Composition and method for transdifferentiating non-neuronal cells into neurons

Abstract

A composition and method for transdifferentiating non-neuronal cells into neurons. Disclosed is a composition for inducing cell transdifferentiation, comprising a Myosin inhibitor, and an isoxazole compound and / or its derivatives. Also disclosed is a method for inducing non-neuronal cells to transdifferentiate into neurons, comprising: culturing non-neuronal cells in an induction culture medium comprising a Myosin inhibitor, and then culturing them in a maturation culture medium comprising a Myosin inhibitor and an isoxazole compound and / or its derivatives until mature neurons are obtained. Also disclosed is a use of the composition in inducing non-neuronal cells to transdifferentiate into neurons, and a method for transdifferentiating non-neuronal cells in a subject into neurons, comprising administering an effective amount of a Myosin inhibitor and an isoxazole compound and / or its derivatives to the subject. The method of the present application is simple, requiring only two simple small molecule combination treatments, without the need for regulation by overexpression of specific genes, and can be performed efficiently both in vivo and in vitro, thereby achieving simple and efficient neuronal regeneration.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a composition and method for transdifferentiating non-neuronal cells into neurons. Background Art

[0002] Neurodegenerative diseases are a class of diseases caused by the loss of neurons and / or their myelin sheaths, which worsen over time and develop functional impairments. Common neurodegenerative diseases include Parkinson's disease (PD), Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), different types of spinocerebellar ataxia (SCA), epilepsy, stroke (also known as stroke), brain injury, and spinal cord injury. Promoting neuronal regeneration is a key link and important means for treating such diseases. How to achieve neuronal regeneration simply and efficiently has long been a hot topic of concern. At present, the existing technology has disclosed some methods for the transdifferentiation of non-neuronal cells into neurons.

[0003] Patent document 1 discloses a method for inducing the transdifferentiation of fibroblasts into neuronal cells and its application. The transformation process uses a retroviral system to stably and efficiently overexpress the above-mentioned miRNA-302 / 367 cluster, miRNA-9 and miRNA-124 in human fibroblasts, thereby regulating a series of biochemical reactions of the cells and transdifferentiating fibroblasts into neuronal cells.

[0004] Patent Document 2 discloses a pharmaceutical composition for inducing direct conversion of fibroblasts into neurons and its use. This composition achieves non-lineage-specific transdifferentiation of neurons through a combination of small molecule compounds without exogenous genes. The composition also discloses a pharmaceutical composition for inducing direct conversion of fibroblasts into neurons, the main active ingredients of which include VPA, CHIR-99021, RepSox, Forskolin, SP600125, Go6983, and Y-27632.

[0005] Patent document 3 discloses a method for inducing the reprogramming of spinal cord astrocytes into motor neurons, which selects seven small molecule drugs: SB431542, LDN193189, RA, bFGF, Purmorphamine, Forskolin, and VPA. The small molecule drugs induce the reprogramming of astrocytes in vitro, inducing the reprogramming of rat astrocytes into motor neurons.

[0006] Existing technologies can achieve neuronal transdifferentiation of non-neuronal cells such as fibroblasts or astrocytes by overexpressing transcription factors in vitro, but these methods have not yet been able to be safely applied in vivo. Furthermore, due to the advantages of small chemical molecules, such as ease of cell handling, good permeability, non-immunogenicity, and ease of local or systemic administration, several existing studies have achieved neuronal transdifferentiation of human fibroblasts through complex combinations of multiple small molecules. However, due to low transdifferentiation efficiency and the excessive number of small molecules, neuronal transdifferentiation in vivo is difficult to achieve.

[0007] Prior art literature

[0008] Patent document 1CN103849601B announcement text

[0009] Patent document 2CN106337037A public text

[0010] Patent document 3CN110283788A public text

[0011] Application Contents

[0012] To achieve safe transdifferentiation of non-neuronal cells into neurons in vivo and improve transdifferentiation efficiency, this application provides a highly efficient neuronal transdifferentiation method mediated by a simple combination of small molecule compounds. This method provides a simpler and more feasible approach for transdifferentiating non-neuronal cells from humans or animals into neurons, achieving groundbreaking and unexpected technical results. The technical solutions of this application are as follows:

[0013] The present application provides a composition for inducing cell transdifferentiation, characterized by comprising:

[0014] Myosin inhibitors,

[0015] and isoxazole compounds and / or their derivatives.

[0016] The present application provides a use of a composition comprising a Myosin inhibitor and an isoxazole compound and / or its derivatives in inducing cell transdifferentiation.

[0017] Preferably, the transdifferentiation is inducing transdifferentiation of non-neuronal cells into neurons.

[0018] The present application provides a use of a composition comprising a Myosin inhibitor and an isoxazole compound and / or its derivatives in the preparation of a medicament for treating neurodegenerative diseases.

[0019] The present application provides a method for inducing transdifferentiation of non-neuronal cells into neurons, characterized in that the method comprises treating the non-neuronal cells with a Myosin inhibitor and an isoxazole compound and / or its derivatives.

[0020] Preferably, the method provided herein for inducing non-neuronal cell transdifferentiation into neurons comprises: culturing the non-neuronal cells in an induction culture medium for 1 to 7 days, and then culturing them in a maturation culture medium for 7 to 45 days, preferably 21 to 45 days.

[0021] The present application provides a culture medium for inducing non-neuronal cells to transdifferentiate into neurons, characterized in that it comprises an induction culture medium and a maturation culture medium.

[0022] Preferably, the induction culture medium contains a Myosin inhibitor.

[0023] Preferably, the induction culture medium comprises: N2B27 culture medium and Myosin inhibitor, wherein the N2B27 culture medium is prepared by mixing DMEM / F12 and Neurobasal in a ratio of 1:1, and then adding N2 cell culture additives, B27 cell culture additives, β-mercaptoethanol, Glutamax, insulin and penicillin-streptomycin.

[0024] Preferably, the maturation culture medium comprises a Myosin inhibitor and an isoxazole compound and / or its derivatives.

[0025] Preferably, the maturation culture medium comprises: a Myosin inhibitor, an isoxazole compound and / or its derivatives, an N2B27 culture medium, a neurotrophic factor, forskolin, a Myosin inhibitor, and an isoxazole compound and / or its derivatives, wherein the N2B27 culture medium is prepared by mixing DMEM / F12 and Neurobasal in a ratio of 1:1, and then adding N2 cell culture additives, B27 cell culture additives, β-mercaptoethanol, Glutamax, insulin, and penicillin-streptomycin;

[0026] Preferably, the neurotrophic factors include: neurotrophic factor 3, brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor;

[0027] Preferably, the maturation culture medium comprises: a Myosin inhibitor, an isoxazole compound and / or its derivatives, and the N2B27 culture medium;

[0028] Preferably, the maturation culture medium consists of a Myosin inhibitor, an isoxazole compound and / or its derivatives, and the N2B27 culture medium;

[0029] Preferably, the maturation culture medium comprises: a Myosin inhibitor, an isoxazole compound and / or its derivatives, the N2B27 culture medium, neurotrophic factor 3, brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor;

[0030] Preferably, the maturation culture medium consists of a Myosin inhibitor, an isoxazole compound and / or its derivatives, the N2B27 culture medium, neurotrophic factor 3, brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor.

