Compositions and methods for transdifferentiating non-neuronal cells into neurons

Abstract

A composition for inducing cell transdifferentiation and its use in inducing the transdifferentiation of non-neuronal cells into neurons is provided, the composition comprising a myosin inhibitor and an isoxazole compound and / or a derivative thereof. Also provided is a method for inducing the transdifferentiation of non-neuronal cells into neurons, comprising culturing non-neuronal cells in an induction medium comprising the myosin inhibitor, followed by culturing the non-neuronal cells in a maturation medium comprising the myosin inhibitor and the isoxazole compound and / or a derivative thereof until mature neurons are obtained. Also provided is a method for transdifferentiating non-neuronal cells into neurons in a subject, comprising administering to the subject an effective amount of the myosin inhibitor and the isoxazole compound and / or a derivative thereof.

Description

[Technical Field]

[0001] TECHNICAL FIELD This application relates to the field of biotechnology, and more particularly to compositions and methods for transdifferentiating non-neuronal cells into neurons. [Background technology]

[0002] Neurodegenerative diseases are caused by the loss of neurons and / or their myelin, which deteriorates and becomes dysfunctional over time. Common neurodegenerative diseases include Parkinson's disease (PD), Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), various types of spinocerebellar ataxia (SCA), epilepsy, stroke, brain injury, and spinal cord injury. Promoting neuronal regeneration is an important part and tool in the treatment of such diseases. A simple and efficient method for achieving neuronal regeneration has long been a hotspot of interest. Currently, the prior art has disclosed several methods for transdifferentiation of non-neuronal cells into neurons.

[0003] Patent Document 1 discloses a method for inducing fibroblasts to transdifferentiate into neurons and its application. In the transformation process, a retroviral system is used to stably and efficiently overexpress the aforementioned miRNA-302 / 367 cluster, miRNA-9, and miRNA-124 in human fibroblasts, thereby regulating a series of intracellular biochemical reactions and transdifferentiating the fibroblasts into neurons.

[0004] Patent Document 2 discloses a pharmaceutical composition that induces the direct transformation of fibroblasts into neurons and its use, and by combining low-molecular-weight compounds, lineage-free transdifferentiation of neurons was achieved without the use of exogenous genes. A pharmaceutical composition that induces the direct transformation of fibroblasts into neurons is disclosed, and its main active ingredients 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. Seven small molecule drugs, SB431542, LDN193189, RA, bFGF, Purmorphamine, Forskolin, and VPA, were selected to induce astrocyte reprogramming in vitro, and the small molecule drugs induced the reprogramming of rat astrocytes into motor neurons.

[0006] In prior art, overexpression of transcription factors in vitro has been shown to achieve neuronal transdifferentiation of non-neuronal cells such as fibroblasts or astrocytes, but these methods have not been able to achieve safe application in vivo. Furthermore, chemical small molecules have the advantages of convenient cell processing, high permeability, non-immunogenicity, and ease of local or systemic administration. Numerous prior art studies have achieved neuronal transdifferentiation of human fibroblasts using complex combinations of multiple small molecules. However, due to low transdifferentiation efficiency and the excessive number of small molecules involved, neuronal transdifferentiation in vivo is difficult. [Prior art documents] [Patent documents]

[0007] Patent Document 1: Chinese Patent No. 103849601 Specification Patent Document 2: Chinese Patent Application Publication No. 106337037 Patent Document 3: Chinese Patent Application Publication No. 110283788 Summary of the Invention

[0008] In order to achieve safe transdifferentiation of non-neuronal cells into neurons in vivo and improve transdifferentiation efficiency, the present application provides a highly efficient neuronal transdifferentiation method mediated by a simple combination of small molecule compounds, proposes a more convenient and simple method for transdifferentiating human or animal non-neuronal cells into neuronal cells, and achieves pioneering and unexpected technical effects. The technical proposal of the present application is as follows:

[0009] The present application relates to a myosin inhibitor and Isoxazole and / or its derivatives The present invention provides a composition for inducing cell transdifferentiation, comprising:

[0010] The present application provides the use of a composition comprising a myosin inhibitor and an isoxazole compound and / or a derivative thereof in inducing cell transdifferentiation.

[0011] Preferably, said transdifferentiation induces the transdifferentiation of a non-neuronal cell into a neuron.

[0012] The present application provides the use of a composition comprising a myosin inhibitor and an isoxazole compound and / or a derivative thereof in the preparation of a medicament for the treatment of a neurodegenerative disease.

[0013] The present application provides a method for inducing transdifferentiation of non-neuronal cells into neurons, comprising treating the non-neuronal cells with a myosin inhibitor and an isoxazole compound and / or a derivative thereof.

[0014] Preferably, the method provided herein for inducing transdifferentiation of non-neuronal cells into neurons comprises culturing the non-neuronal cells in an induction medium for 1 to 7 days, followed by culturing them in a maturation medium for 7 to 45 days, preferably 21 to 45 days.

[0015] The present application provides a medium for inducing transdifferentiation of non-neuronal cells into neurons, comprising an induction medium and a maturation medium.

[0016] Preferably, the induction medium contains a myosin inhibitor.

[0017] Preferably, the induction medium comprises N2B27 medium and a myosin inhibitor, and the N2B27 medium is prepared by mixing DMEM / F12 and Neurobasal in a 1:1 ratio, and then adding N2 cell culture supplement, B27 cell culture supplement, β-mercaptoethanol, Glutamax, insulin, and penicillin.

[0018] Preferably, the maturation medium comprises a myosin inhibitor and an isoxazole compound and / or a derivative thereof.

[0019] Preferably, the maturation medium comprises a myosin inhibitor, an isoxazole compound and / or a derivative thereof, an N2B27 medium, a neurotrophic factor, forskolin, a myosin inhibitor, an isoxazole compound and / or a derivative thereof, and the N2B27 medium is prepared by mixing DMEM / F12 and Neurobasal in a 1:1 ratio, and then adding N2 cell culture supplement, B27 cell culture supplement, β-mercaptoethanol, Glutamax, insulin, and penicillin; Preferably, the neurotrophic factors include neurotrophic factor 3, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor; Preferably, the maturation medium comprises a myosin inhibitor, an isoxazole compound and / or a derivative thereof, and the N2B27 medium; Preferably, the maturation medium comprises a myosin inhibitor, an isoxazole compound and / or a derivative thereof, and the N2B27 medium; Preferably, the maturation medium comprises a myosin inhibitor, an isoxazole compound and / or a derivative thereof, the N2B27 medium, neurotrophic factor 3, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor; Preferably, the maturation medium consists of a myosin inhibitor, an isoxazole compound and / or its derivative, the N2B27 medium, neurotrophic factor 3, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor.

