Use of TGF-β inhibitors in neural stem cell induction and organoid formation

Abstract

The present invention provides a novel application of TGF-β small molecule inhibitors in the field of neural regeneration, providing a method for the in vitro regeneration and directed differentiation of various neural cells and brain organoids. By adding these inhibitors to a series of basal media containing defined chemical components, pluripotent stem cells can be converted into multiple neural stem cell-derived adult stem cells, significantly improving the number of neural cells and organoid size obtained by induction. The induction system provided by the present invention expands the novel capabilities of a single small molecule in the field of ectodermal cell induction and differentiation, while avoiding the use of serum substitutes such as B27, completely eliminating the potential risks associated with the presence of animal-derived components in the cell culture process and significantly expanding the clinical prospects for multiple neural cell transplants.

Description

[Technical field]

[0001] The present invention relates to the field of biology, specifically to the use of small molecule inhibitors of TGF-β in the induction of neural stem cells and organoid formation. [Background technology]

[0002] The ectoderm is the outermost layer formed during embryonic development. When organogenesis begins, ectodermal cells gradually differentiate into important systems such as the brain, spinal cord, and sensory organs. Among them, the nervous system is an important system that controls functions such as thinking, emotion, perception, and movement. Compared with diseases such as tumors, there are currently fewer drugs available for neurological diseases and the development period is long. One of the most important reasons for this is the specificity of various progenitor cells in the ectodermal lineage, for example, the inability of progenitor neural cells to regenerate, which leads to a lack of in vitro screening platforms for drugs for neurological diseases. Summary of the Invention [Problem to be solved by the invention]

[0003] In vitro regeneration of ectodermal cells can be used for drug screening and for the treatment of many types of degenerative diseases. For example, it can be used to treat neurodegenerative diseases, which are currently common aging-related diseases that require a great deal of cost for treatment and nursing care and have no effective drugs. Neurodegenerative diseases include amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), and Alzheimer's disease (AD). According to the World Health Organization (WHO), in 2050, there will be more than 30 million people suffering from neurodegenerative diseases in China, and the expected medical expenses will exceed 1 trillion RMB. Currently, the main treatments include drug therapy that supplements or stimulates the lack of levodopa in the brain, neuronuclear ablation therapy, and deep brain electrical stimulation therapy, but good effects have not been obtained. There is a possibility that it will cause "abnormal involuntary movements" and "fluctuations in drug efficacy", which will have a significant impact on the quality of life.

[0004] Besides neurodegenerative diseases, spinal cord injury (SCI) is a well-known traumatic neurological disease. According to overseas statistics, the average lifetime cost of treatment and rehabilitation for SCI patients is more than US$750,000, and more than US$6 billion is spent annually on SCI patients in the United States. The prevalence of SCI in China is higher than that in developed Western countries, and the high treatment costs, long rehabilitation periods, and loss of working ability have a significant impact on individuals and their families, and also place a heavy burden on society.

[0005] The difficulty in studying neurodegenerative diseases and spinal cord injury is that neurons in the central nervous system cannot regenerate. The lack of in vitro disease models is a limiting factor for basic research. These diseases are caused by irreversible damage to the central nervous system. Neurons include various types of neural cells, such as neural stem cells, mature neural neurons, astrocytes, and oligodendrocytes. Currently, there are only a limited number of neural cell lines, most of which are cell lines of nervous system tumors, and the presence of EB virus is an unstable factor in the process of system construction. Neurons are not easy to passage in vitro, and embryonic and fetal neural stem cells cannot be widely used due to ethical constraints. Currently, although allogeneic transplantation of neural stem cells has achieved a certain degree of clinical efficacy, all of the above factors hinder further clinical progress of related diseases.

[0006] In 2006, Shinya Yamanaka's team invented the "Yamanaka cocktail" of four transcription factors, OCT4, SOX2, KLF4, and c-Myc, and succeeded in reprogramming differentiated and mature skin fibroblasts into pluripotent stem cells. These stem cells are called induced pluripotent stem cells (iPSCs) (Takahashi K, et al., Cell, 2006, 126(4) pp.663-676; Takahashi K and Yamanaka S, Cell, 2007, 131(5) pp.861-872). These stem cells have the same differentiation ability as embryonic stem cells, and have the ability to form the three most basic embryonic layers in human development: ectoderm, mesoderm, and endoderm, and ultimately various adult cells. This invention overcomes the ethical restrictions on the medical use of human embryonic stem cells, resolves the problem of immune rejection in cell transplantation therapy, and greatly expands the potential of stem cell technology in clinical medicine. The use of totipotent stem cells or pluripotent stem cells, such as embryonic stem cells or induced pluripotent stem cells (iPSCs), as the source of induced differentiation of ectodermal cells will provide a new idea for clinical treatment and greatly expand the potential of ectodermal cells in clinical medicine.

[0007] Taking the nervous system as an example, the field of regenerative medicine currently employs "dual SMAD inhibition" (Chambers SM, et.al., Nat Biotechnol, 2009, 27(3):275-80) as a method for inducing neural stem cells and neural neurons. The neural stem cells obtained by this method can be differentiated into other types of neural neuron cells. The principle is to mimic the signaling pathways of early embryonic development by inhibiting BMP and TGF-β, and to induce the development of neural stem cells. LDN-193189 and SB431542 are two widely used small chemical molecule inhibitors that act on ALK2 and ALK3 in the BMP4 pathway and ALK5 in the TGF-β pathway, respectively, to form implantation endoderm and mesoderm, which induces ectoderm development and neural development. This method allows the production of neural cells induced in the presence of serum components (Knockout Serum Replacement) (Chambers SM, et.al., Nat Biotechnol,2013,30(7): 715-720). However, neural cells obtained using the Dual SMAD inhibition method are often mixed with other incompletely differentiated cells. This is the result of asynchronous cell differentiation, and the presence of such cells may have a certain effect on the efficacy of transplantation therapy, and at the same time, it is also a safety issue. Therefore, how to obtain highly pure differentiated cells is an urgent technical issue in cell transplantation.

