Method for preparing motor neurons expressing snap-25, and botulinum neurotoxin potency measuring method using prepared motor neurons

Abstract

The present invention relates to a method for preparing motor neurons expressing SNAP-25, and a botulinum neurotoxin potency measuring method using motor neurons prepared by the method. iPSC-derived motor neurons expressing SNAP-25 prepared by the method of the present invention have been confirmed to highly express SNAP-25 and neuronal and motor neuronal markers in a stable manner. When such motor neurons of the present invention are applied to a botulinum toxin potency test, a potency similar to that shown in a LD50 assay measured in mice has been confirmed to be exhibited. In addition, unlike conventional botulinum toxin potency test methods, the neurons of the present invention are used for analysis, and thus the advantage of being able to represent actual neuron responses is achieved. The method and the motor neurons prepared by the method of the present invention may be used in various ways in the field of botulinum toxin potency research.

Description

Method for producing motor neurons expressing SNAP-25 and method for measuring botulinum neurotoxin titer using the produced motor neurons

[0001] The present invention relates to a method for producing motor neurons expressing SNAP-25 and a method for measuring botulinum neurotoxin titer using motor neurons produced by the method.

[0002] Botulinum neurotoxin (BoNT) is a family of structurally similar but antigenically distinct protein neurotoxins that act on the peripheral nervous system to block neuromuscular transmission. These neurotoxins are highly potent, with a lethal dose of approximately micrograms, causing botulism, a rare but frequently fatal disease. Currently, assays for botulinum neurotoxin are used in both the food and pharmaceutical industries. The food industry employs assays for botulinum neurotoxin to validate new food packaging methods and ensure food safety. As the clinical use of botulinum toxin increases, the pharmaceutical industry requires accurate assays for this toxin for both product formulation and quality control.

[0003] Currently, the mouse LD50 assay is used to measure botulinum neurotoxin potency, and it is a highly sensitive and accurate test method that is currently used as a global standard. In fact, the World Health Organization (WHO) and the U.S. Centers for Disease Control and Prevention utilize the mouse LD50 assay to measure botulinum neurotoxin potency. However, the mouse LD50 assay requires at least 3-5 days to measure botulinum neurotoxin potency and animal sacrifice. Furthermore, because it uses animals, it is difficult to test multiple samples simultaneously. Furthermore, the mouse LD50 assay has limitations in that the results cannot fully represent human responses due to differences in structure and response between species.

[0004] Accordingly, the present inventors developed a method for differentiating motor neurons expressing SNAP-25 from induced pluripotent stem cells, and completed the present invention by confirming that botulinum neurotoxin titer can be measured using motor neurons produced according to the present invention.

[0005] Accordingly, the purpose of the present invention is to provide a method for producing motor neurons and motor neurons produced by the method.

[0006] Another object of the present invention is to provide a method for measuring botulinum neurotoxin potency using motor neurons.

[0007] Another object of the present invention is to provide a composition for measuring botulinum neurotoxin potency including motor neurons.

[0008] Another object of the present invention is to provide a kit for measuring the potency of botulinum neurotoxin including motor neurons.

[0009] In order to achieve the above object, the present invention provides a method for producing motor neurons, comprising: (a) culturing stem cells in a culture medium containing at least one selected from the group consisting of Y27632, CHIR99021, SB431542, and DMH1; (b) culturing the cells of step (a) in a culture medium containing at least one selected from the group consisting of DMH1, SB431542, retinoic acid (RA), and purmorphamine (Pur); and (c) culturing the cells of step (b) in a medium containing retinoic acid and purmorphamine.

[0010] The present invention also provides motor neurons manufactured by the above method.

[0011] In addition, the present invention provides a method for measuring botulinum neurotoxin titer, comprising the steps of (a) treating the motor neurons with botulinum neurotoxin and then culturing them; and (b) measuring the expression of SNAP-25 in the cultured cells of (a).

[0012] The present invention also provides a composition for measuring botulinum neurotoxin potency including the above motor neurons.

[0013] The present invention also provides a kit for measuring the potency of botulinum neurotoxin including the above motor neurons.