[0031] The present application provides a method for transdifferentiating non-neuronal cells in a subject into neurons, characterized in that the method comprises administering an effective amount of a Myosin inhibitor and an isoxazole compound and / or its derivatives to the subject.

[0032] Preferably, the method comprises: administering an effective amount of a Myosin inhibitor and an isoxazole compound and / or a derivative thereof to a subject via intraperitoneal injection.

[0033] Preferably, the method comprises: culturing non-neuronal cells in an induction culture medium and a maturation culture medium in sequence, injecting the cultured non-neuronal cells into the body, and finally administering an effective amount of a Myosin inhibitor and an isoxazole compound and / or its derivatives to the subject by intraperitoneal injection.

[0034] Preferably, the method comprises: culturing the non-neuronal cells in an induction culture medium for 1 to 7 days and a maturation culture medium for 5 to 10 days, injecting the cultured non-neuronal cells into the body, and finally administering an effective amount of a Myosin inhibitor and an isoxazole compound and / or its derivatives to the subject by intraperitoneal injection for more than 14 consecutive days.

[0035] The present application provides a method for treating a neurodegenerative disease in a subject, characterized in that the method comprises administering to the subject an effective amount of a Myosin inhibitor and an isoxazole compound and / or a derivative thereof.

[0036] Preferably, the neurodegenerative diseases include: Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, spinocerebellar ataxia, epilepsy, stroke, brain injury and spinal cord injury.

[0037] The present application provides neurons obtained by the method of inducing transdifferentiation of non-neuronal cells into neurons.

[0038] The present application provides a product or kit for transdifferentiating non-neuronal cells into neurons, characterized in that the product or kit comprises an induction culture medium and a maturation culture medium, wherein:

[0039] The induction culture medium contains a Myosin inhibitor;

[0040] The mature culture medium contains a Myosin inhibitor and an isoxazole compound and / or a derivative thereof.

[0041] The present application provides the use of Myosin inhibitors in promoting neuronal morphogenesis and initiating neural fate.

[0042] The present application provides the use of isoxazole compounds or their derivatives in promoting efficient expression of neuronal genes.

[0043] Preferably, the Myosin inhibitor is (-)-Blebbistatin and / or (-)-Blebbistatin O-Benzoate.

[0044] Preferably, the isoxazole compound or its derivative has a structure shown in formula (I),

[0045]

[0046] In formula (I), the R1 group is selected from any one of thienyl, furyl, pyrrolyl, phenyl and pyridyl; the R2 group is selected from any one of isoxazolyl, isothiazolyl, pyrazolyl, oxazolyl, thiazolyl and imidazolyl, and the R3 group is selected from any one of methyl, ethyl, cyclopropane, cyclobutane and cyclopentane; wherein the attachment site of the R1 group is any carbon atom, the two attachment sites of the R2 group are two meta carbon atoms, and the attachment site of the R3 group is any carbon atom.

[0047] Preferably, the isoxazole compound and / or its derivative is selected from any one or more of isoxazole 9 (ISX9), N-methyl-5-phenylisoxazole-3-carboxamide (ISX-PCA), N,5-dimethylisoxazole-3-carboxamide, N-methyl-5-(pyridin-4-yl)isoxazole-3-carboxamide, N-methyl-5-phenylisothiazole-3-carboxamide, N-methyl-5-phenyl-1H-pyrazole-3-carboxamide, N-methyl-2-phenyloxazole-4-carboxamide, N-methyl-2-phenylthiazole-4-carboxamide, N-methyl-2-phenyl-1H-imidazole-4-carboxamide, N-methyl-5-(thiophen-2-yl)isoxazole-3-carboxamide, 5-(furan-2-yl)-N-methylisoxazole-3-carboxamide, and N-methyl-2-(thiophen-2-yl)-1,3-thiazole-4-carboxamide.

[0048] Preferably, the non-neuronal cells are fibroblasts or astrocytes.

[0049] Effect of application

[0050] According to the present application, by using a combination of a composition comprising a Myosin inhibitor and an isoxazole and / or its derivatives, it is possible to efficiently achieve in vitro transdifferentiation of non-neuronal cells such as fibroblasts or astrocytes into neurons, providing a new cell source for obtaining neurons in vitro in regenerative medicine; and it is possible to achieve in vivo transdifferentiation into neurons by intraperitoneal injection of the composition, thereby achieving neuronal regeneration and helping to treat neurodegenerative diseases. There have been no reports of achieving cell fate changes to obtain neurons by the method of the present application. At the same time, compared with previously reported cell fate regulation methods, this method is simpler to apply, requiring only two simple small molecule combination treatments, without the need for regulation through specific gene overexpression, and achieving cell fate changes only by changing the cell culture matrix. It can be performed efficiently both in vivo and in vitro, thereby achieving simple and efficient neuronal regeneration, providing a new method for treating neurodegenerative diseases caused by aging and pathological damage in vivo.

[0051] The characteristics of the present application are: 1. Simple operation. Non-neuronal cells are added to an induction culture medium containing a Myosin inhibitor and cultured. Then, a mature culture medium containing a Myosin inhibitor and an isoxazole compound and / or its derivatives is used for culture to perform fate conversion. 2. High efficiency and rapidity. During the transdifferentiation of non-neuronal cells to neurons by the combination of Myosin inhibitors and isoxazole compounds and / or their derivatives, obvious neuronal morphology can appear on the 1st to 7th day. 3. Universality. This method has universal adaptability in the process of transdifferentiation of different types of starting cells of different species to neurons. 4. The method of the present application has stronger in vivo induction than the existing technology. 5. Safety. The method of the present application has higher safety than traditional viral vector-mediated genetic means. 6. Controllability. The sustained-release system of two simple small molecules is more feasible than the transdifferentiation mediated by a combination of multiple complex small molecules, and is convenient for metering control. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1A Microscopic photograph of human foreskin fibroblasts (HFF1y, Beijing Stem Cell Bank) (left) and microscopic photograph of induced neurons after culture of said human foreskin fibroblasts (HFF1y, Beijing Stem Cell Bank) with induction medium containing the Myosin II inhibitor (-)-Blebbistatin for 7 days and then with maturation medium containing (-)-Blebbistatin and the isoxazole compound ISX9 for 21 days (right).

[0053] Figure 1B For Figure 1A (Right) Staining results of neuronal markers in induced neurons.

[0054] Figure 1C For Figure 1A (Left) Gene expression of neuronal markers in induced neurons of human foreskin fibroblasts (HFF1y, Beijing Stem Cell Bank) cultured for 7 days in an induction medium containing (-)-Blebbistatin and then for 38 days in a maturation medium containing (-)-Blebbistatin and the isoxazole compound ISX9.

[0055] Figure 1D For Figure 1A (Left) Heatmap of the expression of neural-related genes in human foreskin fibroblasts (HFF1y, Beijing Stem Cell Bank) during the neuronal induction process of cultured in induction medium for 7 days and then in maturation medium for 38 days.