[0008] Galunisertib (LY2157299) used in the present invention has the standard chemical name 4-(2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)quinoline-6-carboxamide. quinoline-6-carboxamide), other chemical names 2-(6-Methyl-pyridin-2-yl)-3-(6-Carbamoyl-quinolin-4-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole ), or 4-[5,6-Dihydro-2-(6-methyl-2-pyridinyl)-4H-pyrrolo[1,2-b]pyrazol-3-yl]-6-quinolinecarboxamide, or LY-2157299, LY2157299. It is a small molecule inhibitor of TGF-β receptor I (TGF-βRI, ALK5). LY2157299 is currently in Phase II evaluation for its anti-cancer activity against hepatocellular carcinoma and glioblastoma cancer (Giannelli G1, Villa E, Lahn M. Transforming Growth Factor-β as a Therapeutic Target in Hepatocellular Carcinoma. Cancer Res. 2014 Apr 1;74(7):1890-4 ). It inhibits tumor growth, invasion and metastasis by blocking the TGF-β signaling pathway. Furthermore, studies have revealed that LY2157299 inhibits cancer cell growth by inhibiting the production of CTGF and suppressing the formation of new blood vessels.Currently, the main use of this molecule is in the development and treatment of therapeutics for lung cancer, liver cancer, and glioblastoma (Pharmaceutics. 2020 May 18;12(5):459), but no applications in the field of nerve regeneration have been disclosed.

[0009] The present invention has the following advantages. Since one chemical small molecule is used for the induction of neural cells, instead of multiple chemical small molecules, it is possible to significantly reduce production costs compared to the induction method combining multiple small molecules that is currently popular internationally, and it also has great advantages in terms of purity and yield. In addition, the present invention pioneers a completely new function of this small molecule in the field of ectoderm induction. In addition, since the present invention does not use substitute serum such as B27, the potential risks associated with the presence of animal-derived components in the cell culture process are completely eliminated, greatly expanding the clinical prospects of neural cell transplantation. The state is stable between multiple lots, the purity is high, and the problems of low purity and long cycles in the manufacturing process of cellular pharmaceuticals are solved. In particular, it can be used for in vitro screening of therapeutic drugs for neurological diseases and treatment of neurodegenerative diseases, so it is expected to have a great economic and social effect. [Means for solving the problem]

[0010] Contents of the invention In order to solve the shortcomings of the prior art, the present invention provides a serum-free medium and a differentiation induction method for inducing neural stem cells and brain organoids, and the medium for inducing differentiation of the neural stem cells and brain organoids comprises a basal medium and a neural induction compound.

[0011] To achieve the above objectives, the present invention provides the following technical solutions: The present invention provides the use of a TGF-β inhibitor in inducing neural stem cells and inducing the formation of organoids, the TGF-β inhibitor being 4-[2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]quinoline-6-carboxamide.

[0012] Preferably, the above-mentioned TGF-β inhibitor is added to a basal medium to form a neural stem cell induction medium.

[0013] Preferably, the basal medium is composed of DMEM / F12 medium, minimal essential medium non-essential amino acids, sodium chloride, sodium selenite, insulin and recombinant human transferrin.

[0014] Preferably, the basal medium is composed of DMEM / F12 medium, 1% minimum essential medium non-essential amino acids, 0.1 to 0.8 g / L sodium chloride, 13.6 μg / L sodium selenite, 20 ng / ml to 42 μg / ml insulin, and 50 to 180 ng / ml recombinant human transferrin.

[0015] More preferably, the basal medium is composed of 0.5 g / L sodium chloride, 13.6 μg / L sodium selenite, 22 ug / ml insulin and 100 ng / ml recombinant human transferrin.

[0016] Preferably, the concentration of the TGF-β inhibitor is 10 nM to 100 μM.

[0017] More preferably, the concentration of the TGF-β inhibitor is 12.5 μM.

[0018] In one embodiment, inducing neural stem cell formation comprises the steps of: culturing pluripotent stem cells in an adherent manner using a neural stem cell induction medium.

[0019] Preferably, the pluripotent stem cells are mammalian pluripotent stem cells.

[0020] More preferably, the pluripotent stem cells are human pluripotent stem cells.

[0021] Preferably, the adherent culture is carried out in the presence of a basement membrane preparation.

[0022] More preferably, the basement membrane preparation is one or a combination of two or more of basement membrane gel, laminin and vitronectin.

[0023] In one embodiment, the neural stem cells differentiate into one or more neural neurons selected from the group consisting of pain receptor neurons, photoreceptor neurons, and dopaminergic neurons.

[0024] The present invention also provides the application of neural stem cells obtained by the above method in the preparation of a therapeutic drug for nerve damage.

[0025] In another embodiment, the organoid formation comprises the steps of: (1) culturing a pluripotent stem cell floating cell group using the neural stem cell induction medium, adding 10 μM of ROCK inhibitor Y-27632, (2) replacing the neural stem cell induction medium the next day, and replacing the medium every day until the 10th day, (3) replacing the medium with the basic medium on the 10th day, further adding 2% B27 cell culture additive, and culturing until the 120th day.

[0026] The present invention also provides the application of the organoids obtained by the above-mentioned method to screening for therapeutic agents for nervous system diseases.

[0027] The present invention provides the application of antitumor small molecule inhibitors in the in vitro development of ectoderm cells, the combination of said compounds with a basal medium, and the culture of various neural cells and their clinical application.