[0020] The present application provides a method for transdifferentiating non-neuronal cells into neurons in a subject, comprising administering to the subject an effective amount of a myosin inhibitor and an isoxazole compound and / or its derivative.

[0021] Preferably, the method comprises administering to a subject an effective amount of a myosin inhibitor and an isoxazole and / or a derivative thereof by intraperitoneal injection.

[0022] Preferably, the method comprises sequentially culturing non-neuronal cells in an induction medium and a maturation medium, then injecting the cultured non-neuronal cells into the body, and finally administering an effective amount of a myosin inhibitor and an isoxazole and / or its derivative to the subject by intraperitoneal injection.

[0023] Preferably, the method comprises culturing the non-neuronal cells in an induction medium for 1 to 7 days and in a maturation medium for 5 to 10 days, injecting the cultured non-neuronal cells into the body, and finally administering effective amounts of a myosin inhibitor and an isoxazole compound and / or its derivatives to the subject by intraperitoneal injection for 14 or more consecutive days.

[0024] The present application provides a method for treating a neurodegenerative disease in a subject, comprising administering to the subject an effective amount of a myosin inhibitor and an isoxazole compound and / or a derivative thereof.

[0025] Preferably, the neurodegenerative disease comprises Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, spinocerebellar ataxia, epilepsy, stroke, brain injury, and spinal cord injury.

[0026] The present application provides neurons obtained by the above-described method of inducing transdifferentiation of non-neuronal cells into neurons.

[0027] The present application provides a product or kit for transdifferentiating non-neuronal cells into neurons, the product or kit comprising an induction medium and a maturation medium, the induction medium comprises a myosin inhibitor; The maturation medium is characterized by containing a myosin inhibitor and an isoxazole compound and / or a derivative thereof.

[0028] The present application provides the use of myosin inhibitors in promoting neuronal morphogenesis and initiation of neural fates.

[0029] The present application provides the use of an isoxazole compound or a derivative thereof in promoting high expression of neuronal genes.

[0030] Preferably, the myosin inhibitor is (-)-Blebbistatin and / or (-)-Blebbistatin O-Benzoate.

[0031] Preferably, the isoxazole compound or derivative thereof has a structure represented by formula (I):

[0032] [ka]

[0033] In formula (I), the R1 group is any one selected from thienyl, furyl, pyrrolyl, phenyl, and pyridyl; the R2 group is any one selected from isoxazolyl, isothiazolyl, pyrazolyl, oxazolyl, thiazolyl, and imidazolyl; and the R3 group is any one selected from methyl, ethyl, cyclopropyl, cyclobutyl, and cyclopentyl, wherein the linking site of the R1 group is any carbon atom, the two linking sites of the R2 group are two meta-position carbon atoms, and the linking site of the R3 group is any carbon atom.

[0034] Preferably, the isoxazole compound and / or derivative thereof is 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- Any one or more selected from 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.

[0035] Preferably, the non-neuronal cells are fibroblasts or astrocytes. [Effects of the Invention]

[0036] According to the present application, the combined treatment of a composition containing a myosin inhibitor and an isoxazole and / or its derivatives allows for efficient in vitro transdifferentiation of non-neuronal cells, such as fibroblasts or astrocytes, into neurons, providing a new cell source for in vitro neuronal generation in regenerative medicine. Furthermore, intraperitoneal injection of the composition can achieve in vivo neuronal transdifferentiation, leading to neuronal regeneration and potentially useful for the treatment of neurodegenerative diseases. This method has not been reported to achieve cell fate change to obtain neurons. At the same time, compared to previously reported cell fate control methods, this method is simpler to apply. By combining two simple small molecules, cell fate change can be achieved simply by modifying the cell culture substrate, without the need for specific gene overexpression control. This method can be efficiently performed in vitro and in vivo, enabling simple and efficient neuronal regeneration. This provides a new approach for the in vivo treatment of neurodegenerative diseases caused by aging or pathological damage.

[0037] The present invention has the following features: 1. It is simple to operate: non-neuronal cells are cultured in an induction medium containing a myosin inhibitor, followed by the use of a maturation medium containing a myosin inhibitor and an isoxazole compound and / or its derivative for fate conversion. 2. It is efficient and rapid: during the process of transdifferentiation from non-neuronal cells to neurons using the combination of a myosin inhibitor and an isoxazole compound and / or its derivative, clear neuronal morphology can appear within 1 to 7 days. 3. It is universally applicable: this method can be generally applied to the process of transdifferentiation of different types of initiating cells from different species into neurons. 4. Compared to prior art methods, the present method has stronger in vivo induction. 5. It is safe: the present method is safer than conventional viral vector-mediated gene methods. 6. It is controllable: compared to transdifferentiation mediated by a combination of multiple complex small molecules, a sustained release system of two simple small molecules is more feasible and easier to control in terms of dosage. [Brief explanation of the drawings]