[0056] Figure 1E For Figure 1A (Right) The results of the patch clamp experiment of the induced neurons. The left picture shows the detected sodium and potassium currents, and the right picture shows the induced action potentials.

[0057] Figure 1F For Figure 1A (Left) Comparison of single-cell sequencing results of human foreskin fibroblasts (HFF1y, Beijing Stem Cell Bank) cultured in an induction medium containing (-)-Blebbistatin for 7 days and then in a maturation medium containing (-)-Blebbistatin and the isoxazole compound ISX9 for 38 days (3rd hour, 6th hour, 1st day, 2nd day, 7th day, 24th day, 30th day, 45th day) with the sequencing results of neurons in vivo.

[0058] Figure 1G For Figure 1A (Left) Comparison of gene expression of neuronal markers in induced neurons of human foreskin fibroblasts (HFF1y, Beijing Stem Cell Bank) cultured for 7 days in an induction medium containing (-)-Blebbistatin and then for 45 days in a maturation medium containing (-)-Blebbistatin and the isoxazole compound ISX9; cultured for 7 days in an induction medium containing (-)-Blebbistatin and then for 45 days in a maturation medium not containing (-)-Blebbistatin and the isoxazole compound ISX9; and a blank control group.

[0059] Figure 2AMicroscopic photograph of human foreskin fibroblasts (HFF1y, Beijing Stem Cell Bank) (left) and microscopic photograph of induced neurons after culture of said human foreskin fibroblasts (HFF1y, Beijing Stem Cell Bank) with an induction medium containing (-)-Blebbistatin for 7 days and then with a maturation medium containing (-)-Blebbistatin and ISX-PCA, a derivative of the isoxazole compound ISX9, for 21 days (right).

[0060] Figure 2B For Figure 2A (Left) Gene expression of neuronal markers in induced neurons of human foreskin fibroblasts (HFF1y, Beijing Stem Cell Bank) cultured for 7 days in an induction medium containing (-)-Blebbistatin and then for 45 days in a maturation medium containing (-)-Blebbistatin and ISX-PCA, a derivative of the isoxazole compound ISX9.

[0061] Figure 3 Figure 2. Human foreskin fibroblasts (HFF1y, Beijing Stem Cell Bank) were cultured in an induction medium containing (-)-Blebbistatin, then in a maturation medium containing (-)-Blebbistatin and the isoxazole compound ISX9, before being transplanted in vivo. The staining results for neuronal transdifferentiation following intraperitoneal injection of (-)-Blebbistatin and the isoxazole compound ISX9 are shown (transplanted cells are marked with GFP).

[0062] Figure 4A Microscopic photographs of human foreskin fibroblasts (HFF1y, Beijing Stem Cell Bank) (left) and induced neurons after culturing the human foreskin fibroblasts (HFF1y, Beijing Stem Cell Bank) with an induction medium containing (-)-Blebbistatin for 7 days (right).

[0063] Figure 4B For Figure 4A (Right) GO clustering analysis results after RNA-seq of induced neurons.

[0064] Figure 5A Microscopic photograph of human foreskin fibroblasts (HFF1y, Beijing Stem Cell Bank) (left) and microscopic photograph of induced cells after culturing the human foreskin fibroblasts (HFF1y, Beijing Stem Cell Bank) for one day with an induction medium that does not contain the Myosin II inhibitor (-)-Blebbistatin and then for six days with a maturation medium that contains the isoxazole compound ISX9 and does not contain (-)-Blebbistatin (right).

[0065] Figure 5B For Figure 5A (Right) Gene expression of neuronal markers in induced cells.

[0066] Figure 6 For Figure 5A (Left) Gene expression of classic neuronal markers in cells cultured for one day in induction medium and then for six days in neural maturation medium lacking forskolin, or lacking both neurotrophin-3, brain-derived neurotrophic factor, glial cell-derived neurotrophic factor, and forskolin. (F stands for forskolin, and FBGN stands for a mixture of forskolin, brain-derived neurotrophic factor, glial cell-derived neurotrophic factor, and neurotrophin-3.)

[0067] Figure 7A Figure 3. Morphology of induced neurons and staining results of neuronal markers in mouse astrocytes cultured for 1 day in an induction medium containing (-)-Blebbistatin and then for 13 days in a maturation medium containing (-)-Blebbistatin and the isoxazole compound ISX9.

[0068] Figure 7B For Figure 7A Expression of neuronal marker genes in induced neurons.

[0069] Figure 8 The results are a comparison of the neuronal marker gene expression of neurons obtained by induction culture using different induction culture media and mature culture media. DETAILED DESCRIPTION

[0070] The following describes and illustrates the implementation of the present application in detail through specific examples, but the following content should not be understood as limiting the present application in any way.

[0071] The terms used in this document have the following meanings:

[0072] High-glucose DMEM: A high-glucose DMEM medium (Dulbecco's modified eagle medium, DMEM), which is a commercial medium containing various glucose and amino acids, developed based on MEM medium.

[0073] N2B27 culture medium: a cell culture medium with clear ingredients, which is a mixture of DMEM / F12 basal medium and Neurobasal basal medium in a ratio of 1:1, and contains N2 cell culture additives and B27 cell culture additives. Depending on the cells to be cultured, the other ingredients in the N2B27 culture medium besides the above-mentioned ingredients are also different. It is reported that it is beneficial for mouse embryonic stem cells to differentiate in the direction of nerves. The N2B27 culture medium of the present application is prepared by mixing DMEM / F12 and Neurobasal in a ratio of 1:1, and then adding N2 cell culture additives, B27 cell culture additives, β-mercaptoethanol, Glutamax, insulin and penicillin-streptomycin.

[0074] DMEM / F12: A commercial basal culture medium made by mixing DMEM and F12 in a 1:1 ratio, suitable for culturing at high clonal densities.

[0075] Neurobasal: A commercial basal medium that facilitates the culture of neural cells.

[0076] Glutamax: A cell culture supplement that can directly replace L-glutamine in cell culture media.

[0077] Penicillin and streptomycin: Penicillin and streptomycin are two antibiotics commonly used in cell culture to prevent bacterial contamination during cell culture.

[0078] N2 Cell Culture Supplement: A commercial serum-free cell culture supplement.

[0079] B27 Cell Culture Supplement: A commercial serum-free cell culture supplement.

[0080] Neurotrophin 3: Neurotrophins (NTs) are a class of protein molecules produced by neurally innervated tissues (such as muscle) and astrocytes and are essential for neuronal growth and survival. NT-3 is a neurotrophic factor that is primarily distributed in the dorsal root ganglia, spinal cord, brainstem, cerebellum, and hippocampus of the nervous system, where it maintains the survival of sympathetic, sensory, basal forebrain cholinergic, and motor neurons.

[0081] Brain-derived neurotrophic factor (BDNF): A neurotrophic factor, it is the most abundant in the body and exerts its effects by binding to TrkB (tyrosine kinase B). It is distributed widely throughout the central nervous system, peripheral nervous system, endocrine system, bone, and cartilage, but is primarily expressed in the central nervous system, with the highest concentrations in the hippocampus and cortex.