[0014] It was confirmed that the iPSC-derived motor neurons expressing SNAP-25 produced by the method of the present invention stably and highly express SNAP-25, neuronal markers, and motor neuron markers. In addition, when the motor neurons of the present invention were applied to a botulinum toxin titer test, it was confirmed that they showed titers similar to the LD50 assay measured in mice. In addition, unlike existing botulinum toxin titer test methods, since the neurons of the present invention are used for analysis, there is an advantage in that they can represent the response of actual neurons. Therefore, the method of the present invention and the motor neurons produced by the method can be utilized in various fields of botulinum toxin titer research.

[0015] Figure 1 is a schematic diagram showing the differentiation of iPSC-derived motor neurons expressing SNAP-25.

[0016] Figure 2 is a diagram showing the results of measuring the expression of SNAP-25, TuJ1, and MAP2 in iPSC-derived motor neurons expressing SNAP-25 according to culture conditions.

[0017] Figure 3 is a diagram showing the results of measuring the expression of SNAP-25 and TRA-1-60 in iPSC-derived motor neurons expressing SNAP-25 according to the differentiation period.

[0018] Figure 4 is a diagram showing the results of measuring the expression of SNAP-25, an undifferentiated marker (TRA-1-60), neuronal markers (TuJ1, MAP2), and a motor neuron-specific marker (ChAT) in iPSC-derived motor neurons expressing SNAP-25.

[0019] Figure 5 is a diagram showing the results of measuring the expression of SNAP-25 in iPSC-derived motor neurons expressing SNAP-25 using flow cytometry.

[0020] Figure 6 is a diagram showing the results of measuring the protein expression of SNAP-25 in iPSC-derived motor neurons expressing SNAP-25 through Western blot.

[0021] Figure 7 is a diagram showing the results of quantifying the protein of SNAP-25 in iPSC-derived motor neurons expressing SNAP-25 through ELISA.

[0022] Figure 8 is a diagram showing the results of confirming neurite outgrowth and expression of related markers in iPSC-derived motor neurons expressing SNAP-25 according to the coating method.

[0023] Figure 9 is a diagram showing the results of measuring botulinum neurotoxin titer using iPSC-derived motor neurons expressing SNAP-25.

[0024] Hereinafter, the present invention will be described in detail.

[0025] According to an aspect of the present invention, the present invention provides a method for producing motor neurons and motor neurons produced by the method. Specifically, the method for producing motor neurons comprises: (a) culturing stem cells in a culture medium containing at least one selected from the group consisting of Y27632, CHIR99021, SB431542, and DMH1; (b) culturing the cells of step (a) in a culture medium containing at least one selected from the group consisting of DMH1, SB431542, retinoic acid (RA), and purmorphamine (Pur); and (c) culturing the cells of step (b) in a medium containing retinoic acid or purmorphamine.

[0026] In a specific embodiment of the present invention, the stem cell may be a pluripotent stem cell, an induced pluripotent stem cell, or an embryonic stem cell, and preferably an induced pluripotent stem cell.

[0027] In the present invention, stem cells refer to cells that have the ability to self-replicate and differentiate into two or more cells, and include pluripotent stem cells, induced pluripotent stem cells, embryonic stem cells, etc.

[0028] In the present invention, induced pluripotent stem cells (iPSCs) refer to cells that have been reprogrammed from already differentiated cells to a pre-differentiated state. Induced pluripotent stem cells possess morphological and functional characteristics similar to embryonic stem cells, can indefinitely self-replicate in a culture environment, and have the ability to differentiate into all cell types.

[0029] In a specific example of the present invention, the step (a) is preferably a step of culturing stem cells in a culture medium containing at least one selected from the group consisting of Y27632, CHIR99021, SB431542, and DMH1, and culturing the cultured stem cells in a culture medium containing at least one selected from the group consisting of CHIR99021, SB431542, and DMH1.

[0030] In the most preferred embodiment of the present invention, the method for producing motor neurons of the present invention preferably sequentially performs the following steps (a) to (c):

[0031] (a) a step of culturing stem cells in a culture medium containing at least one selected from the group consisting of Y27632, CHIR99021, SB431542, and DMH1, and culturing the cultured stem cells in a culture medium containing at least one selected from the group consisting of CHIR99021, SB431542, and DMH1;

[0032] (b) a step of culturing the cells of step (a) with a culture medium containing at least one selected from the group consisting of DMH1, SB431542, retinoic acid (RA), and purmorphamine (Pur); and

[0033] (c) A step of culturing the cells of step (b) in a medium containing retinoic acid or permorphamine.