[0028] The present invention provides a process for producing human artificial neural stem cells: pluripotent stem cells are cultured in a monolayer in a serum-free sensory nerve induction medium that contains components such as small chemical molecules, amino acids, and inorganic salts used in the present invention, but does not contain serum, substances of the BMP and TGF signaling pathways, and the pluripotent stem cells are cultured in this serum-free nerve induction medium for 20 days, resulting in neural stem cells that are uniformly arranged in a rosette shape.

[0029] In a specific embodiment, the substance acting on the BMP signaling pathway includes BMP2, BMP4, Smad1, Smad5, Smad8, and one or more proteins in a freely arranged combination, while the substance acting on the TGF signaling pathway includes activin, TGF-β, Nodal, Smad2, Smad3, and one or more proteins in a freely arranged combination.

[0030] In a specific embodiment, the adhesion culture is performed in the basement membrane preparation. The basement membrane preparation of the present invention forms a thin film of extracellular matrix molecules on the surface of the culture vessel, and can support parameters such as cell morphology, growth differentiation, and motility in an environment close to that in vivo. In a specific embodiment, the basement membrane is a combination of one or more of basement gel (Matrigel, STEMCELL Technologies), laminin, and vitronectin.

[0031] The serum-free medium in the present invention refers to a medium that does not contain serum directly separated from blood. Serum is the transparent liquid portion of plasma that does not contain fibrinogen or blood cells, and remains liquid even after blood coagulation. The serum-free medium may contain a serum substitute, and examples of serum substitutes include purified substances such as serum albumin, transferrin, and fatty acids.

[0032] In a specific embodiment, the adhesion culture in the first to third steps is preferably carried out in the presence of a basement membrane. The basement membrane of the present invention forms a thin film of extracellular matrix molecules on the surface of the culture vessel, and can support parameters such as cell morphology, growth differentiation, and motility in an environment close to that in vivo. In a specific embodiment, the basement membrane is a combination of one or more of basement gel (Matrigel, STEMCELL Technologies), laminin, and vitronectin.

[0033] The serum-free medium in the present invention refers to a medium that does not contain serum directly separated from blood. Serum is the clear liquid portion of plasma that does not contain fibrinogen or blood cells, and remains liquid even after blood has been coagulated. The serum-free medium may contain a serum substitute. Examples of serum substitutes include purified substances such as serum albumin, transferrin, and fatty acids, which are well known in the art as serum substitutes.

[0034] For the preparation of a drug for treating nerve damage, reference can be made to, for example, the method published by Okada et al. in which embryonic stem cells are used as a drug for treating nerve damage (Okada Y, Matsumoto A, Shimazaki T, Enoki R, Koizumi A, Ishii S, Itoyama Y, Sobue G, Okano H. Stem Cells. 2008 vol. 26, pp. 3086-98).

[0035] In addition to human-derived pluripotent cells, drugs used for treating nerve damage can include components such as buffer solutions containing salts and / or antibiotics, and nerve tissues to be treated (central nervous system such as brain and spinal cord, peripheral nervous system, etc.). Furthermore, diseases to be treated are not limited to specific symptoms, and include traumatic diseases (spinal cord injury, etc.), neurodegenerative diseases (atrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease, progressive supranuclear palsy, Huntington's disease, multiple system atrophy, spinocerebellar degeneration, etc.), and neuronal necrosis due to cerebral infarction or cerebral hemorrhage. In addition, the etiology is not particularly limited, and includes not only main causes related to injury, cerebral infarction, etc., but also secondary causes such as infections and tumors, and diseases or pathological symptoms caused by nerve cell damage.

[0036] definition Neural stem cells (NSCs) Neural stem cells have the ability to differentiate into neural neurons, astrocytes, and oligodendrocytes, and have the ability of self-renewal, and can provide a sufficient number of brain tissue cells. They are a kind of parent cells with the ability of division and self-renewal, and can produce various cells of neural tissues such as neural neurons, astrocytes, and oligodendrocytes through asymmetric division. The neural stem cells according to the present invention can be induced neural stem cells (iNSCs).

[0037] Neural rosette Neurulation is an early neural development process in which the neurula develops into the nervous system, including the brain and spinal cord, under precise control of various factors and is considered to be a critical stage in human embryonic development. Neurulation is the production of rosette-like neural stem cell structures within a group of cell tissues, i.e., one neural rosette structure represents one neurula. Effect of the Invention

[0038] (1) The medium provided by the present invention is serum-free, which solves the problem that animal-derived culture methods limit the clinical use of stem cells, and is suitable for culturing neural stem cells, including but not limited to induced pluripotent stem cells. (2) The medium provided by the present invention is characterized by its simplicity, safety, and efficiency in that it enables directed differentiation of induced stem cells into neural stem cells by using a single small molecule inhibitor. (3) The method provided by the present invention enables the induction of differentiated neural stem cells capable of differentiating into neural neurons or organoids. These cells can be used not only as materials for clinical research and clinical treatment, but also in screening research for drugs to treat nervous system diseases, and are expected to have significant economic and social benefits. [Brief description of the drawings]