[0082] Glial cell line-derived neurotrophic factor: It can support the survival of midbrain dopaminergic neurons in in vitro experiments, and can increase the survival rate of dopaminergic neurons and the density of nerve endings in various Parkinson's disease animal models, thereby improving its symptoms.

[0083] GABA: It is the abbreviation of gamma-aminobutyric acid, an important neurotransmitter in the central nervous system. It is an inhibitory neurotransmitter that affects learning, memory and sleep.

[0084] GABAN: It is the abbreviation of GABAergic neurons, which refers to the part of nerve cells that mainly use GABA as a neurotransmitter.

[0085] The present application provides a composition for inducing cell transdifferentiation, comprising:

[0086] Myosin inhibitors, and

[0087] Isoxazole compounds and / or their derivatives.

[0088] The derivatives in this application refer to derivatives of isoxazole compounds.

[0089] In a specific embodiment, the transdifferentiation is inducing transdifferentiation of non-neuronal cells into neurons, and the non-neuronal cells are fibroblasts or astrocytes; and the Myosin inhibitor is (-)-Blebbistatin and / or (-)-Blebbistatin O-Benzoate.

[0090] (-)-Blebbistatin used in the present invention, abbreviated as Ble, has a structure represented by formula (II).

[0091]

[0092] (-)-Blebbistatin O-Benzoate used in the present invention, abbreviated as Ble-OB, has a structure represented by formula (IIb).

[0093]

[0094] The present application also provides a use of a composition comprising a Myosin inhibitor and an isoxazole compound and / or its derivatives in inducing cell transdifferentiation.

[0095] The present application also provides a use of a composition comprising a Myosin inhibitor and an isoxazole compound and / or its derivatives in the preparation of a medicament for treating neurodegenerative diseases.

[0096] In a specific embodiment, the neurodegenerative disease includes but is not limited to Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, spinocerebellar ataxia, epilepsy, stroke, brain injury, spinal cord injury and other diseases.

[0097] The present application provides a method for inducing the transdifferentiation of non-neuronal cells into neurons in vitro, characterized in that the method comprises treating the non-neuronal cells with a Myosin inhibitor and an isoxazole compound and / or its derivatives.

[0098] In one embodiment, the method comprises: culturing the non-neuronal cells in an induction medium for 1 to 7 days, optionally 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days, and then culturing them in a maturation medium for 7 to 45 days, preferably 21 to 45 days, more preferably 30 to 45 days, optionally 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days or 45 days, etc.; wherein the induction culture medium comprises a Myosin inhibitor, and the maturation culture medium comprises a Myosin inhibitor and an isoxazole compound and / or its derivatives.

[0099] The present application also provides a method for transdifferentiating non-neuronal cells in a subject into neurons, characterized in that the method comprises administering an effective amount of a Myosin inhibitor and an isoxazole compound and / or a derivative thereof to the subject.

[0100] In one embodiment, the method comprises: placing the non-neuronal cells in an induction culture medium for 1 to 7 days, optionally 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, etc., and then culturing them in a maturation culture medium for 5 to 10 days, optionally 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, etc., preferably 7 days, and then injecting the cultured non-neuronal cells into the body, and finally administering an effective amount of a Myosin inhibitor and an isoxazole compound and / or its derivatives to the subject by intraperitoneal injection for more than 14 consecutive days (optionally 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, etc.).

[0101] The present application also provides a method for treating a neurodegenerative disease in a subject, characterized in that the method comprises administering an effective amount of a Myosin inhibitor and an isoxazole compound and / or a derivative thereof to the subject.

[0102] The present application also provides a culture medium for inducing non-neuronal cells to transdifferentiate into neurons, characterized in that it comprises an induction culture medium and a maturation culture medium.

[0103] In a specific embodiment, the induction culture medium comprises: N2B27 culture medium and a Myosin inhibitor, and the maturation culture medium comprises: N2B27 culture medium, a neurotrophic factor, forskolin, a Myosin inhibitor, and an isoxazole compound and / or its derivative; wherein the N2B27 culture medium is prepared by mixing DMEM / F12 and Neurobasal in a ratio of 1:1, and then adding N2 cell culture additives, B27 cell culture additives, β-mercaptoethanol, Glutamax, insulin, and penicillin-streptomycin.

[0104] In one embodiment, the neurotrophic factors include: neurotrophin 3, brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor.

[0105] In a specific embodiment, the maturation culture medium comprises: a Myosin inhibitor, an isoxazole compound and / or its derivatives, and the N2B27 culture medium, but does not comprise neurotrophic factors and forskolin.

[0106] In a specific embodiment, the maturation culture medium consists of a Myosin inhibitor, an isoxazole compound and / or its derivatives, and the N2B27 culture medium.

[0107] In a specific embodiment, the maturation culture medium comprises: a Myosin inhibitor, an isoxazole compound and / or its derivatives, the N2B27 culture medium, neurotrophic factor 3, brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor, but does not comprise forskolin.

[0108] In a preferred embodiment, the maturation culture medium consists of a Myosin inhibitor, an isoxazole compound and / or its derivatives, the N2B27 culture medium, neurotrophic factor 3, brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor.

[0109] In a specific embodiment, the concentration of neurotrophic factor 3 is 0-25 ng / mL, optionally 0 ng / mL, 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, 21 ng / mL, 22 ng / mL, 23 ng / mL. / mL, 24ng / mL, 25ng / mL, etc., the brain-derived neurotrophic factor is 0-25ng / mL, optionally 0ng / mL, 1ng / mL, 2ng / mL, 3ng / mL, 4ng / mL, 5ng / mL, 6ng / mL, 7ng / mL, 8ng / mL, 9ng / mL, 10ng / mL, 11ng / mL, 12ng / mL, 13ng / mL, 14ng / mL, 15ng / mL, 16ng / mL, 17ng / mL, 18ng / mL, 19ng / mL, 20ng / mL, 21ng / mL, 22ng g / mL, 23ng / mL, 24ng / mL, 25ng / mL, etc., the glial cell line-derived neurotrophic factor is 0-25ng / mL, optionally 0ng / mL, 1ng / mL, 2ng / mL, 3ng / mL, 4ng / mL, 5ng / mL, 6ng / mL, 7ng / mL, 8ng / mL, 9ng / mL, 10ng / mL, 11ng / mL, 12ng / mL, 13ng / mL, 14ng / mL, 15ng / mL, 16ng / mL, 17ng / mL, 18ng / mL, 19ng / mL, 20ng / mL , 21 ng / mL, 22 ng / mL, 23 ng / mL, 24 ng / mL, 25 ng / mL, etc., forskolin 0-20 μM, optionally 0 ng / mL, 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, etc. The concentrations described are the final concentrations of the three different nutrient factors in the maturation medium.

[0110] In a specific embodiment, the concentration of the Myosin inhibitor in the induction medium is 5 to 25 μM, optionally 5 μM, 10 μM, 15 μM, 20 μM, 21 μM, 22 μM, 23 μM, 24 μM or 25 μM. The concentration is the final concentration of the Myosin inhibitor in the induction medium used to treat non-neuronal cells.