[0038] [Figure 1A] Figure 1A shows a micrograph (left) of human foreskin fibroblasts (HFF1y, Beijing Stem Cell Bank) and a micrograph (right) of induced neurons after culturing the human foreskin fibroblasts (HFF1y, Beijing Stem Cell Bank) in induction medium containing the myosin II inhibitor (-)-Blebbistatin for 7 days and then in maturation medium containing (-)-Blebbistatin and the isoxazole compound ISX9 for 21 days. [Figure 1B] FIG. 1B shows staining results for neuronal markers of the induced neurons in FIG. 1A (right). [Figure 1C] Figure 1C shows the gene expression profiles of neuronal markers in induced neurons after culturing the human foreskin fibroblasts (HFF1y, Beijing Stem Cell Bank) shown in Figure 1A (left) in induction medium containing (-)-Blebbistatin for 7 days, followed by culturing in maturation medium containing (-)-Blebbistatin and the isoxazole compound ISX9 for 38 days. [Figure 1D] Figure 1D shows a heat map of the expression of neural-related genes during neuronal induction of human foreskin fibroblasts (HFF1y, Beijing Stem Cell Bank) shown in Figure 1A (left), which were cultured in induction medium for 7 days followed by maturation medium for 38 days. [Figure 1E] Figure 1E shows the results of a patch clamp experiment on the induced neuron in Figure 1A (right). The left panel shows detected sodium and potassium currents, and the right panel shows evoked action potentials. [Figure 1F] Figure 1F shows a comparison of single-cell sequencing results and in vivo neuron sequencing results for the entire process (3 hours, 6 hours, day 1, day 2, day 7, day 24, day 30, and day 45) of culturing human foreskin fibroblasts (HFF1y, Beijing Stem Cell Bank) shown in Figure 1A (left) in induction medium containing (-)-Blebbistatin for 7 days, followed by maturation medium containing (-)-Blebbistatin and the isoxazole compound ISX9 for 38 days. [Figure 1G]Figure 1G is a comparative graph of gene expression of neuronal markers in induced neurons of human foreskin fibroblasts (HFF1y, Beijing Stem Cell Bank) shown in Figure 1A (left) cultured in induction medium containing (-)-Blebbistatin for 7 days, then cultured in maturation medium containing (-)-Blebbistatin and the isoxazole compound ISX9 for 45 days, and then cultured in induction medium containing (-)-Blebbistatin for 7 days, then cultured in maturation medium without (-)-Blebbistatin and the isoxazole compound ISX9 for 45 days, and in the blank control group. [Figure 2A] Figure 2A shows a micrograph (left) of human foreskin fibroblasts (HFF1y, Beijing Stem Cell Bank) and a micrograph (right) of induced neurons after culturing the human foreskin fibroblasts (HFF1y, Beijing Stem Cell Bank) for 7 days in induction medium containing (-)-Blebbistatin and then for 21 days in maturation medium containing (-)-Blebbistatin and ISX-PCA, a derivative of the isoxazole compound ISX9. [Figure 2B] Figure 2B shows the gene expression profile of neuronal markers in induced neurons after culturing the human foreskin fibroblasts (HFF1y, Beijing Stem Cell Bank) shown in Figure 2A (left) for 7 days in induction medium containing (-)-Blebbistatin, followed by culturing for 45 days in maturation medium containing (-)-Blebbistatin and ISX-PCA, a derivative of the isoxazole compound ISX9. [Figure 3] Figure 3 shows the staining results of neuronal transdifferentiation after human foreskin fibroblasts (HFF1y, Beijing Stem Cell Bank) were sequentially cultured in induction medium containing (-)-Blebbistatin, then cultured in maturation medium containing (-)-Blebbistatin and the isoxazole compound ISX9, and then transplanted in vivo. Subsequently, (-)-Blebbistatin and the isoxazole compound ISX9 were intraperitoneally injected. [Figure 4A]Figure 4A shows a micrograph of human foreskin fibroblasts (HFF1y, Beijing Stem Cell Bank) (left) and induced neurons (right) after culturing the human foreskin fibroblasts (HFF1y, Beijing Stem Cell Bank) in induction medium containing (-)-Blebbistatin for 7 days. [Figure 4B] Figure 4B shows the results of GO cluster analysis after RNA-seq of the induced neurons in Figure 4A (right). [Figure 5A] Figure 5A shows a micrograph (left) of human foreskin fibroblasts (HFF1y, Beijing Stem Cell Bank) and a micrograph (right) of induced neurons after culturing the human foreskin fibroblasts (HFF1y, Beijing Stem Cell Bank) for 1 day in induction medium without the myosin II inhibitor (-)-Blebbistatin, followed by culturing them for 6 days in maturation medium containing the isoxazole compound ISX9 but without (-)-Blebbistatin. [Figure 5B] FIG. 5B shows gene expression of neuronal markers in the induced neurons of FIG. 5A (right). [Figure 6] Figure 6 shows gene expression of classical neuronal markers after 1 day of culture in induction medium (Figure 5A (left)) and subsequent removal of forskolin, or after 6 days of culture in neural maturation medium from which neurotrophic factor 3, brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor, and forskolin were simultaneously removed. (Here, F stands for forskolin, and FBGN stands for a mixture of forskolin, brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor, and neurotrophic factor 3.) [Figure 7A] Figure 7A shows the morphology of induced neurons and the results of neuronal marker staining after mouse astrocytes were cultured for 1 day in induction medium containing (-)-Blebbistatin and then for 13 days in maturation medium containing (-)-Blebbistatin and the isoxazole compound ISX9. [Figure 7B] FIG. 7B shows gene expression of neuronal markers in the induced neurons of FIG. 7A. [Figure 8]FIG. 8 shows the results of comparing the expression of neuronal marker genes in neurons obtained by induction culture in different induction and maturation media. DETAILED DESCRIPTION OF THE INVENTION

[0039] Hereinafter, the embodiments of the present application will be described in detail with reference to specific examples, but the following content should not be construed as any limitation on the present application.

[0040] The terms used in this text have the following meanings: High-glucose DMEM: High-glucose DMEM medium (Dulbecco's modified eagle medium, DMEM), a commercially available medium developed based on MEM medium and containing various glucose and amino acids. N2B27 medium: A cell culture medium with defined components, consisting of a 1:1 mixture of DMEM / F12 basal medium and Neurobasal basal medium, containing N2 cell culture supplement and B27 cell culture supplement. The components in N2B27 medium vary depending on the cell being cultured. It has been reported to contribute to the differentiation of mouse embryonic stem cells into the nervous system. The N2B27 medium of the present application is prepared by mixing DMEM / F12 and Neurobasal in a 1:1 ratio, followed by the addition of N2 cell culture supplement, B27 cell culture supplement, β-mercaptoethanol, Glutamax, insulin, and penicillin. DMEM / F12: A commercially available basal culture medium that is a 1:1 mixture of DMEM and F12 media, suitable for clonal density culture. Neurobasal: A commercially available basal medium suitable for neuronal culture. GlutaMAX: A cell culture supplement that can directly replace L-glutamine in cell culture media. Penicillin and Streptomycin: Penicillin and streptomycin are two antibiotics commonly used in cell culture to prevent bacterial contamination during cell culture. N2 Cell Culture Supplement: A commercially available serum-free cell culture additive. B27 cell culture supplement: A commercially available serum-free cell culture additive. Neurotrophin 3: Neurotrophins (NTs) are a class of protein molecules produced by innervated tissues (e.g., muscles) and astrocytes and are required for the growth and survival of neurons. NT-3 (NT-3) is a type of neurotrophic factor that is distributed mainly in the dorsal root ganglia, spinal cord, brainstem, cerebellum, and hippocampus in the nervous system, and can maintain the survival of sympathetic, sensory, cholinergic, and motor neurons in the basal forebrain. Brain-derived neurotrophic factor: A type of neurotrophic factor, it is the most abundant neurotrophic factor in the body and acts by binding to TrkB (tyrosine kinase B). It is widely distributed in the central nervous system, peripheral nervous system, endocrine system, bone and cartilage tissue, etc., but is mainly expressed in the central nervous system, with the highest concentration in the hippocampus and cortex. Glial cell line-derived neurotrophic factor: It can support the survival of midbrain dopaminergic neurons in vitro and improve the survival rate and density of dopaminergic neurons and symptoms in various animal models of Parkinson's disease. GABA: stands for gamma-aminobutyric acid, an important neurotransmitter in the central nervous system, is an inhibitory neurotransmitter, and influences learning, memory, and sleep. GABAN: An abbreviation for GABAergic neuron, which refers to nerve cells that primarily use GABA as a transmitter.