[0034] In a specific embodiment of the present invention, the culture in steps (a) to (c) is preferably a 3D culture. In an embodiment of the present invention, when culturing motor neurons using 2D culture, two peaks were observed, indicating the possibility of the existence of two cell subpopulations. In the case of 3D culture, one uniform peak was observed, indicating that the cell purity is high during 3D culture.

[0035] In a preferred embodiment of the present invention, the 3D culture is preferably performed in a non-adhesive culture vessel, preferably an aggrewell plate. When stem cells are 3D cultured in a non-adhesive culture vessel, 3D spheroids of uniform size can be mass-produced. Furthermore, since the 3D spheroids are uniformly sized, cell differentiation can be uniformly induced, and drug testing can also be performed under uniform conditions.

[0036]

[0037] The method for producing motor neurons of the present invention can be performed as follows.

[0038]

[0039] -Stage 0: This stage is a pre-culture stage and may be performed prior to step (a). In this stage, it is preferable to pre-culture the cells for 3 to 5 days, and more preferably, for 4 days. The culture medium used for the pre-culture may be Stem MACS.

[0040]

[0041] - Stage 1: This stage is a stage for forming neural stem spheroids, and corresponds to stage (a) in the claims. It is preferable that the stage (a) be cultured for 1 to 7 days. This stage may be culturing stem cells in a culture medium containing at least one selected from the group consisting of Y27632, CHIR99021, SB431542, and DMH1.

[0042] More preferably, this step can be divided into Stage 1-1 and 1-2.

[0043] The culture medium of Stage 1-1 above preferably contains at least one selected from the group consisting of Y27632 5 to 15 μM, CHIR99021 1 to 5 μM, SB431542 1 to 5 μM, and DMH 1 to 5 μM, and more preferably may contain Y27632 8 to 12 μM, CHIR99021 1 to 3 μM, SB431542 1 to 3 μM, and DMH1 1 to 3 μM. In addition, it is preferable that Stage 1-1 is cultured for 1 to 2 days, and more preferably may be cultured for 1 day.

[0044] In addition, the culture medium of Stage 1-2 preferably includes at least one selected from the group consisting of CHIR99021 1 to 5 μM, SB431542 1 to 5 μM, and DMH1 1 to 5 μM, and more preferably may include CHIR99021 1 to 3 μM, SB431542 1 to 3 μM, and DMH1 1 to 3 μM. In addition, the culture medium of Stage 1-2 is preferably cultured for 2 to 6 days, and more preferably may be cultured for 4 days.

[0045] The basic culture medium of the above stages 1-1 and 1-2 may be a mixed culture medium (1:1) of Advanced DMEM / F12 and Neurobasal media containing MEM Non-Essential Amino Acids, N2, B27, Glutamax, p / s, and Ascorbic acid.

[0046]

[0047] -Stage 2: This step is a step for forming motor neuron progenitor spheroids, and corresponds to step (b) in the claims. In this step, the cells cultured in Stage 1 can be cultured in a culture medium containing at least one selected from the group consisting of DMH1, SB431542, retinoic acid, and permorphamine, and preferably, the culture medium can contain at least one selected from the group consisting of 1 to 5 μM DMH1, 1 to 5 μM SB431542, 0.1 to 2 μM retinoic acid, and 0.1 to 2 μM permorphamine, and more preferably, it can contain 1 to 3 μM DMH1, 1 to 3 μM SB431542, 0.1 to 1 μM retinoic acid, and 0.1 to 1 μM permorphamine. Additionally, the basic culture medium of Stage 2 may be Neurobasal media containing MEM Non-Essential Amino Acids, N2, B27, Glutamax, and p / s.

[0048] It is preferable to culture the above Stage 2 for 1 to 3 days, and more preferably for 2 days.

[0049]

[0050] -Stage 3: This stage is a stage for forming motor neuron spheroids, and corresponds to stage (c) in the claims. In this stage, the cells cultured in Stage 2 may be cultured in a medium containing retinoic acid or permorphamine, preferably in a medium containing 0.1 to 2 μM retinoic acid and 0.1 to 2 μM permorphamine, and more preferably in a medium containing 0.1 to 1 μM retinoic acid and 0.1 to 1 μM permorphamine. In addition, the basic culture medium of Stage 3 may be Neurobasal media containing MEM Non-Essential Amino Acids, N2, B27, Glutamax, p / s.

