L relative to the total volume of the medium. The medium according to any one of claims 1 to 3, wherein the neuronal differentiation induction factor is ISX-9. The medium according to any one of claims 1 to 4, further comprising an AMP-activated protein kinase inhibitor. A method for producing nerve cells, comprising a transdifferentiation step of culturing macrophages and directly transdifferentiating them into nerve cells using a medium containing a glycogen synthase kinase 3 inhibitor, an adenylate cyclase activator, a Rho kinase inhibitor, a neuronal differentiation inducer, and a PU.1 inhibitor. A method for directly transdifferentiating macrophages into neural cells, comprising:
A method comprising a transdifferentiation step of culturing macrophages using a medium containing a glycogen synthase kinase 3 inhibitor, an adenylate cyclase activator, a Rho kinase inhibitor, a neuronal differentiation factor, and a PU.1 inhibitor, and directly transdifferentiating the macrophages into neurons.

Images