[0111] In a specific embodiment, the concentration of the Myosin inhibitor in the maturation culture medium is 0-25 μM, optionally 0 μM, 5 μM, 10 μM, 15 μM, 20 μM, or 25 μM; the concentration of the isoxazole compound or its derivative in the maturation culture medium is 20-50 μM, optionally 20 μM, 22 μM, 24 μM, 26 μM, 28 μM, 30 μM, 32 μM, 34 μM, 36 μM, 40 μM, 42 μM, 44 μM, 46 μM, 48 μM, 50 μM, etc., wherein the above concentrations are respectively the final concentrations of the Myosin inhibitor and the isoxazole compound or its derivative in the induction medium used to treat non-neuronal cells.

[0112] In one embodiment, the non-neuronal cells are first cultured using a basal culture medium, and then cultured using an induction culture medium and a maturation culture medium in sequence.

[0113] In a specific embodiment, the basal culture medium is prepared by high-glucose DMEM plus 10% fetal bovine serum.

[0114] In a specific embodiment, the isoxazole compound is isoxazole 9 (abbreviated as ISX9), which has an isoxazole ring amide bond skeleton structure. Specifically, it has a structure shown in formula (III).

[0115]

[0116] In one embodiment, isoxazole 9 or its derivative has the structure shown in formula (I),

[0117]

[0118] Wherein, R1 can be selected from any one of thienyl, furyl, pyrrolyl, phenyl and pyridyl; R2 can be selected from any one of isoxazolyl, isothiazolyl, pyrazolyl, oxazolyl, thiazolyl and imidazolyl, and R3 can be selected from any one of methyl, ethyl, cyclopropane, cyclobutane and cyclopentane; wherein, the connection site of the R1 group is any carbon atom, the two connection sites of the R2 group are two meta carbon atoms, and the connection site of the R3 group is not limited.

[0119] The present application includes a method for inducing the transdifferentiation of non-neuronal cells into neurons using a composition comprising a Myosin inhibitor and an isoxazole compound and / or its derivatives, wherein the non-neuronal cells are cultured in an induction culture medium and a maturation culture medium, wherein the induction culture medium comprises a Myosin inhibitor; the maturation culture medium comprises a Myosin inhibitor and an isoxazole compound and / or its derivatives; during the culture process, the cells show obvious neuronal morphology, with prominent cell bodies and obvious synapses; and the positive rate of neuronal marker staining is 99.6%. Neural transcription factors are upregulated, synapse-related gene expression is upregulated, and the transdifferentiated neurons are mainly GABAergic neurons. As the culture time increases from D0 to D45, the neural-related genes become closer to neurons, and from D14 to D45, the color depth is close to that of neurons, indicating that the induced neurons have similar expression patterns to stem cell-derived neurons and primary isolated neurons. Single-cell sequencing results show that the overall transcriptional levels of neurons on days 30 and 45 of the culture process are closer to those of neurons isolated in vivo. Compared with induction culture using induction medium and mature culture medium without Ble and ISX9, the induction culture method of the present application showed higher gene expression of neuronal markers in neurons obtained. When the induced cultured cells were injected into the left hippocampus of mice, GFP-positive induced neurons were visible in the hippocampus on the side of the injected induced neurons, expressing the classic neuronal markers MAP2 and NEUN. Compared with culture medium supplemented with Ble and Ble-OB mixture and culture medium supplemented with Ble-OB, the culture medium supplemented with Ble showed the highest gene expression of neuronal markers in neurons obtained.

[0120] Example

[0121] Example 1: The combination of the small molecule Myosin II inhibitor (-)-Blebbistatin and isoxazole 9 effectively achieves neuronal transdifferentiation in vitro

[0122] Taking a 10 cm dish (Corning, 430167) as an example, 3 mL of 20 μg / mL fibronectin solution (Millipore, fc010, prepared in 1×PBS) was used for each dish for coating for 6 hours. The fibronectin solution was removed and human foreskin fibroblasts (HFF1y, Beijing Stem Cell Bank) were evenly seeded at 2×10 cells per dish. 6 The cells were cultured in basal medium (high-glucose DMEM (Gibco, C12430500BT) plus 10% fetal bovine serum (Gibco, 16000-044)) for 12 hours, the basal medium was removed, and the cells were washed once with PBS.

[0123] The cell transformation kit of the present application is used for neuronal transdifferentiation, and the kit includes the following induction culture medium and maturation culture medium.

[0124] Add induction culture medium to the HFF1y culture dish treated as above and culture for 1 to 7 days. The induction medium was prepared by adding 25 μM Ble (MCE, HY-13441) to N2B27 culture medium, which was prepared by mixing DMEM / F12 (Gibco, 10565018) and Neurobasal (Gibco, 21103-049) in a 1:1 ratio, and then adding N2 cell culture supplement (100×, Gibco, 17502048), B27 cell culture supplement (50×, Gibco, 17504044), β-mercaptoethanol (1000×, Gibco, 21985023), Glutamax (100×, Gibco, 35050-061), 1 μg / mL insulin (Roche, 11376497001), and penicillin-streptomycin (100×, Gibco, REF 15140-122). After 1 to 7 days of culture in the induction medium, HFF1y showed obvious neuronal morphology.

[0125] After 1-7 days of induction, HFF1y cells are cultured in maturation medium for 7-45 days. The maturation medium is prepared by adding 20 ng / mL neurotrophin 3 (Peprotech, 450-03), 20 ng / mL brain-derived neurotrophic factor (Peprotech, 450-02), 20 ng / mL glial cell line-derived neurotrophic factor (Peprotech, 450-10), 10 μM forskolin (Stemgent, 04-0025), 20 μM Ble (MCE, HY-13441), and 30 μM ISX9 (MCE, HY-12323) to the above-mentioned N2B27 culture medium.

[0126] To further illustrate the neural transdifferentiation effects of Ble and ISX9, the applicant provided the attached Figures 1A to 1F .

[0127] The micrograph of HFF1y after culture in high glucose DMEM plus 10% fetal bovine serum is shown in FIG. Figure 1A The micrograph of the induced neurons after culturing the HFF1y with an induction medium containing Ble for 7 days and then with a maturation medium containing Ble and ISX9 for 21 days is shown in the left figure. Figure 1A As shown in the right figure. Figure 1A As shown in the right figure, after 21 days of culture, the cells showed obvious neuronal morphology, with protruding cell bodies and obvious synapses.

[0128] right Figure 1A The cells in (right) were stained for classic neuronal markers. The staining results are shown in Figure 1BNeuronal markers included TUJ1 (Covance, MRB-435P), MAP2 (Santa Cruz Biotechnology, sc-20172), NF2000 (Abcam, ab4680), and NEUN (Chemicon, MAB377). All of these neuronal markers stained positive, with a positivity rate approaching 100%, specifically 99.6%.