[0039] attached [Figure 1]This is a diagram comparing the difference between the method of the present invention (LY induction method) and the control method (LSB induction method) in the neural stem cell induction process. Figure 1a shows the structure of neural rosettes induced and produced using multiple small molecule inhibitors for induction (LSB induction method). Figure 1b shows the structure of neural rosettes induced and produced using only LY2157299 (LY induction method). Figure 1c shows the difference in cell number of neural stem cells obtained by the two methods. [Diagram 2] This is a diagram showing the results of screening the core components of the basal medium in the method of the present invention. Figure 2(a) shows the effect of different concentrations of sodium chloride on the osmotic pressure of the culture system, and if the concentration is too high, the osmotic pressure exceeds the tolerance of the cells. Figure 2(b) shows the effect of different concentrations of insulin on cell proliferation (Cyquant experiment), and there is a positive correlation between the insulin concentration and the cell activity. Figure 2(c) shows the effect of different concentrations of recombinant human serum albumin on cell proliferation (Cyquant experiment), and there is a positive correlation between the recombinant human serum albumin concentration and the cell activity. [Diagram 3] 3 shows molecular identification of cells obtained by the method of the present invention (LY induction method) and the control method (LSB induction method). FIG. 3a shows neural stem cells obtained using the LSB induction method reconstructing the structure of neural rosettes after in vitro passage. FIG. 3b shows neural stem cells obtained using the LSB induction method, with the expression of the specific marker PAX6. FIG. 3c shows neural stem cells obtained using the LSB induction method, with the expression of the specific marker Nestin. FIG. 3d shows the pathway integration of FIG. 3a-3c. FIG. 3e shows neural stem cells obtained using the LY induction method reconstructing the structure of neural rosettes after in vitro passage. FIG. 3f shows neural stem cells obtained using the LY induction method, with the expression of the specific marker PAX6. FIG. 3g shows neural stem cells obtained using the LY induction method, with the expression of the specific marker Nestin. FIG. 3h shows the pathway integration of FIG. 3e. FIG. 3k shows a comparison of the difference in expression of neural stem cell markers during neural stem cell formation between the LY induction method and the LSB control induction method using Q-PCR. [Figure 4]Chemical induction of neural stem cells by different concentrations of LY2157299. Figure 4a shows neural stem cells obtained using polymolecular control (LSB induction method, indicated as CK). Figure 4b shows chemical induction with 0 nM LY2157299. Figure 4c shows chemical induction with 20 nM LY2157299. Figure 4d shows chemical induction with 12.5 μM LY2157299. Figure 4e shows chemical induction with 25 μM LY2157299. Figure 4f shows comparative analysis of the regulation of Pax6 during the induction process of different concentrations of LY2157299 using Q-PCR. Figure 4g shows comparative analysis of the regulation of cell apoptosis gene CASP3 during the induction process of different concentrations of LY2157299 using Q-PCR. [Diagram 5]The identification of the differentiation ability of neural stem cells obtained by the LY induction method is shown. Figure 5 a to c shows the induced neural stem cells obtained by LY2157299 further differentiated to obtain nociceptor neural neurons. Figure 5 a shows the nociceptor neural neurons obtained by this method, with the expression of the nociceptor neural neuron specific marker SCN11A. Figure 5 b shows the nociceptor neural neurons obtained by this method, with the expression of the nociceptor neural neuron specific marker Nestin. Figure 5 c shows the integration of two fluorescent passages, Figure 5 a and Figure 5 b. Figure 5 df shows the induced neural stem cells obtained by LY2157299 further differentiated to obtain dopaminergic neural neurons. Figure 5 d shows the dopaminergic neural neurons obtained by this method, with the expression of the mature dopaminergic neural neuron specific marker Pitx3. FIG. 5e shows dopaminergic neurons obtained by this method, with expression of the mature dopaminergic neuron specific marker TH. FIG. 5f shows the integration of two fluorescent passages, FIG. 5d and FIG. 5e. FIG. 5g-i shows the induced neural stem cells obtained by LY2157299 further differentiated to obtain photoreceptor neurons. FIG. 5g shows photoreceptor neurons obtained by this method, with expression of the photoreceptor neuron specific marker OPSIN. FIG. 5h shows photoreceptor neurons obtained by this method, with expression of the photoreceptor neuron specific marker CRX. FIG. 5i shows the integration of two fluorescent passages, FIG. 5g and FIG. 5h. [Figure 6] Analysis of MHC-related gene expression in neural stem cells obtained by the LY induction method. Figure 6 a-d shows that the expression of CD4, HLA-A, HLA-C, HLA-F, and HLA-DPB1 is significantly lower in neural stem cells obtained with LY2157299 than in human induced pluripotent stem cells. [Figure 7]Flow cytometry was used to detect HLA-DR antigens in neural stem cells obtained by induction with LY2157299. Figure 7a shows a negative control without antibody staining. Figure 7b shows the results of HLA-DR detection in neural stem cells obtained by induction with LY2157299. Figure 7a-1 shows the use of two passages, SSC and FSC, to eliminate cell debris and other particulate impurities and to set gates to determine cell populations for analysis. Figure 7a-2 and Figure 7a-3 show cell analysis using blank fluorescence passages and cell morphology, respectively. Figure 7a-4 shows cell analysis using two blank fluorescence passages. Figure 7b-1 shows the use of two passages, SSC and FSC, to eliminate cell debris and other particulate impurities and to set gates to determine cell populations for analysis. Figure 7b-2 and Figure 7b-3 show cell analysis using CD45 PerCP (Abcam, item no. ab157309) and HLA-DR FITC (CST, item no. 54126) passages, and cell morphology, respectively. Figure 7b-4 shows cell analysis using two fluorescent passages, CD45 PerCP and HLA-DR FITC. The results showed that the neural stem cells obtained by induction of LY2157299 were negative for the expression of HLA-DR antigen. [Figure 8] LY2157299 can induce the development of cerebral organoids. Figure 8a shows cerebral organoids (D50) formed by the LSB induction method. Figure 8b shows cerebral organoids (D50) formed by the LY induction method. Figure 8c shows the number of spontaneous discharges by the multichannel electrode system comparing cerebral organoids prepared by different methods. Figure 8d shows the frequency of spontaneous discharges by the multichannel electrode system comparing cerebral organoids prepared by different methods. Figure 8e shows the diameters of cerebral organoids prepared by different methods (n=10). Figure 8f-g show the analysis of gene expression differences obtained by different cerebral organoid preparation methods using Q-PCR. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the scope of the claims of the present invention is not limited to the following embodiments. In addition, it should be understood that the terms used in the embodiments of the present invention are intended to describe specific specific embodiments and are not intended to limit the scope of the claims of the present invention. Modifications and advantages that a person skilled in the art may envision without departing from the concept and scope of the concept of the present invention are included in the present invention, and the appended claims and their equivalents are also included in the scope of the claims of the present invention. In the specification and claims of the present invention, the singular forms "one", "one" and "this" include the plural forms unless the context clearly dictates otherwise. The experimental methods in the following examples are generally known and common knowledge of those skilled in the art or follow the conditions recommended by the manufacturer unless specific conditions are indicated. All materials and reagents used in the examples are commercially available unless otherwise specified.