[0041] This application is Myosin inhibitors, Isoxazole and / or its derivatives The present invention provides a composition for inducing cell transdifferentiation, comprising:

[0042] Derivatives in this application refer to derivatives of isoxazole.

[0043] In a specific embodiment, the transdifferentiation is inducing transdifferentiation of a non-neuronal cell into a neuron, wherein the non-neuronal cell is a fibroblast or an astrocyte, and the myosin inhibitor is (-)-Blebbistatin and / or (-)-Blebbistatin O-Benzoate.

[0044] The (-)-Blebbistatin used in the present invention is abbreviated as Ble and has the structure shown in formula (II).

[0045] [ka]

[0046] The (-)-Blebbistatin O-Benzoate used in the present invention is abbreviated as Ble-OB and has the structure shown in formula (IIb).

[0047] [ka]

[0048] The present application also provides the use of a composition comprising a myosin inhibitor and an isoxazole compound and / or a derivative thereof in inducing cell transdifferentiation.

[0049] The present application also provides the use of a composition comprising a myosin inhibitor and an isoxazole compound and / or a derivative thereof in the preparation of a medicament for the treatment of a neurodegenerative disease.

[0050] In one particular embodiment, the neurodegenerative disease includes, but is not limited to, diseases such as Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, spinocerebellar ataxia, epilepsy, stroke, brain injury, and spinal cord injury.

[0051] The present application provides a method for inducing transdifferentiation of non-neuronal cells into neurons in vitro, comprising treating the non-neuronal cells with a myosin inhibitor and an isoxazole compound and / or a derivative thereof.

[0052] In one particular embodiment, the method comprises culturing non-neuronal cells in induction medium for 1 to 7 days, optionally 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days, followed by culturing with 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. Here, the induction medium contains a myosin inhibitor, and the maturation medium contains a myosin inhibitor and an isoxazole compound and / or a derivative thereof.

[0053] The present application also provides a method for transdifferentiating non-neuronal cells into neurons in a subject, comprising administering to the subject an effective amount of a myosin inhibitor and an isoxazole compound and / or its derivative.

[0054] In one particular embodiment, the method comprises culturing the non-neuronal cells in an induction medium for 1-7 days, optionally 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days, etc., followed by culturing in a maturation medium for 5-10 days, optionally 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, etc., preferably 7 days, followed by 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 derivative thereof by intraperitoneal injection to the subject for 14 or more consecutive days (optionally 14, 15, 16, 17, 18, 19 days, etc.).

[0055] The present application also provides a method for treating a neurodegenerative disease in a subject, comprising administering to the subject an effective amount of a myosin inhibitor and an isoxazole compound and / or a derivative thereof.

[0056] The present application also provides a medium for inducing transdifferentiation of non-neuronal cells into neuronal cells, comprising an induction medium and a maturation medium.

[0057] In one specific embodiment, the induction medium comprises N2B27 medium and a myosin inhibitor, and the maturation medium comprises N2B27 medium, a neurotrophic factor, forskolin, a myosin inhibitor, an isoxazole compound and / or a derivative thereof. The N2B27 medium is prepared by mixing DMEM / F12 and Neurobasal in a 1:1 ratio, followed by the addition of N2 cell culture supplement, B27 cell culture supplement, β-mercaptoethanol, glutamax, insulin, and penicillin.

[0058] In one particular embodiment, the neurotrophic factors include neurotrophic factor 3, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor.

[0059] In one particular embodiment, said maturation medium comprises a myosin inhibitor, an isoxazole compound and / or a derivative thereof, said N2B27 medium, but does not contain neurotrophic factors and forskolin.

[0060] In one particular embodiment, the maturation medium consists of a myosin inhibitor, an isoxazole compound and / or a derivative thereof, and the N2B27 medium.

[0061] In one particular embodiment, the maturation medium comprises a myosin inhibitor, an isoxazole compound and / or its derivatives, the N2B27 medium, neurotrophic factor 3, brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor, but does not comprise forskolin.

[0062] In a preferred embodiment, the maturation medium consists of a myosin inhibitor, an isoxazole compound and / or its derivative, the N2B27 medium, neurotrophic factor 3, brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor.

[0063] In one particular embodiment, the concentration of the neurotrophic factor 3 is 0 to 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, 24 ng / mL, 25 ng / mL, etc. The brain-derived neurotrophic factor is 0 to 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, 24 ng / mL, 25 ng / mL, etc. The glial cell line-derived neurotrophic factor is 0 to 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, 24 ng / mL, 25 ng / mL, etc. Forskolin is 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., where the concentrations are the final concentrations of the three different trophic factors in the maturation medium.

[0064] In one particular embodiment, the concentration of the myosin inhibitor in the induction medium is 5-25 μM, optionally 5 μM, 10 μM, 15 μM, 20 μM, 21 μM, 22 μM, 23 μM, 24 μM, or 25 μM, wherein the concentration is the final concentration of the myosin inhibitor in the induction medium used to treat non-neuronal cells.

[0065] In one particular embodiment, the concentration of the myosin inhibitor in the maturation medium is 0-25 μM, optionally 0 μM, 5 μM, 10 μM, 15 μM, 20 μM, or 25 μM, and the concentration of the isoxazole compound or derivative thereof in the maturation 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 concentrations are the final concentrations of the myosin inhibitor and isoxazole compound or derivative thereof, respectively, in the induction medium used to treat non-neuronal cells.