[0051] The above Stage 3 is preferably cultured for 20 to 60 days, more preferably for 30 to 50 days, and even more preferably for 30 to 35 days.

[0052]

[0053] In a specific embodiment of the present invention, it is preferable that the motor neurons produced by the method of the present invention express SNAP-25.

[0054] In a specific example of the present invention, it is preferable that the motor neurons produced by the method of the present invention express at least one selected from the group consisting of ChAT, TuJ1, and MAP2.

[0055] In a specific example of the present invention, it is preferable that the motor neurons produced by the method of the present invention in the examples of the present invention do not express TRA-1-60.

[0056] Motor neurons produced by the method of the present invention were confirmed to stably and highly express SNAP-25, as well as motor neuron markers (ChAT) and neuronal markers (TuJ1, MAP2). Simultaneously, undifferentiated markers were confirmed to be underexpressed. This indicates that motor neurons produced by the method of the present invention are highly pure.

[0057]

[0058] According to another aspect of the present invention, the present invention provides a method for measuring botulinum neurotoxin potency, comprising the steps of: (a) treating motor neurons produced by the above method with botulinum neurotoxin and then culturing them; and (b) measuring the expression of SNAP-25 in the cultured cells of (a).

[0059] In a specific example of the present invention, the culturing in step (a) is preferably performed for 12 to 60 hours, more preferably 48 hours, but the scope of the present invention is not limited thereto.

[0060] In a specific example of the present invention, the culturing in step (a) is preferably performed in (i) a culture vessel coated with a mixture containing poly-L-ornithine and laminin; or (ii) a culture vessel sequentially coated with poly-L-ornithine and laminin; and more preferably, it may be performed in the culture vessel of (i). In an example of the present invention, it was confirmed that when poly-L-ornithine and laminin were sequentially coated, cells were strongly attached, and nerve axons were better extended.

[0061] In a specific embodiment of the present invention, the measurement of the expression of SNAP-25 in step (b) may be Western blotting using an antibody capable of identifying the 25 kD, uncleaved form of SNAP-25.

[0062] The method for measuring botulinum neurotoxin potency according to the present invention, although a cell-based test, was confirmed to exhibit a potency similar to the mouse LD50 assay of 1 Unit of botulinum neurotoxin. This indicates that the motor neurons produced by the method of the present invention are cells applicable to the cell-based potency test of botulinum neurotoxin. Furthermore, the motor neurons produced by the method of the present invention have the advantage of being able to represent the responsiveness of actual nerve cells because they can reproduce the characteristics and mechanisms of actual motor neurons.

[0063]

[0064] According to another aspect of the present invention, the present invention provides a composition for measuring botulinum neurotoxin potency, including the motor neurons. The present invention also provides a kit for measuring botulinum neurotoxin potency, including the motor neurons.

[0065] The composition for measuring botulinum neurotoxin potency of the present invention may further include a known substance for preserving and maintaining motor neurons, such as a culture medium. Additionally, the composition may further include a known reagent used in an assay for measuring botulinum neurotoxin potency.

[0066] The kit of the present invention may additionally include reagents known in the art for use in measuring botulinum neurotoxin titer. Furthermore, the kit of the present invention may further include a user guide describing optimal performance conditions. The guide is a printed document explaining how to use the kit, such as the proposed reaction conditions. The guide includes instructions in the form of a pamphlet or leaflet, a label attached to the kit, and descriptions on the surface of the package containing the kit. Furthermore, the guide includes information disclosed or provided through electronic media, such as the Internet.

[0067]

[0068] Duplicate contents are omitted in consideration of the complexity of this specification, and terms not otherwise defined in this specification have meanings commonly used in the technical field to which the present invention belongs.

[0069] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0070]

[0071] [Experimental Example]