[0129] Will Figure 1A (Left) The expression of neuronal marker genes in HFF1y cells after 7 days of induction with induction medium and 38 days of culture with maturation medium. Figure 1C As shown in the figure, the fibroblast marker FSP1 is downregulated; classic neural markers such as GFAP, DCX, TUJ1, MAP2, and NEUN are significantly upregulated; at the same time, neural transcription factors such as ASCL1, BRN2, and NEUROD1 are upregulated, indicating that the induced cells have acquired a neural fate; synapse-related genes such as NEFH, PSD95, SYN1, and SYT are upregulated, laying the foundation for the induction of neuronal function; in addition, the expression of PVALB, GAD, and GABBR3 genes is upregulated, indicating that the transdifferentiated neurons are mainly GABAergic neurons.

[0130] Heat map of the expression of neural-related genes during neuronal induction Figure 1D As shown. D0 represents the initial cell state of human foreskin fibroblasts (HFF1y, Beijing Stem Cell Bank) on day 0 of induction; h represents hours; D represents days; 3h, 6h, D1, D2, D7, D14, D30, and D45 represent different induction culture stages, i.e., Figure 1A (Left) HFF1y cells were induced with induction medium at 3 hours, 6 hours, day 1, day 2, and day 7 (corresponding to 3 hours, 6 hours, D1, D2, and D7 in the figure, respectively), and then cultured in maturation medium on days 7, 23, and 38 (corresponding to D14, D30, and D45 in the figure, respectively). HN cDNA represents primary human neurons; GABAN represents GABAergic neurons differentiated from human pluripotent stem cells. The vertical axis on the right side of the figure indicates the gene name, and the horizontal axis indicates the sample name. Each colored square indicates the expression level of the gene, and the color scale is in the upper left corner. Each row represents the expression level of each gene in different samples, and each column represents the expression level of all genes in each sample. The dendrogram on the left shows the clustering analysis results of different genes from different samples. Figure 1D It can be clearly seen that as the induction time increases from D0 to D45, the neural-related genes become closer to neurons, indicating that the induced neurons have similar expression patterns to the stem cell-derived neurons and the primary isolated neurons.

[0131] right Figure 1A (Right) The induced neurons were subjected to patch clamp experiments, and the results are as follows Figure 1E As shown. Patch clamp is an electrophysiological technique primarily used to monitor cell membrane ion channel activity and evoked action potentials. Electrophysiological results showed that the induced neurons had sodium and potassium currents and evoked action potentials typical of mature neurons, indicating that the induced neurons had developed electrophysiological properties.

[0132] Figure 1F For Figure 1A (Left) Single-cell sequencing results of human foreskin fibroblasts (HFF1y, Beijing Stem Cell Bank) induced with the induction medium of this example for 7 days and then cultured with the maturation medium of this example for 38 days. D0 represents the initial cell state on day 0 of induction, 3h, 6h, D1, D2, D7, D14, D30, and D45 represent the 3rd hour, 6th hour, 1st day, 2nd day, 7th day, 24th day, 30th day, and 45th day of culture, respectively, using this method. "Neuron 1-6" represents neurons isolated in vivo. The analysis results show that the overall transcriptional levels of neurons on days 30 and 45 of the culture process are closer to those of neurons isolated in vivo.

[0133] Comparative Example 1

[0134] The experimental operation of Example 1 was performed with the exception that the cells were cultured with the induction medium for 7 days and then cultured with the maturation medium (compared to the maturation medium in Example 1, the maturation medium in this comparative example did not contain Ble and ISX9) for 38 days. The gene expression of neuronal markers in the cells was as follows: Figure 1G As shown, Figure 1G Also shown are the gene expression of neuronal markers in cells cultured for 7 days using the induction medium of Example 1 and then cultured for 38 days using the maturation medium of Example 1, as well as the gene expression of neuronal markers in cells of the blank control. Figure 1G This indicates that the gene expression levels of neuronal markers in neurons obtained using the induction culture method of the present application are higher.

[0135] Example 2: Ble combined with isoxazole compounds or their derivatives to achieve efficient neuronal transdifferentiation

[0136] A derivative of the isoxazole compound ISX9, N-methyl-5-phenylisoxazole-3-carboxamide (ISX-PCA, 20-50 μM, TCI, BD399148), was selected. The R1 group was a benzene ring, and the R3 group was a methyl group. The resulting ISX-PCA structure is shown in Formula (IV). Neuronal transdifferentiation was performed using ISX-PCA in place of ISX9 according to the method of Example 1.

[0137]

[0138] Figure 2A The figure shows a micrograph of HFF1y (left) and a micrograph of induced neurons after culturing HFF1y with an induction medium containing Ble for 7 days and then with a maturation medium containing Ble and ISX-PCA for 21 days (right). Figure 2A (Right) As shown, the cells induced by the combination of Ble and ISX-PCA Figure 1A (Right) The cell morphology is similar, with obvious neuronal morphology, prominent cell bodies, and obvious synapses.

[0139] Further targeting Figure 2A (Right) Detection of gene expression of classic neuronal markers. Figure 2B As shown, classic neuronal markers such as GFAP, DCX, TUBB3, MAP2, NEUN, MAPT, and NEFH were significantly upregulated; at the same time, neural transcription factors such as ASCL1, BRN2, and NEUROD1 were upregulated, indicating that the transdifferentiated cells acquired neural fate.

[0140] Example 3: Ble combined with isoxazoles and their derivatives promotes neural transdifferentiation of human fibroblasts in vivo

[0141] Taking a 10 cm dish (Corning, 430167) as an example, 3 mL of 20 μg / mL fibronectin solution (millipore, fc010, prepared in 1×PBS) was used for each dish for coating for 6 hours. The fibronectin solution was removed and human foreskin fibroblasts (HFF13y-GFP, Beijing Stem Cell Bank) were evenly seeded at 2×10 per dish. 6 The cells were cultured in basal medium (high-glucose DMEM (Gibco, C12430500BT) plus 10% fetal bovine serum (Gibco, 16000-044)) for 12 hours, the basal medium was removed, and the cells were washed once with PBS.

[0142] After the above treatment, the induction medium of Example 1 was added to the HFF13y-GFP culture dish and induced for 7 days. Then, the mature culture medium of Example 1 was used for 7 days. Then, 2.5×10 5The number of cells per mouse was injected into the left hippocampus of 5-week-old SCID mice, and the transplanted cells were labeled with green fluorescent protein (GFP). Then, the mice were intraperitoneally injected with 3mg / kg Ble + 10mg / kg ISX9 every day for 14 consecutive days. The injection solvent was dimethyl sulfoxide (DMSO) (2% final volume) + PEG400 (40% final volume) + Tween 80 (2% final volume) + sterile water. After 14 days, the hippocampus was taken for paraffin section staining to detect the expression of GFP and classic neural markers. The results are as follows Figure 3 As shown, from top to bottom, the first and second rows are the control groups, which are the staining results of neuronal marker proteins MAP2 (first row), NEUN (second row) and GFP (derived from transplanted induced cells) in the brain side of mice without transplanted cells after in vivo induction; Figure 3 The third and fourth rows are the staining results of neuronal marker proteins MAP2 (third row), NEUN (fourth row) and GFP (derived from transplanted fibroblasts to be induced) on one side of the brain of the same mouse after in vivo induction of transplanted cells. Merge represents overlapping images. The third and fourth rows show the presence of GFP-positive cells expressing MAP2 (cells with GFP signals co-localized with MAP2 signals in the third row overlapping image) and NEUN (cells with GFP signals co-localized with NEUN signals in the fourth row overlapping image), indicating that the transplanted GFP cells have become neurons after in vivo induction. Figure 3 It can be seen that in the hippocampus on the side of the injected induced neurons, GFP-positive induced neurons were visible, and the classic neuronal markers MAP2 and NEUN were expressed.