[0066] In one particular embodiment, the non-neuronal cells are first cultured in a basal medium, and then sequentially cultured in an induction medium and a maturation medium.

[0067] In one particular embodiment, said basal medium is prepared from high glucose DMEM+10% fetal bovine serum.

[0068] In one particular embodiment, the isoxazole compound is isoxazole 9 (abbreviated as ISX9) having an isoxazole ring amide bond backbone structure, specifically having the structure shown in formula (III).

[0069] [ka]

[0070] In one particular embodiment, the isoxazole 9 or a derivative thereof has the structure shown in formula (I):

[0071] [ka]

[0072] In the formula, R1 can be any one selected from thienyl, furyl, pyrrolyl, phenyl, and pyridyl; R2 can be any one selected from isoxazolyl, isothiazolyl, pyrazolyl, oxazolyl, thiazolyl, and imidazolyl groups; and R3 can be any one selected from methyl, ethyl, cyclopropyl, cyclobutyl, and cyclopentyl, where the linking site of the R1 group is any carbon atom, the two linking sites of the R2 group are two meta-position carbon atoms, and the linking site of the R3 group is not limited.

[0073] This application provides a method for inducing transdifferentiation of non-neuronal cells into neurons using a composition containing a myosin inhibitor and an isoxazole compound and / or its derivative. The non-neuronal cells are cultured in an induction medium and a maturation medium, the induction medium containing a myosin inhibitor, and the maturation medium containing a myosin inhibitor and an isoxazole compound and / or its derivative. During the culture process, the cells exhibited clear neuronal morphology, prominent cell bodies, and distinct synapses. The positive rate of neuronal marker staining was 99.6%. Neuronal transcription factors and synapse-related gene expression were upregulated, and the transdifferentiated neurons were primarily GABAergic neurons. As culture time progressed from D0 to D45, neural-related genes increasingly resembled those of neurons, and from D14 to D45, the color depth approached that of neurons, indicating that the induced neurons had expression patterns similar to those of stem cell-derived neurons and primary isolated neurons. Single-cell sequencing results showed that the overall transcriptional levels of neurons at days 30 and 45 of the culture process were close to those of isolated neurons in vivo. Gene expression levels of neuronal markers were higher in neurons obtained using our culture method than in cultures using induction medium or maturation medium lacking Ble and ISX9. When cells from the culture were injected into the left hippocampus of mice, GFP-positive induced neurons were observed in the hippocampus adjacent to the injected induced neurons, and the classical neuronal markers MAP2 and NEUN were expressed. Compared to cultures containing Ble and a Ble-OB mixture, and cultures containing Ble-OB, the gene expression levels of neuronal markers were highest in neurons obtained from cultures containing Ble. [Example]

[0074] Example 1 The combination of the small molecule myosin inhibitor (-)-Blebbistatin and isoxazole 9 efficiently achieves neuronal transdifferentiation in vitro For example, 10 cm dishes (Corning, 430167) were coated with 3 mL of 20 μg / mL fibronectin solution (Millipore, FC010, prepared with 1x PBS) for 6 hours. The fibronectin solution was removed, and human foreskin fibroblasts (HFF1y, Beijing Stem Cell Bank) were added at 2 × 10 per dish. 6 The cells were uniformly seeded and cultured in basal medium (high-glucose DMEM (Gibco, C12430500BT) and 10% fetal bovine serum (Gibco, 16000-044)) for 12 hours. The basal medium was removed and the cells were washed once with PBS.

[0075] The cell transformation kit of the present application was used for transdifferentiation of neurons. The kit contains the following induction medium and maturation medium:

[0076] After the above treatment, induction medium was added to the HFF1y culture dish (Petri dish) and cultured for 1 to 7 days. The induction medium was prepared by adding 25 μM Ble (MCE, HY-13441) to N2B27 medium. N2B27 medium was prepared by mixing DMEM / F12 (Gibco, 10565018) with Neurobasal (Gibco, 21103-049) at a 1:1 ratio, followed by the addition of N2 cell culture supplement (100x, Gibco, 17502048), B27 cell culture supplement (50x, Gibco, 17504044), β-mercaptoethanol (1000x, Gibco, 21985023), Glutamax (100x, Gibco, 35050), 1 μg / mL insulin (Roche, 11376497001), and penicillin (100x, Gibco, REF 15140-122). After 1–7 days of culture in induction medium, HFF1y cells developed clear neuronal morphology.

[0077] After 1 to 7 days of induction, HFF1y cells were cultured in maturation medium for 7 to 45 days. The maturation medium was prepared by adding 20 ng / mL neurotrophic factor 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 N2B27 medium.

[0078] To further illustrate the neurotransdifferentiation effects of Ble and ISX9, Applicants provide Figures 1A-1F.

[0079] The left side of Figure 1A shows a micrograph of HFF1y cells cultured in high-glucose DMEM and 10% fetal bovine serum. The right side of Figure 1A shows a micrograph of induced neurons after culturing the HFF1y cells in induction medium containing Ble for 7 days and then in maturation medium containing Ble and ISX9 for 21 days. As shown on the right side of Figure 1A, the cells after 21 days of culture exhibited clear neuronal morphology, prominent cell bodies, and distinct synapses.

[0080] The cells in Figure 1A (right) were stained with classical neuronal markers, and the staining results are shown in Figure 1B. The neuronal markers included TUJ1 (Covance, MRB-435P), MAP2 (Santa Cruz Biotechnology, sc-20172), NF2000 (Abcam, ab4680), and NEUN (Chemicon, MAB377). Staining for all of the above neuronal markers was positive, with a positive rate close to 100%, specifically 99.6%.

[0081] Figure 1C shows the gene expression profile of neuronal markers in HFF1y cells (Figure 1A, left) induced in induction medium for 7 days and then cultured in maturation medium for 38 days. The figure shows that the fibroblast marker FSP1 was downregulated, while classical neuronal markers such as GFAP, DCX, TUJ1, MAP2, and NEUN were significantly upregulated. Concomitantly, neuronal transcription factors such as ASCL1, BRN2, and NEUROD1 were upregulated. This indicates the acquisition of a neuronal fate in the induced cells. The expression of synapse-related genes such as NEFH, PSD95, SYN1, and SYT was upregulated, laying the foundation for the induction of neuronal function. Furthermore, the expression levels of PVALB, GAD, and GABBR3 genes were upregulated, indicating that the transdifferentiated neurons were primarily GABAergic neurons.