[0072] Experimental Example 1. Flow Cytometry

[0073] After collecting cells in one tube, they were reacted with 10X TLPLE at 37°C for 30 minutes. After completion of the TLPLE reaction, they were centrifuged at 1,200 rpm for 5 minutes. After centrifugation, the supernatant was removed. The cell pellet was suspended with Cytofix / Cytoperm™ Solution and incubated at room temperature for 20 minutes. After completion of the reaction, they were centrifuged at 1,200 rpm for 5 minutes. After removal of the supernatant, the cell pellet was suspended with Perm / Wash Buffer. The cells were centrifuged at 1,200 rpm for 5 minutes to obtain a cell pellet, and 1 mL of Perm / Wash Buffer was added and resuspended. After cell counting, 1x10 5Cells were dispensed into each tube at a volume of 50 μL. Isotype Ab and primary Ab antibodies were added to the dispensed cell suspension at a ratio of 1:50, and the cells were incubated at 4°C for 2 hours. Washing was repeated 2–3 times with Perm / Wash Buffer. Secondary antibody and cell suspension were added to Perm / Wash Buffer at a ratio of 1:50, and the cells were incubated at room temperature for 1 hour. After the reaction, the cells were washed 2–3 times with Perm / Wash Buffer. The washed cells were analyzed using a SONY SH100 flow cytometer.

[0074]

[0075] Antibody information for flow cytometry analysis is as follows.

[0076] - Isotype Control Antibody: Alexa Fluor™ 488

[0077] - Primary antibodies: Anti-SNAP25 antibody, Anti-MAP2 antibody, Anti-beta III Tubulin antibody, Anti-Choline Acetyltransferase antibody

[0078] - Secondary antibody: Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488

[0079]

[0080] The expression rate of the fluorescent signal of the target antibody (Alexa Fluor™ 488 primary Ab) was calculated compared to the expression rate of the non-expressed phenotype analysis antibody (Alexa Fluor™ 488 Isotype Control).

[0081]

[0082] Experimental Example 2. Western Blot

[0083] After collecting cells in one tube, the cells were reacted with 10X TPLE for 30 minutes at 37°C. After completion of the TLPLE reaction, the cells were centrifuged at 1,200 rpm for 3 minutes. After centrifugation, the supernatant was removed. The cells were suspended in Cell Extraction Buffer PR containing Halt Protease and Phosphatase Inhibitor Cocktail 1X and reacted on ice for 20 minutes. Centrifuged at 4°C and 18,000xg for 20 minutes. The supernatant was transferred to a new tube and the precipitate was discarded. The protein concentration was measured using the BCA quantitative analysis method, and samples were prepared according to the quantified values. The cells were heated at 90–100°C for 5 minutes. The prepared proteins were loaded onto a 4–20% SDS page gel. The proteins were developed under the conditions of 70 volts, 0.02 A, 2 W, and 2 hours 30 minutes to 3 hours. After transfer, the membrane was transferred under the condition of 70 volts, 2 hours (wet transfer). After transfer, the membrane was stained with Ponceau S Staining Solution and cut to the size of the target band. After washing once or twice with TBS-T, 10 mL of EveryBlotBlocking Buffer was added and blocked for 10 minutes. The primary antibody was diluted 1:1000 in TBS-T and reacted at 4°C for 24 hours. The membrane was washed 2-3 times for 10 minutes with TBS-T. The secondary antibody was added at a ratio of 1:5000 in 10 mL of EveryBlotBlocking Buffer and reacted at room temperature for 1 hour. The membrane was washed 2-3 times for 10 minutes with TBS-T. After mixing the ECL substrate in a 1:1 ratio, 2-3 mL was dispensed onto the membrane. Images were taken on a ChemiDOC MP Imaging System.

[0084]

[0085] Experimental Example 3. ELISA

[0086] Reagent components were brought to room temperature (18-25℃) before use. Capture antibody and Detector antibody were diluted with Antibody Diluent 4BI (a component of the SNAP-25 SimplestepELISA Kit (ab256394)) to prepare an antibody cocktail. The standard was prepared by adding 368 μL of 1X Cell Extraction Buffer PTR to tube 1 and 150 μL of 1X Cell Extraction Buffer PTR to tubes 2 to 8, respectively. 50 μL of the standard and quantified analyte protein were dispensed into each 96-well well, and 50 μL of the antibody cocktail was added. The plate was sealed and incubated on a plate shaker at 400 rpm for 1 hour. Each well was washed three times with 350 μL of 1X Wash Buffer PT. 100 μL of TMB Substrate (a component of the SNAP-25 SimplestepELISA Kit (ab256394)) was added and incubated on a plate shaker at 400 rpm in the dark. The reaction was allowed to proceed for 10 minutes. 100 μL of stop solution was added to each well, mixed on a plate shaker for 1 minute, and measurement was performed at 450 nm.