[0041] Example 1: Preparation of medium for neural stem cell induction The composition of the neural induction basal medium (hereafter referred to as NouvNeu001) is: DMEM / F12 medium, 1% minimum essential medium non-essential amino acids (MEM non-essential amino acids, Thermo Fisher, lot number 11140076), sodium chloride (0.5 g / L), sodium selenite (13.6 μg / L), insulin (22 μg / ml), and recombinant human transferrin (100 ng / ml).

[0042] The method for preparing the neural stem cell induction medium used in the present invention is to add 55nM to 24μM of LY2157299 (Selleck, S2230) to NouvNeu001 basal medium, and the final concentration is preferably 300nM, 500nM, 700nM, 900nM, 1μM, 2.5μM, 5μM, 7.5μM, 10μM, 12.5μM, 15μM, 17.5μM, 20μM, or 22μM, and the most preferred concentration is 12.5μM. The above medium is called LY induction medium, and the following experimental set using this medium is called LY induction method.

[0043] The medium used in the control experiment was prepared by adding 100 nM LDN-193189 (Selleck, S2618) and 10 μM SB431542 (Selleck, S1067) to NouvNeu001 basal medium. The above medium is called LSB induction medium, and the following experimental set using this medium is called LSB induction method.

[0044] Example 2: Derivation and identification of neural stem cells 2.1 Chemical induction of neural stem cells Human pluripotent stem cells include embryonic pluripotent stem cells such as the H9 cell line and human induced pluripotent stem cells. The human induced pluripotent stem cells used in the present invention are CD34 reprogrammed cells according to the "Reprogramming medium and method for culturing reprogrammed induced pluripotent stem cells" (method of patent ZL201910050800.7). + It is obtained from cells.

[0045] Human pluripotent stem cells are cultured using a T25 cell culture flask coated with Matrigel (STEMCELL Technologies), the cell type is placed in the medium, and incubated in a 37°C incubator for 1 hour or more. 6 The cells are seeded into a T25 culture flask for expansion and passage.

[0046] At the time of neural induction, 6-well culture plates are coated with 50 μg / ml polylysine (SIGMA, item P6407) and the plates are placed in a 37°C incubator and incubated for at least 3 hours. Then, the plates are further coated with 5 μg / ml laminin (Laminin, SIGMA ALDRICH, lot number I2020), the cell types are placed in the medium, and the plates are placed in a 37°C incubator and incubated for at least 3 hours. When the pluripotent stem cells reach a coverage rate of 70%, they are digested with EDTA at 37°C for 5 minutes, and cell digestion is terminated with DMEM. After washing and centrifuging the cells, 2 × 10 cells are placed in a T25 culture plate. 5 The cells are reseeded in a flask / flask. The medium and neural induction compound of the present invention are used, and the medium is replaced daily until the neural stem cell rosettes are formed. The single molecule induction method (LY induction method) used in the present invention formed small and uniform neural rosette structures as shown in Fig. 1a and Fig. 1b, compared with the multi-molecule induction method (LSB induction method) used in the control experiment. This result indicates that the single molecule induction method provided by the present method produced more uniform induction results compared with the multi-molecule induction method used in the control experiment. The multi-molecule induction method used in the control experiment produced rosette structures of different sizes, and there was also local non-formation of neural rosette structures during the same culture cycle. Thus, it was shown that the single molecule induction method provided by the present method can produce induction results such as more efficient and uniform formation of neural rosette structures in pluripotent stem cells.

[0047] 2.2 Identification of the concentrations of major components in the induction system Using the LSB method as a control, the Cyquant test was used to quantitatively measure cell activity and to examine the effect of LY2157299 on the cell physiological state during the long-term cell culture process. 96-well light-shielding cell culture plates were coated, and after coating was completed, 5X10 cell types were added. 41 well, and three sets were placed in parallel (the average value of the three sets was used as the data for calculation). The process of Example 2.1 was repeated, and the culture conditions were 37 degrees and 5% carbon dioxide. Samples were taken on the 1st, 5th, 10th, 15th, and 20th days, and the cell activity was detected using a CyQuant Kit (Invitrogen, item no. X12223), and the data was read using a SpectraMax i3 Multi-Mode Microplate Reader (VWR, model ID3-STD) according to the instruction manual. The results are shown in Figure 1c. In the Cyquant experiment, LY2157299 induced more neurons than the LSB method, indicating that LY2157299 had higher proliferation activity during the induction process.

[0048] Similarly, Cyquant assay was used to quantitatively measure cell activity and to investigate the effects of different concentrations of recombinant insulin and recombinant serum albumin on the physiological state of cells during long-term cell culture. The results are shown in Figure 2b and Figure 2c, and both the concentrations of recombinant insulin and recombinant serum albumin are positively correlated with the cell proliferation rate. Considering the cost factor, the recombinant insulin and recombinant serum albumin used in the present invention are in the optimal range.

[0049] An automatic freezing point osmometer (FM-8P, Shanghai Medical University Instrument Co., Ltd.) was used to test the effect of different concentrations of sodium chloride on the osmotic pressure of the culture system. The specific operation was referred to the product manual. The detailed test results are shown in Figure 2a. The results show that the osmotic pressure of the culture system increases linearly in proportion to the sodium chloride concentration. However, at a concentration of 1g / ml, the osmotic pressure of the culture system already exceeds the cell culture limit osmotic pressure of 320mOsm / kg. This indicates that the sodium chloride concentration used in the present invention is the most suitable concentration for cell culture.