[0082] Figure 1D shows a heatmap of neural-related gene expression during neuronal induction. DO represents the initial cell state on day 0 of human foreskin fibroblast (HFF1y, Beijing stem cell) induction, h represents the number of hours, and D represents the number of days. 3h, 6h, D1, D2, D7, D14, D30, and D45 represent different induction culture stages. HFF1y cells in Figure 1A (left) were induced in induction medium at 3 h, 6 h, 1 day, 2 days, and 7 days (corresponding to 3 h, 6 h, D1, D2, and D7, respectively), and then cultured in maturation medium at 7 days, 23 days, and 38 days (corresponding to D14, D30, and D45, respectively). HNcDNA represents primary human neurons, and GABAN represents GABAergic neurons differentiated from human pluripotent stem cells. The vertical axis on the right side of the figure shows the gene name, the horizontal axis shows the sample name, and each small colored box represents the magnitude of gene expression. The upper left corner shows the color scale. Each row represents the expression of each gene in a different sample, and each column represents the expression of all genes in each sample. The dendrogram on the left shows the cluster analysis results of various genes from different samples. In Figure 1D, it can be clearly seen that as the induction time progresses from D0 to D45, neural-related genes become increasingly similar to those of neurons. This indicates that the induced neurons have an expression pattern similar to that of stem cell-derived neurons and primary isolated neurons.

[0083] A patch clamp experiment was performed on the induced neuron in Figure 1A (right), and the results are shown in Figure 1E. Patch clamp is an electrophysiological technique primarily used to detect the activity of cell membrane ion channels and induce action potentials. The electrophysiological results showed that the induced neuron possessed the sodium currents, potassium currents, and evoked action potentials of mature neurons, indicating that the induced neuron possessed the electrophysiological properties of mature neurons.

[0084] Figure 1F shows the results of single-cell sequencing of the human foreskin fibroblasts (HFF1y, Beijing Stem Cell Bank) shown in Figure 1A (left), cultured for 7 days in the induction medium of this example, followed by 38 days in the maturation medium of this example. In the figure, D0 represents the initial cell state on day 0 of induction, while 3 h, 6 h, D1, D2, D7, D14, D30, and D45 represent days 3, 6, 1, 2, 7, 24, 30, and 45 of culture using this method, respectively. "Neurons 1-6" represent neurons isolated in vivo. Analysis results show that the overall transcription levels of neurons on days 30 and 45 of the culture process are closer to those of neurons isolated in vivo.

[0085] Comparative Example 1 The experimental procedures in Example 1 were followed, except that after 7 days of culture in the induction medium, the cells were cultured for 38 days using a maturation medium (compared to the maturation medium in Example 1, the maturation medium in this comparative example was not supplemented with Ble and ISX9). Figure 1G shows the gene expression of neuronal markers in the cells after 7 days of culture in the induction medium of Example 1 and then 38 days of culture in the maturation medium of Example 1, as well as the gene expression of neuronal markers in the blank control cells. Figure 1G demonstrates that the neuronal marker gene expression levels of neurons obtained by the induction culture method of the present application were higher.

[0086] Example 2 Combination of Ble with an isoxazole compound or its derivative efficiently achieves neuronal transdifferentiation N-methyl-5-phenylisoxazole-3-carboxamide (abbreviated as ISX-PCA, 20-50 μM, TCI, BD399148), a derivative of the isoxazole compound ISX9, was selected. The R group is a benzene ring and the R group is methyl. The structure of the resulting ISX-PCA is shown in formula (IV). Neuronal transdifferentiation was performed according to the method of Example 1 using ISX-PCA instead of ISX9.

[0087] [ka]

[0088] Figure 2A shows a micrograph of HFF1y (left) and a micrograph of induced neurons (right) after culturing the HFF1y in Ble-containing induction medium for 7 days and then in maturation medium containing Ble and ISX-PCA for 21 days. As shown in Figure 2A (right), the cells induced by the combination of Ble and ISX-PCA are morphologically similar to the cells in Figure 1A (right), with clear neuronal morphology, prominent cell bodies, and obvious synapses.

[0089] Furthermore, gene expression of classical neuronal markers was detected for Figure 2A (right), and the results are shown in Figure 2B. Here, classical neuronal markers such as GFAP, DCX, TUBB3, MAP2, NEUN, MAPT, and NEFH were significantly upregulated, and neural transcription factors such as ASCL1, BRN2, and NEUROD1 were also upregulated, indicating that the transdifferentiated cells had acquired a neural fate.

[0090] Example 3 Combination of Ble with isoxazole or its derivatives promotes neuronal transdifferentiation of human fibroblasts in vivo For example, 10 cm dishes (Corning, 430167) were coated with 3 mL of 20 μg / mL fibronectin solution (Millipore, FC010, prepared with 1x PBS) for 6 hours. The fibronectin solution was removed, and human foreskin fibroblasts (HFF1y, Beijing Stem Cell Bank) were added at 2 × 10 per dish. 6 The cells were uniformly seeded and cultured in basal medium (high-glucose DMEM (Gibco, C12430500BT) and 10% fetal bovine serum (Gibco, 16000-044)) for 12 hours. The basal medium was removed and the cells were washed once with PBS.

[0091] The induction medium of Example 1 was added to the HFF13y-GFP culture dish treated as above, and the dish was cultured for 7 days. After that, the dish was cultured for 7 days in the maturation medium of Example 1, and then 2.5 × 105 Cells were transplanted into the left hippocampus of 5-week-old immunodeficient (SCID) mice at a dose of 3 mg / kg Ble + 10 mg / kg ISX9 intraperitoneally for 14 consecutive days. The injection solvent was dimethyl sulfoxide (DMSO) (final volume 2%) + PEG400 (final volume 40%) + Tween 80 (final volume 2%) + sterile water. After 14 days, the hippocampi were harvested and paraffin sections were stained to detect the expression of GFP and classical neuronal markers. The results are shown in Figure 3. The first and second rows are the control group, and the second row is the staining results for the neuronal marker proteins MAP2 (row 1), NEUN (row 2), and GFP (derived from the transplanted induced cells) after in vivo induction of the non-transplanted brain side of the mice. The third and fourth rows of Figure 3 show the staining results for the neuronal marker proteins MAP2 (row 3), NEUN (row 4), and GFP (derived from the induced transplanted fibroblasts) after in vivo induction of the brain side of the transplanted cells in the same mouse. "Merge" indicates an overlay. Rows 3 and 4 show the presence of GFP-positive cells expressing MAP2 (row 3 of the overlay confirms that cells with GFP signal also have MAP2 signal by signal colocalization) and NEUN (row 4 of the overlay confirms that cells with GFP signal also have NEUN signal by signal colocalization), indicating that the GFP-transplanted cells became neurons after in vivo induction. Figure 3 shows that GFP-positive induced neurons are observed in the hippocampus on the side where the induced neurons were injected, and that the classical neuronal markers MAP2 and NEUN are expressed.