[0143] Example 4: Ble promotes neuronal morphogenesis and upregulation of neural fate-related genes

[0144] Take a 10 cm dish (Corning, 430167) as an example, use 3 mL of 20 μg / mL fibronectin solution (millipore, fc010, prepared in 1× PBS) per dish and coat for 6 hours. Remove the fibronectin solution and evenly inoculate HFF1y at 2×10 per dish. 6 The cells were cultured in basal medium (high-glucose DMEM (Gibco, C12430500BT) plus 10% fetal bovine serum (Gibco, 16000-044)) for 12 hours, the basal medium was removed, and the cells were washed once with PBS.

[0145] After adding the induction culture medium of Example 1 to the HFF1y culture dish treated as above and culturing for 1 to 7 days, the cell morphology changes are shown as follows: Figure 4AAfter induction, the cells have obvious neuronal morphology, with small and round cell bodies and abundant and slender synapses.

[0146] RNA-seq and GO cluster analysis were performed on the above cells, and the results were as follows Figure 4B As shown, the length of red represents the negative logarithm of the significant p-value enriched to the process (p<0.05). Figure 4B It shows that in the neural induction culture system containing only Ble, the upregulated genes in the induced cells are enriched in pathways related to neuronal fate, which further indicates that Ble can alone promote the development of neuronal morphology and the upregulation of genes related to neural fate.

[0147] Example 5: Isoxazole compounds and their derivatives potently promote neural fate conversion at the transcriptional level

[0148] The experimental operation of Example 1 was referred to, except that, for HFF1y, the cells were cultured with an induction medium (compared to the induction medium in Example 1, the induction medium in this example did not contain Ble) for 7 days, and then cultured with a maturation medium (compared to the maturation medium in Example 1, the maturation medium in this example did not contain Ble) for 7 days. The cell morphology changes were shown in FIG. Figure 5A (right), Figure 5A (Left) is HFF1y. By comparison, the induced cell morphology does not show a neuron-like morphology.

[0149] Further targeting Figure 5A (Right) Detection of gene expression of classic neuronal markers. Figure 5B As shown, the fibroblast marker FSP1 was significantly downregulated, while the expression of classic neuronal markers such as GFAP, TUJ1, MAP2, MAPT, STMN1, NCAM and synaptic-related proteins NEFH, PSD95, SYN1, and SYT was significantly upregulated; at the same time, neural transcription factors such as ASCL1, BRN2, and NEUROD1 were upregulated, indicating that the transdifferentiated cells acquired a neural fate.

[0150] Based on the results of Example 4 and Example 5, it can be inferred that in the process of promoting the efficient conversion of fibroblasts to neurons by the combination of Ble and isoxazole compounds or their derivatives, Ble is responsible for the occurrence of neural morphology and initiating the conversion of neural fate, while isoxazole compounds or their derivatives strongly promote the complete transdifferentiation of neuronal fate.

[0151] Example 6 Ble combined with isoxazoles or their derivatives has a potent neuronal induction effect

[0152] To further verify the potent neuronal induction effect of Ble+isoxazole or its derivatives. Figure 5A(Left) After culturing for 7 days in the induction medium of Example 1, we used the neural maturation medium of Example 1 without forskolin, or without neurotrophic factor 3, brain-derived neurotrophic factor, glial cell-derived neurotrophic factor, and forskolin, and cultured for 7 days to detect the gene expression of classical neuronal markers. The results are shown in Figure 1. Figure 6 As shown, in cells treated with neural maturation culture medium without forskolin, or without three neurotrophic factors and forskolin at the same time, classic neuronal markers such as DCX, TUBB3, MAP2, NEUN, MAPT, STMN1, and synaptic-related proteins NEFH, SYT1, SYN1, and PSD95 were significantly upregulated. At the same time, neural transcription factors such as ASCL1, BRN2, and NEUROD1 were upregulated, indicating that the transdifferentiated cells acquired a neural fate.

[0153] Example 7: Ble combined with isoxazole compounds or their derivatives to achieve efficient transdifferentiation of mouse astrocytes into neurons

[0154] Astrocytes are one of the main cells in the brain environment and are involved in the physiological functions of the brain. Transdifferentiation of astrocytes into neurons has important guiding significance for the treatment of neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease.

[0155] The experimental procedures of Example 1 were referred to, except that HFF1y was replaced with astrocytes from mouse cerebral cortex. Mouse astrocytes were induced with the induction medium of Example 1 for 1 day and then cultured with the maturation medium of Example 1 for 13 days. After 13 days, the cell morphology and staining of classical neuronal markers were as follows: Figure 7A The cells induced from mouse astrocytes have a morphology similar to neurons, with small and round cell bodies, abundant synapses, and expression of classic neuronal markers TUJ1, MAP2, and NEUN.

[0156] Figure 7A The neuronal marker gene expression of induced cells is as follows Figure 7B The results showed that classic neural markers such as DCX, TUBB3, MAP2, NEUN, MAPT, and NEFH were significantly upregulated; the expression of postsynaptic density protein PSD95 was upregulated; at the same time, neural transcription factors such as ASCL1, BRN2, and MYTL1 were upregulated, indicating that the transdifferentiated cells acquired a neural fate.

[0157] Example 8

[0158] The experimental procedure was the same as in Example 1, except that the cells were cultured with the induction medium for 7 days and then with the maturation medium for 7 days.

[0159] Example 9

[0160] Refer to the experimental operation of Example 1, except that the cells were cultured with induction culture medium (relative to the induction culture medium in Example 1, the induction culture medium in this example used Ble-OB instead of Ble) for 7 days, and then cultured with maturation culture medium (relative to the maturation culture medium in Example 1, the maturation culture medium in this example used Ble-OB instead of Ble) for 7 days.

[0161] Example 10

[0162] Refer to the experimental operation of Example 1, except that the cells were cultured with induction culture medium (relative to the induction culture medium in Example 1, the induction culture medium in this example used Ble-OB instead of Ble) for 7 days, and then cultured with maturation culture medium (relative to the maturation culture medium in Example 1, the maturation culture medium in this example used ISX-PCA instead of ISX9, and Ble-OB instead of Ble) for 7 days.

[0163] Example 11

[0164] Refer to the experimental operation of Example 1, except that the cells were cultured for 7 days with induction culture medium (relative to the induction culture medium in Example 1, the induction culture medium of this example used a 1:1 mixture of Ble-OB and Ble instead of Ble, and the total concentration of Ble-OB and Ble in the induction culture medium of this example was equal to the concentration of Ble in the induction culture medium of Example 1) and then cultured for 7 days with maturation culture medium (relative to the maturation culture medium in Example 1, a 1:1 mixture of Ble-OB and Ble was used instead of Ble, and the total concentration of Ble-OB and Ble in the maturation culture medium was equal to the concentration of Ble in the maturation culture medium of Example 1).