[0050] 2.3 Fluorescent immunoassay of neural stem cell markers Neural stem cells obtained by LY induction and LSB induction are collected separately and identified by immunofluorescence staining. The cells are fixed with 4% formaldehyde for 40 min at room temperature and washed twice with DPBS buffer. The cells are permeabilized with 0.1% Triton X-100 for 5 min and washed twice with DPBS buffer. The cells are then incubated overnight at 4°C with DPBS buffer containing 10% horse serum and 0.1% Triton X-100. The cells are then washed with DPBS buffer, and the primary antibody is diluted in DPBS buffer containing 2% horse serum and 0.1% Triton X-100 and incubated at 37°C for 2 h. After washing with DPBS buffer, the secondary antibody is diluted in DPBS buffer containing 2% horse serum and 0.1% Triton X-100, incubated at 37°C for 2 h, washed three times, and photographed. Photography is performed using a Leica DMi8. Details of the antibody usage are shown in Table 1. The results, shown in Figure 3a-h, confirmed that, compared with the LSB induction method, the LY induction method enabled pluripotent stem cells to form neural rosette structures and express neural stem cell markers Pax6 and Nestin.

[0051] [Table 1]

[0052] 2.4 Identification of transcription levels in neural stem cells The transcriptional changes of various marker genes during the induction process from pluripotent stem cells to neural stem cells are detected using Q-PCR. The induced pluripotent stem cells used in this example were neural stem cells obtained by the LY induction method and the LSB induction method, and total RNA was extracted using Rneasy Mini or Micro Kit (QIAGEN), respectively. 1 mg of RNA was used to perform cDNA synthesis using SuperScript III First-Strand Synthesis System (Invitrogen). Quantitative PCR labeling and reaction were performed using SYBR Premix Ex Taq (TaKaRa) and Thermal Cycler Dice Real Time System (TaKaRa), and β-actin was used as an internal reference. All data were analyzed by the delta-Ct method. Each set of experiments was tested repeatedly using three sets, and variance statistics were performed. The primer sequences used to identify genes encoding different cell markers are shown in Table 2. The results are shown in Figure 3 ik. Neural stem cells obtained by the LY and LSB induction methods expressed neural stem cell-specific markers Sox2, Pax6, and Nestin, and the expression of neural stem cell markers was increased by the LY induction method compared to the LSB induction method.

[0053] The results of concentration screening by adding different concentrations of LY2157299 to NouvNeu001 basal medium using the above method are shown in Figure 4b-e. Figure 4b shows that when LY2157299 is not used, cells do not differentiate into ectodermal cells. Figure 4c shows that when chemical induction is performed using 20 nM LY2157299, a mixed cell population including neural cells is obtained. Figure 4d shows that when chemical induction is performed using 12.5 μM LY2157299, a morphologically homogenous cell population is obtained, and all cells are similar to the control (4a) and have the same typical neural stem cell morphology. Figure 4e shows that when chemical induction is performed using 25 μM LY2157299, a mixed neuronal-like cell population is obtained, with some cells forming longer axons and showing signs of stress with intracellular vacuoles. These results reveal that differentiation is incomplete under low concentration conditions, and a wide variety of different cell types are observed. It was also found that neural stem cells obtained under high concentration conditions easily differentiated and showed signs of intracellular stress. Transcription levels further indicated that the concentration used in the present invention was optimal (Fig. 4 fg). Fig. 4 f shows a positive correlation between the concentration of LY2157299 and the expression of Pax6. Fig. 4 g shows a positive correlation between the concentration of LY2157299 and apoptosis. These results revealed that either too low or too high a concentration of LY2157299 may have a negative effect on neural induction.

[0054] [Table 2]

[0055] Example 3: Identification of differentiation function of neural stem cells Directed differentiation was performed in NouvNeu001 basal medium using neural stem cells obtained by the LY induction method in Example 2. When performing neural cell differentiation, 6-well culture plates were coated with 50 μg / ml polylysine (SIGMA ALDRICH, lot number P6407) and plated, and incubated in a 37° C. incubator for 3 hours or more until the cells were seeded.

[0056] 3.1 Differentiation of nociceptive neurons The neural stem cells obtained in Example 2 were cultured at 1 × 10 5 The cells were inoculated into a T25 culture flask at a ratio of 1000 / flask, 3 μM CHIR99021 (Selleck, lot number S2924), 10 μM SU5402 (Tocris, lot number 3300 / 1), and 10 μM DAPT (Selleck, lot number S2215) were added to NouvNeu001 basal medium, and fresh medium was replaced every 3 days until the 21st day. The culture conditions were 37°C and 5% carbon dioxide. As a result of the experiment, the neural stem cells obtained in Example 2 had axon structures after differentiation and expressed the nociceptor neuron markers SCN11A and Nestin (Figure 5a-c). Specifically, Figure 5a shows the nociceptor neuron expressing the nociceptor neuron specific marker SCN11A obtained by further differentiating the induced neural stem cells obtained by LY2157299. Figure 5b shows the nociceptor neuron expressing the nociceptor neuron specific marker Nestin obtained by this method. Figure 5c is a merged photograph of the two fluorescent passages in Figure 5a and Figure 5b.