[0087]

[0088] To measure protein concentration, BSA was quantified. A 2 mg / mL BSA standard was serially diluted to produce seven concentration levels ranging from 0 to 2,000 μg / mL. Ten μL of the prepared BSA standard and the diluted protein were dispensed into a 96-well plate. After dispensing 200 μL of the A+B mixed reagent (50 mL (A) + 1 mL (B)) in a ratio of 1 mL, the mixture was incubated at 37°C in a 5% CO₂ incubator for 30 minutes. After the reaction, the OD value for each sample was measured. The mean OD value was calculated through repeated measurements at least three times for each sample (BCA Protein Assay Kits: 562 nm). A standard curve for each concentration level was generated using the BSA standard, and the equation was derived through linear regression analysis. The standard curve equation is expressed in the form y = ax + b, where y represents OD and x represents concentration. The OD Mean value was substituted into the standard curve equation to calculate the protein concentration of each sample. Calculate 10 μg / mL (protein concentration to be injected) / x value. In order to match the final calculated values ​​to the same concentration, the remaining samples were diluted with 1X Cell Extraction Buffer PR based on the lowest concentration to equalize the concentration. After ELISA analysis with the SNAP-25 ELISA kit (450 nm), the x value and the concentration of SNAP-25 protein were calculated by substituting the value into the standard curve equation.

[0089]

[0090] Experimental Example 4. ICC (Immunocytochemistry)

[0091] After reacting with 4% PFA for 10-30 minutes at room temperature, the sections were washed 3-4 times with 0.03% Triton X-100. After permeabilization, the sections were reacted with 0.1% Triton X-100 for 10 minutes. After reacting with 5% Normal Goat Serum for 30 minutes at room temperature, the sections were washed 3-4 times with 0.03% Triton X-100. The primary antibodies (SNAP25, Tuj1, MAP2) were diluted 1:100 in 5% Normal Goat Serum, added to the samples, and reacted overnight at 4℃. The next day, the secondary antibodies diluted 1:500 in 5% Normal Goat Serum were added to the samples, reacted at room temperature for 1-2 hours, and washed 3-4 times with 0.03% Triton X-100. DAPI diluted 1:1000 in DPBS was added, reacted for 3–5 minutes, and washed 3–4 times with 0.03% Triton X-100. Fluorescence signals were analyzed using an Olympus confocal laser scanning microscope.

[0092]

[0093] The antibody information used in ICC is as follows.

[0094] ① SNAP25 antibody

[0095] - 1 st antibody:Anti-SNAP25 antibody [EPR3275], ab109105,1:200 dilution, 24 hour (4℃)

[0096] - 2 nd antibody: Goat anti rabbit IgG (Alexa Fluor™ 594, Invitrogen, A11012), 1:2000, 1 hour (RT)

[0097] ② Anti-beta III Tubulin antibody (TuJ1)

[0098] - 1st antibody:Anti-beta III Tubulin antibody [2G10], ab78078, 1:200 dilution, 24 hours (4℃)

[0099] - 2 nd antibody: Goat anti Mouse IgG2a (Alexa Fluor™ 488, Invitrogen, A11001), 1:2000, 1 hour (RT)

[0100] ③ Anti-MAP2 antibody

[0101] - 1 st antibody:[EPR22641-16], ab254264, 1:200 dilution, 24 hours (4℃)

[0102] - 2 nd antibody: Goat anti rabbit IgG (Alexa Fluor™ 594, Invitrogen, A11012), 1:2000, 1 hour (RT)

[0103]

[0104] [Example]

[0105] Example 1. Method for differentiating iPSC-derived motor neurons expressing SNAP-25 (3D differentiation)

[0106] 1.1 Method for differentiating iPSC-derived motor neurons expressing SNAP-25

[0107] A schematic diagram of SNAP-25-expressing motor neuron differentiation is shown in Figure 1. In addition, the specific composition of the culture medium used at each stage is shown in Table 1 below.