[0165] Example 12

[0166] Refer to the experimental procedures of Example 1, except that the cells were cultured for 7 days with induction culture medium (relative to the induction culture medium in Example 1, the induction culture medium of this example used Ble-OB instead of Ble) and then cultured for 7 days with maturation culture medium (relative to the maturation culture medium in Example 1, the maturation culture medium of this example used Ble-OB instead of Ble, and a 1:1 mixture of ISX9 and ISX-PCA was used instead of ISX9, and the total concentration of ISX9 and ISX-PCA in the maturation culture medium of this example was equal to the ISX9 concentration in the maturation culture medium of Example 1).

[0167] Comparative Example 2

[0168] Refer to the experimental operation of Comparative Example 1, except that the cells were cultured with the induction culture medium for 7 days and then cultured with the maturation culture medium for 7 days.

[0169] The gene expression of neuronal markers (MAPT and MAP2) in the neurons obtained in Examples 8 to 12 and Comparative Example 2 is as follows: Figure 8 As shown, the expression levels of neural marker genes in neurons induced by culture medium supplemented with isoxazoles and / or their derivatives were significantly increased compared to culture medium without isoxazoles and / or their derivatives. Furthermore, a comparison of the expression levels of neural marker genes in neurons cultured in medium supplemented with Ble, a mixture of Ble and Ble-OB, and Ble-OB showed that the highest expression levels of neural marker genes were observed in neurons cultured in medium supplemented with Ble, followed by the mixture of Ble and Ble-OB, and then the medium supplemented with Ble-OB.

[0170] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application in any other manner. Any person skilled in the art may utilize the above disclosed technical content to modify or modify the present application into equivalent embodiments with equivalent variations. However, any simple modifications, equivalent variations, and modifications to the above embodiments that do not depart from the technical content of the present application and are based on the technical essence of the present application shall still fall within the scope of protection of the present application.

Claims

1. A composition for inducing cell transdifferentiation, characterized in that: include: Myosin inhibitors, and isoxazole compounds and / or their derivatives; Wherein, the Myosin inhibitor is (-)-Blebbistatin; The isoxazole compound is isoxazole 9 (ISX9), and the derivative of the isoxazole compound is N-methyl-5-phenylisoxazole-3-carboxamide (ISX-PCA); The transdifferentiation is to induce fibroblasts or astrocytes to transdifferentiate into neurons.

2. Use of a composition comprising a Myosin inhibitor and an isoxazole compound and / or its derivatives in inducing cell transdifferentiation in vitro; in, The Myosin inhibitor is (-)-Blebbistatin; The isoxazole compound is isoxazole 9 (ISX9), and the derivative of the isoxazole compound is N-methyl-5-phenylisoxazole-3-carboxamide (ISX-PCA); The transdifferentiation is to induce fibroblasts or astrocytes to transdifferentiate into neurons.

3. Use of a composition comprising a Myosin inhibitor and an isoxazole compound and / or its derivatives in the preparation of a medicament for treating neurodegenerative diseases; in, The Myosin inhibitor is (-)-Blebbistatin; The isoxazole compound is isoxazole 9 (ISX9), and the derivative of the isoxazole compound is N-methyl-5-phenylisoxazole-3-carboxamide (ISX-PCA).

4. The use according to claim 3, characterized in that The neurodegenerative diseases include: Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, spinocerebellar ataxia, epilepsy, stroke, brain injury and spinal cord injury.

5. A method for inducing transdifferentiation of non-neuronal cells into neurons, characterized in that: The method comprises treating non-neuronal cells with a Myosin inhibitor and an isoxazole compound and / or a derivative thereof; Wherein, the Myosin inhibitor is (-)-Blebbistatin; The isoxazole compound is isoxazole 9 (ISX9), and the derivative of the isoxazole compound is N-methyl-5-phenylisoxazole-3-carboxamide (ISX-PCA); The transdifferentiation is to induce fibroblasts or astrocytes to transdifferentiate into neurons.

6. The method according to claim 5, characterized in that The method comprises: culturing non-neuronal cells in an induction culture medium for 1 to 7 days, and then culturing in a maturation culture medium for 7 to 45 days; Wherein, the induction culture medium contains a Myosin inhibitor; The mature culture medium contains a Myosin inhibitor and an isoxazole compound and / or a derivative thereof.

7. The method according to claim 6, comprising: The non-neuronal cells are cultured in an induction medium for 1 to 7 days and then cultured in a maturation medium for 21 to 45 days.

8. The method according to claim 6, wherein The induction culture medium further comprises N2B27 culture medium, wherein the N2B27 culture medium is prepared by mixing DMEM / F12 and Neurobasal in a ratio of 1:1, and then adding N2 cell culture additives, B27 cell culture additives, β-mercaptoethanol, Glutamax, insulin and penicillin-streptomycin.

9. The method according to claim 6, wherein The maturation culture medium comprises: Myosin inhibitor, isoxazole compound and / or its derivative, N2B27 culture medium, neurotrophic factor, forskolin, wherein the N2B27 culture medium is prepared by mixing DMEM / F12 and Neurobasal in a ratio of 1:1, and then adding N2 cell culture additive, B27 cell culture additive, β-mercaptoethanol, Glutamax, insulin and penicillin-streptomycin.

10. The method according to claim 9, wherein The neurotrophic factors include: neurotrophic factor 3, brain-derived neurotrophic factor and glial cell-derived neurotrophic factor.

11. The method according to claim 6, wherein The mature culture medium comprises: a Myosin inhibitor, an isoxazole compound and / or its derivatives, and an N2B27 culture medium.

12. The method according to claim 6, wherein The mature culture medium consists of a Myosin inhibitor, an isoxazole compound and / or its derivatives, and an N2B27 culture medium.

13. The method according to claim 6, wherein The maturation culture medium comprises: Myosin inhibitor, isoxazole compound and / or derivatives thereof, N2B27 culture medium, neurotrophic factor 3, brain-derived neurotrophic factor and glial cell-derived neurotrophic factor.

14. The method according to claim 6, wherein The mature culture medium consists of Myosin inhibitor, isoxazole compound and / or its derivative, N2B27 culture medium, neurotrophic factor 3, brain-derived neurotrophic factor and glial cell-derived neurotrophic factor.

15. Neurons obtained by the method according to any one of claims 5 to 6 and 8 to 14.

16. A product or kit for transdifferentiating non-neuronal cells into neurons, characterized in that: The product or kit includes an induction culture medium and a maturation culture medium, wherein The induction culture medium contains a Myosin inhibitor; The mature culture medium comprises a Myosin inhibitor and an isoxazole compound and / or its derivatives; Wherein, the Myosin inhibitor is (-)-Blebbistatin; The isoxazole compound is isoxazole 9 (ISX9), and the derivative of the isoxazole compound is N-methyl-5-phenylisoxazole-3-carboxamide (ISX-PCA); The transdifferentiation is to induce fibroblasts or astrocytes to transdifferentiate into neurons.

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