[0057] 3.2 Induction of dopaminergic neurons The neural stem cells obtained in Example 2 were cultured in a T25 culture plate at 1 × 10 5The cells were reseeded at a ratio of 1 / flask. The neural stem cells were cultured in NouvNeu001 basal medium supplemented with 1 μM Purmorphamine (Sellek, lot number S3042) and 1 ng / ml TGF-β3 (Novoprotein, lot number CJ44) at 37 °C and 5% carbon dioxide, with medium changes every 3 days until neural neuron formation on the 30th day. As a result, the neural stem cells obtained in Example 2 had axonal structures after differentiation and expressed tyrosine hydroxylase (TH) and Pitx3, which are specific markers of mature dopaminergic neural neurons (Figure 5d-f). Specifically, Figure 5d shows that the induced neural stem cells obtained by LY2157299 further differentiated and expressed the dopaminergic neuron-specific marker Pitx3. Figure 5e shows that the induced neural stem cells obtained by this method expressed the dopaminergic neuron-specific marker TH. Figure 5f shows the integration of two fluorescent passages, Figure 5d and Figure 5e.

[0058] 3.3 Differentiation of photoreceptor neurons The neural stem cells obtained in Example 2 were cultured in a T25 culture plate at 1×10 5 The cells were reseeded at a ratio of 1 / flask. The NouvNeu001 basal medium supplemented with 0.5 μM retinyl acetate (Sigma Aldrich, Lot No. R7882) was used, and the medium was replaced with fresh medium every 3 days until the 21st day. The culture conditions were 37°C and 5% carbon dioxide. As a result of the experiment, the neural stem cells obtained in Example 2 had axonal structures after differentiation and expressed the photoreceptor neuron markers OPSIN and CRX (Figure 5 g-i). Specifically, Figure 5 g shows that the induced neural stem cells obtained by LY2157299 were further differentiated to obtain photoreceptor neuron expressing the photoreceptor neuron specific marker OPSIN. Figure 5 h shows that the photoreceptor neuron obtained by this method obtained photoreceptor neuron expressing the specific marker CRX. Figure 5 i shows the integration of two fluorescent passages of Figure 5 g and Figure 5 h.

[0059] After differentiation induction was completed, cells were harvested for immunofluorescence staining identification. The cells were fixed with 4% paraformaldehyde at room temperature for 40 min and washed twice with DPBS buffer. Then, the cells were permeabilized with 0.1% Triton X-100 for 5 min and washed twice with DPBS buffer, after which the cells were incubated overnight at 4°C in DPBS buffer containing 10% horse serum and 0.1% Triton X-100. After washing the cells with DPBS buffer, the secondary antibody was diluted in DPBS buffer containing 2% horse serum and 0.1% Triton X-100, incubated for 2 h at 37°C, washed three times, and photographed with a Leica Dmi8. The antibody usage details are shown in Table 3.

[0060] [Table 3] These results demonstrated that neural stem cells obtained by the LY induction method have the ability to differentiate into various types of neurons.

[0061] Example 4: Effect of LY2157299 on MHC formation 4.1 Q-PCR measurements According to the method of Example 2.4, major histocompatibility complex (MHC) genes of neural stem cells obtained by induction with LY2157299 are measured by Q-PCR using pluripotent stem cells (iPS) as a control. The primer sequences are shown in Table 4, and the measurement results are shown in Figure 6. It was confirmed that the expression of MHC-related genes CD4, HLA-A, HLA-C, HLA-F, and HLA-DPB1 was extremely low in neural stem cells obtained by introducing LY2157299, compared to human-derived pluripotent stem cells.

[0062] [Table 4-1]

[0063] [Table 4-2]

[0064] 4.2 Flow cytometry measurements The HLA-DR antigen was measured by flow cytometry in neural stem cells obtained by induction of LY2157299. HLA-DR is a kind of MHC class II molecule, which is very important for the antigen presentation ability of cells and plays an important role in adoptive immune response. The induced neural stem cells obtained in Example 2 were first digested with EDTA, washed with DPBS, centrifuged to remove the supernatant, and then 106 cells were resuspended in 100 μl of diluted primary antibody (CD45 PerCP, Abcam, lot number ab157309, HLA-DR FITC, CST, lot number 54126), incubated on ice for 1 hour in the dark, and centrifuged. The supernatant was removed, and the cells were resuspended in 500 μl of antibody dilution buffer (CST) and analyzed by flow cytometry. The sample incubated without primary antibody was used as the control. The results are shown in Figure 7, where a in Figure 7 is the negative control, and b in Figure 7 shows the measurement results of HLA-DR of neural stem cells obtained by induction with LY2157299. This result shows that neural stem cells obtained by the LY induction method do not express HLA-DR. This result is consistent with the results of Example 4.1.

[0065] Example 5: Effect of LY2157299 on organoid formation Brain organoids were cultured using human induced pluripotent stem cells. The induction medium for brain organoids was NouvNeu basal medium supplemented with 12.5 μM LY2157299. The induced pluripotent stem cells were digested into single cells using Accutase, and the brain organoid induction medium was inoculated into a U-bottom-ultra-low attachment 96-well plate at a ratio of 9000 cells / 150 ml. 10 μM of ROCK inhibitor Y-27632 (Selleck, S1049) was added, and the medium was incubated at 37 °C for 24 hours using a cell incubator (Panasonic, model number MCO-18AC) with 5% carbon dioxide. The medium was replaced with fresh brain organoid induction medium without Y-27632 from the next day until the 10th day. On day 10, brain organoids were transferred to ultra-low attachment 6-well plates, changed to NouvNeu001 basal medium supplemented with 2% B27 cell culture additive, and cultured at 37°C, 5% carbon dioxide, and 80 rpm on a horizontal rotator cell culture device until day 120.

[0066] The control experiment used a brain organoid culture basal medium with N2B27 according to the published method (Clair B et.al, Nat Methods. 2019, Nov; 16(11):1169-1175). The control brain organoid induction medium was N2B27 basal medium supplemented with 10 μM SB-431542, 100 nM LDN-193189, and 2 μM XAV-939. The rest of the operation was as described above. In the present invention and figures, this method is referred to as the N2B27 method.