[0092] Example 4 Ble promotes neuronal morphogenesis and upregulation of neural fate-related genes For example, 10 cm dishes (Corning, 430167) were coated with 3 mL of 20 μg / mL fibronectin solution (Millipore, FC010, prepared with 1x PBS) for 6 hours. The fibronectin solution was removed, and 2 × 10 HFF1y cells were added per dish. 6 The cells were uniformly seeded and cultured in basal medium (high-glucose DMEM (Gibco, C12430500BT) and 10% fetal bovine serum (Gibco, 16000-044)) for 12 hours. The basal medium was removed and the cells were washed once with PBS.

[0093] The induction medium of Example 1 was added to the HFF1y culture dish treated as above, and the culture was continued for 1 to 7 days. After this, the morphological changes of the cells were as shown in Figure 4A. After induction, the cells had a clear neuronal morphology, with small and round cell bodies and abundant and thin synapses.

[0094] We performed RNA-seq and GO cluster analysis on the above cells, and the results are shown in Figure 4B. The red bars represent the negative logarithm of the significance p-value (p<0.05) enriched for this process. Figure 4B shows that in neural induction culture systems containing only Ble, up-regulated genes in induced cells were enriched in pathways related to neuronal fate development. This further demonstrates that Ble alone can promote the up-regulation of genes related to neuronal morphogenesis and neural fate development.

[0095] Example 5 Isoxazole compounds and their derivatives potently promote neural fate conversion at the transcriptional level The experimental procedures in Example 1 were repeated, except that HFF1y cells were cultured in induction medium (compared to the induction medium in Example 1, the induction medium in this Example did not contain Ble) for 7 days, followed by maturation medium (compared to the maturation medium in Example 1, the maturation medium in this Example did not contain Ble) for 7 days. The morphological changes of the cells are shown in Figure 5A (right), with Figure 5A (left) showing HFF1y. As can be seen from the comparison, the induced cell morphology did not resemble that of neurons.

[0096] In Figure 5A (right), we further examined gene expression of classical neuronal markers. The results are shown in Figure 5B. The fibroblast marker FSP1 was significantly downregulated, whereas the expression of classical neuronal markers such as GFAP, TUJ1, MAP2, MAPT, STMN1, and NCAM, as well as the synapse-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 had acquired a neural fate.

[0097] From the results of Examples 4 and 5, it can be inferred that in the process of promoting efficient conversion of fibroblasts to neurons by the combination of Ble and an isoxazole compound or its derivative, Ble is involved in neuronal morphogenesis and initiates the conversion of neural fate, while the isoxazole compound or its derivative strongly promotes complete transdifferentiation of neuronal fate.

[0098] Example 6 The combination of Ble with isoxazole or its derivatives has a potent neuronal induction effect To further verify the potent neuronal induction effect of Ble+ isoxazole or its derivatives, as shown in Figure 5A (left), cells were cultured for 7 days in the induction medium described in Example 1, followed by 7 days in maturation medium based on the maturation medium described in Example 1, but lacking forskolin, or lacking neurotrophic factor 3, brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor, and forskolin. Gene expression of classical neuronal markers was then detected. The results are shown in Figure 6. Cells treated with neural maturation medium lacking forskolin or lacking the three neurotrophic factors and forskolin simultaneously showed significant upregulation of classical neuronal markers such as DCX, TUBB3, MAP2, NEUN, MAPT, and STMN1, as well as synapse-related proteins NEFH, SYT1, SYN1, and PSD95. These results also showed upregulation of neural transcription factors such as ASCL1, BRN2, and NEUROD1. This indicates that the transdifferentiated cells had acquired a neural fate.

[0099] Example 7 Combination of Ble with an isoxazole compound or its derivatives results in efficient transdifferentiation of mouse astrocytes into neurons Astrocytes are one of the key cells in the brain environment and are involved in physiological functions of the brain. Transdifferentiation of astrocytes into neurons provides important guidance for the treatment of neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease.

[0100] The experimental procedures in Example 1 were followed, except that mouse cerebral cortex-derived astrocytes were used instead of HFF1y and were treated. Mouse astrocytes were induced in the induction medium of Example 1 for 1 day and then cultured in the maturation medium of Example 1 for 13 days. Figure 7A shows the cell morphology and staining for classic neuronal markers after 13 days. The morphology of the cells induced by mouse astrocytes was similar to that of neurons, with small, round cell bodies, abundant synapses, and expression of classic neuronal markers TUJ1, MAP2, and NEUN.

[0101] Figure 7B shows the expression of neuronal marker genes in the induced cells shown in Figure 7A. We found that classical neuronal markers such as DCX, TUBB3, MAP2, NEUN, MAPT, and NEFH were significantly upregulated, as was the expression of the postsynaptic density protein PSD95. Concurrently, neuronal transcription factors such as ASCL1, BRN2, and MYTL1 were upregulated. This indicates that the transdifferentiated cells had acquired a neural fate.

[0102] Example 8 The experimental procedures in Example 1 were repeated, except that the cells were cultured in the induction medium for 7 days, followed by culture in the maturation medium for 7 days.

[0103] Example 9 The experimental procedures in Example 1 were followed, except that the cells were cultured for 7 days in induction medium (compared to the induction medium in Example 1, the induction medium in this example used Ble-OB instead of Ble) and then for 7 days in maturation medium (compared to the maturation medium in Example 1, the maturation medium in this example used Ble-OB instead of Ble).