[0067] The organoids were photographed using a Leica Dmi8, and the results of photographing and measuring the diameter of the brain organoids are shown in Figure 8a, Figure 8b, and Figure 8e. The spontaneous electrical signals of the cells were measured using a multichannel electrode for the induced brain organoids. The 120-day induced brain organoids were digested with 10% trypsin / EDTA at 37°C for 5-8 minutes. A 96-well MEA system multi-channel electrode plate (AXION Biosystem, US) was coated with 100 ng / ml polylysine (Poly-L-lysine, Sigma-Aldrich, P4707) and placed in a cell incubator (Panasonic, model number MCO-18AC) at 37°C and 5% carbon dioxide for 12 hours. The polylysine-coated MEA was removed, the polylysine was aspirated, and the MEA was washed three times with sterile water. After that, a PBS solution containing 3 μg / ml gelatin (laminin, SIGMA ALDRICH, lot number I2020) was used as a substrate for coating the neural cells, and added to the MEA multichannel electrode plate and placed in a 37°C, 5% carbon dioxide incubator for 3 hours. After coating the MEA multi-passage electrode plate, 5 x 10 digested brain organoid cells were 5 The cells were seeded at 100 cells / well. The seeded MEA multichannel electrode plate was placed in the MEA chamber, and the cell culture conditions were adjusted to 37°C and 5% carbon dioxide using the AxIS Navigator 2.0.2 software. The chamber was operated for 10 minutes until the environment was stable. The spontaneous electrical signals of the cells were recorded using the AxIS Navigator 2.0.2 software (AXION Biosystem, US). The experimental results showed that the neurons induced with the NouNeu system showed good electrophysiological activity, and the cell spontaneous discharge was measured using a multi-passage electromechanical system. The number of cell spontaneous discharges per unit time (Figure 8c) and the average discharge rate (Figure 8d) were compared, and it was revealed that the LY induction method showed more active electrophysiological activity compared to the N2B27 control induction method.

[0068] The transcriptional changes of different marker genes after brain organoid formation were examined by Q-PCR. Total RNA from 120-day brain organoids obtained by different culture methods and human induced pluripotent stem cell control was extracted with Rneasy Mini or Micro Kit (QIAGEN), respectively, and 1 mg of RNA was used to synthesize cDNA using SuperScript III First-Strand Synthesis System (Invitrogen). Quantitative PCR labeling and reactions were performed using SYBR Premix Ex Taq (TaKaRa) and Thermal Cycler Dice Real Time System (TaKaRa), with β-actin used as an internal reference. All data were analyzed by the delta-Ct method. Each set of experiments was repeated in triplicate, and variance statistics were also performed. The primer sequences used to identify genes encoding different cell markers are listed in Table 5. Glial cells, neural progenitor cells, and neural neurons were obtained from brain organoids formed by the LY induction method and the N2B27 control induction method (Figure 8 f-g).

[0069] [Table 5]

[0070] From the above, LY2157299 can induce the generation of brain organoids, which have electrophysiological functions and expression of key genes. Furthermore, the brain organoids formed by the LY induction method were larger in diameter and had more active electrophysiological functions compared to the N2B27 control induction method.

[0071] All documents mentioned in the present invention are incorporated herein by reference in their entirety. Furthermore, it should be understood that various changes or modifications may be made to the present invention by those skilled in the art after viewing the above disclosure of the present invention, and that equivalent modifications and forms thereof are also within the scope defined by the claims of the present application.

Claims

1. the TGF-β inhibitor is only 4-[2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]quinoline-6-carboxamide, the TGF-β inhibitor is added to a basal medium to form a neural stem cell induction medium, and the neural stem cell induction medium does not contain a protein kinase C inhibitor, a SMAD2 / 3 inhibitor other than 4-[2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]quinoline-6-carboxamide, and / or a WNT signaling activator; a TGF-β inhibitor is the only small molecule inhibitor in said neural stem cell induction medium; The basal medium is composed of DMEM / F12 medium, 1% minimum essential medium non-essential amino acids, 0.5 g / L sodium chloride, 13.6 μg / L sodium selenite, 22 μg / ml insulin, and 100 ng / ml recombinant human transferrin. Use of TGF-β inhibitors in inducing neural stem cells and inducing organoid formation.

2. 2. The use according to claim 1, characterized in that the TGF-β inhibitor is present in a concentration between 10 nM and 100 μM.

3. The use according to claim 2, characterized in that the concentration of the TGF-β inhibitor is 12.5 μM.

4. The use according to claim 1 , wherein the formation of neural stem cells comprises a step of culturing pluripotent stem cells in an adherent manner using the neural stem cell induction medium.

5. The use according to claim 4, characterized in that the pluripotent stem cells are mammalian pluripotent stem cells.

6. The use according to claim 5 , characterized in that the pluripotent stem cells are human pluripotent stem cells.

7. The use according to claim 4, characterized in that the adherent culture is carried out in the presence of a basement membrane preparation.

8. The use according to claim 7, characterized in that the basement membrane preparation is one or a combination of two or more of basement membrane gel, laminin and vitronectin.

9. The use according to claim 4, characterized in that the neural stem cells are differentiated into one or more neural neurons selected from the group consisting of pain receptor neural neurons, photoreceptor neural neurons, and dopaminergic neural neurons.

10. 5. Use of neural stem cells obtained by the process according to claim 4 in the preparation of a remedy for nerve damage.

11. Organoid formation involves the following steps: (1) A step of culturing a floating population of pluripotent stem cells using the neural stem cell induction medium according to claim 1 and adding 10 μM of a ROCK inhibitor Y-27632; (2) replacing the neural stem cell induction medium according to claim 1 on the following day, and replacing the medium every day until the 10th day; (3) on the 10th day, replacing the medium with the basal medium according to claim 1, further adding 2% B27 cell culture additive, and culturing until the 120th day; 2. The use according to claim 1, characterized in that it comprises:

12. Use of the organoid obtained by the process described in claim 11 in screening for therapeutic drugs for neurological diseases.

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