[0104] Example 10 The experimental procedures in Example 1 were followed, except that the cells were cultured for 7 days in induction medium (compared to the induction medium in Example 1, the induction medium in this example used Ble-OB instead of Ble) and then for 7 days in maturation medium (compared to the maturation medium in Example 1, the maturation medium in this example used ISX-PCA instead of ISX9 and Ble-OB instead of Ble).

[0105] Example 11 The experimental procedures in Example 1 were followed, except that the cells were cultured for 7 days in an induction medium (compared to the induction medium in Example 1, the induction medium in this example uses a 1:1 mixture of Ble-OB and Ble instead of Ble, and the total concentration of Ble-OB and Ble in the induction medium in this example is equal to the Ble concentration in the induction medium in Example 1), and then for 7 days in a maturation medium (compared to the maturation medium in Example 1, the maturation medium in this example uses a 1:1 mixture of Ble-OB and Ble instead of Ble, and the total concentration of Ble-OB and Ble in the maturation medium is equal to the Ble concentration in the maturation medium in Example 1).

[0106] Example 12 The experimental procedures in Example 1 were followed, except that the cells were cultured for 7 days in an induction medium (compared to the induction medium in Example 1, the induction medium in this example uses Ble-OB instead of Ble) and then for 7 days in a maturation medium (compared to the maturation medium in Example 1, the maturation medium in this example uses Ble-OB instead of Ble, and uses a 1:1 mixture of ISX9 and ISX-PCA instead of ISX9, and the total concentration of ISX9 and ISX-PCA in the maturation medium in this example is equal to the ISX9 concentration in the maturation medium in Example 1).

[0107] Comparative Example 2 The experimental procedures were the same as those in Comparative Example 1, except that the cells were cultured in the induction medium for 7 days and then in the maturation medium for 7 days.

[0108] Figure 8 shows the gene expression of neuronal markers (MAPT and MAP2) in neurons obtained in Examples 8 to 12 and Comparative Example 2. As can be seen from the figure, the gene expression levels of neuronal markers in neurons obtained by induction culture in a medium supplemented with isoxazoles and / or their derivatives were significantly higher than in a medium without isoxazoles and / or their derivatives. Furthermore, when the gene expression levels of neuronal markers in neurons obtained in a medium supplemented with Ble, a medium supplemented with a mixture of Ble and Ble-OB, and a medium supplemented with Ble-OB were compared, the gene expression levels of neuronal markers in neurons obtained in a medium supplemented with Ble were highest, whereas the gene expression levels of neuronal markers in neurons obtained in a medium supplemented with Ble and a mixture of Ble-OB were low, and the gene expression levels of neuronal markers in neurons obtained in a medium supplemented with Ble-OB were even lower.

[0109] The above is only a preferred embodiment of the present application, and is not intended to limit the present application to other forms, and those skilled in the art can use the technical content disclosed above to make changes or modifications to equivalent embodiments of equivalent changes, however, any simple modifications, equivalent changes, and improvements made to the above embodiments in accordance with the technical essence of the present application without departing from the content of the technical solution of the present application still fall within the protection scope of the technical solution of the present application.

Claims

1. Myosin inhibitors, Isoxazole compounds and Including, the myosin inhibitor is (-)-blebbistatin, the isoxazole compound is isoxazole 9 (ISX9); A composition for inducing cell transdifferentiation, wherein the cell transdifferentiation is the transdifferentiation of fibroblasts or astrocytes into neurons.

2. Use of a composition comprising a myosin inhibitor and an isoxazole compound in inducing cell transdifferentiation in vitro, wherein the transdifferentiation induces the transdifferentiation of fibroblasts or astrocytes into neurons, and the myosin inhibitor is (-)-blebbistatin; The use, wherein the isoxazole compound is isoxazole 9 (ISX9).

3. 1. An in vitro method for inducing transdifferentiation of non-neuronal cells into neurons, comprising treating the non-neuronal cells with a myosin inhibitor and an isoxazole compound, wherein the myosin inhibitor is (-)-blebbistatin; the isoxazole compound is isoxazole 9 (ISX9), The method, wherein the cell transdifferentiation is the transdifferentiation of fibroblasts or astrocytes into neurons.

4. culturing the non-neuronal cells in induction medium for 1-7 days, followed by culturing with maturation medium for 7-45 days; the induction medium comprises a myosin inhibitor; 4. The method of claim 3, wherein the maturation medium comprises a myosin inhibitor and an isoxazole compound.

5. 5. The method of claim 4, comprising culturing the non-neuronal cells in induction medium for 1 to 7 days, followed by culturing them using maturation medium for 21 to 45 days.

6. 5. The method of claim 4, wherein the induction medium further comprises N2B27 medium, which is prepared by mixing DMEM / F12 and Neurobasal in a 1:1 ratio, and then adding N2 cell culture supplement, B27 cell culture supplement, β-mercaptoethanol, glutamax, insulin, and penicillin.

7. 5. The method of claim 4, wherein the maturation medium comprises a myosin inhibitor, an isoxazole compound, an N2B27 medium, a neurotrophic factor, and forskolin, and the N2B27 medium is prepared by mixing DMEM / F12 and Neurobasal in a 1:1 ratio, and then adding N2 cell culture supplement, B27 cell culture supplement, β-mercaptoethanol, glutamax, insulin, and penicillin.

8. 8. The method of claim 7, wherein the neurotrophic factors include neurotrophic factor 3, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor.

9. 5. The method of claim 4, wherein the maturation medium comprises a myosin inhibitor, an isoxazole compound, and N2B27 medium.

10. 10. The method of claim 9, wherein the maturation medium consists of a myosin inhibitor, an isoxazole compound, and N2B27 medium.

11. 5. The method of claim 4, wherein the maturation medium comprises a myosin inhibitor, an isoxazole compound, N2B27 medium, neurotrophic factor 3, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor.

12. 12. The method of claim 11, wherein the maturation medium comprises a myosin inhibitor, an isoxazole compound, N2B27 medium, neurotrophic factor 3, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor.

13. 1. A product or kit for transdifferentiating non-neuronal cells into neurons, the product or kit comprising an induction medium and a maturation medium; the induction medium comprises a myosin inhibitor; the maturation medium comprises a myosin inhibitor and an isoxazole compound; the myosin inhibitor is (-)-blebbistatin, the isoxazole compound is isoxazole 9 (ISX9); The product or kit, wherein the cell transdifferentiation is transdifferentiation of fibroblasts or astrocytes into neurons.

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