[0108] stageInductionScheduleFactorMediastage 0iPSCD-4~D-1-Stem MACSstage 1-1Neural stem spheroidD08~12 μM Y276321~3 μM CHIR990211~3 μM SB4315421~3 μM DMH1Advanced DMEM / F12 Neurobasal (1:1) media+ MEM Non-Essential Amino Acids+ AsA(Ascorbic acid)+ N2+ B27+ Glutamax+ p / sstage 1-2D1~3 μM CHIR990211~3 μM SB4315421~3 μM DMH1stage 2Motor neuron progenitor spheroidD5~D61~3 μM DMH11~3 μM SB4315420.1~1 μM Retinoic acid0.1~1 μM PurmorphamineNeurobasal media+ MEM Non-Essential Amino Acids+ N2+ B27+ Glutamax+ p / sstage 3Motor neuron spheroidD7~D400.1~1 μM Retinoic acid0.1~1 μM Purmorphamine

[0109]

[0110] [Stage 0 - iPSC]

[0111] Induced pluripotent stem cells (iPSCs) were treated with 0.5 mM EDTA to dissociate and suspend single cells. After cell counting, the cells were dispensed into aggrewell plates, a non-adhesive culture vessel. The dispensed cells were cultured in Stage 0 medium (D-4 to D-1).

[0112] The above AggreWell plate is an AggreWell™400 6-well plate (Stemcell, 34425), which can produce 3D spheroids of uniform size and ensure uniform cell differentiation. In addition, when using the AggreWell plate, the number and size of the produced spheroids (5900 microwells / well, 400 μm size) can be quantitatively determined, which has the advantage of allowing the drug to be treated under uniform conditions.

[0113]

[0114] [Stage 1 - Neural stem spheroid]

[0115] Cells seeded on Agriwell plates were cultured in Stage 1-1 medium at 37°C in a 5% CO₂ incubator for 1 day to induce embryoid body formation and differentiation into neurospheres (D0). On the second day of differentiation, all neurospheres were collected into a 50 mL tube using a micropipette (1,000 μL), allowed to settle, and the existing medium was removed. The collected neurospheres were then placed in a new petri dish and further cultured in Stage 1-2 medium (D1–D4).

[0116]

[0117] [Stage 2 - Motor neuron progenitor spheroid]

[0118] On the 6th day of differentiation, all neurospheres were collected using a micropipette (1,000 μL), placed in a new petri dish, and cultured with stage 2 culture medium to induce differentiation into motor neuron progenitor spheroids (D5-D6).

[0119]

[0120] [Stage 3 - Motor neuron spheroid]

[0121] On the 8th day of differentiation, all motor neuron progenitor spheroids were collected using a micropipette (1,000 μL), placed in a new petri dish, and cultured in stage 3 medium to induce differentiation into motor neuron spheroids (D7-D40). The medium was replaced with fresh medium every two days, and the process was performed continuously for more than 40 days, the differentiation period.

[0122] Finally, iPSC-derived motor neurons expressing SNAP-25 were obtained.

[0123] The finally obtained iPSC-derived motor neurons expressing SNAP-25 were named 'BS-hiPSC-001-MN, BS-hiPSC-002-MN'.

[0124]

[0125] 1.2 Optimization of differentiation of iPSC-derived motor neurons expressing SNAP-25 according to culture conditions.

[0126] The differentiation protocol established in Example 1.1 above was verified for differentiation into SNAP-25-expressing motor neurons. To this end, BS-hiPSC-001-MN cells were cultured under the following culture conditions.

[0127]

[0128] - Experimental group 1: Differentiation period 24 days, 3D culture

[0129] - Experimental group 2: Differentiation period 43 days, 3D culture

[0130] - Experimental group 3: Differentiation period 43 days, 2D culture

[0131]

[0132] The expression of SNAP-25, TuJ1, and MAP2 in iPSC-derived motor neurons expressing SNAP-25, cultured as described above, was measured using the method of Experimental Example 1. The results of the expression measurement are shown in Fig. 2.

[0133] As shown in Fig. 2, regardless of the culture conditions, it was confirmed that the neural cell markers TuJ1 and MAP2 were expressed by more than 99%, and SNAP-25 was expressed by more than 92%. The above results indicate that the differentiation method of Example 1.1 is capable of differentiating into motor neurons expressing SNAP-25 regardless of the culture conditions.

[0134] However, in the case of experimental group 3, two peaks were confirmed in the expression graph of SNAP-25, and as a result, it was determined that 3D culture would be advantageous in order to show a constant differentiation rate, as there is a possibility that two cell subpopulations exist when differentiation progresses under 2D conditions.

[0135]

[0136] 1.3 Optimization of differentiation of iPSC-derived motor neurons expressing SNAP-25 according to differentiation period

[0137] We investigated the conditions under which SNAP-25-expressing motor neurons, produced using the differentiation protocol of Example 1.1, could stably and highly express SNAP-25. To this end, BS-hiPSC-001-MN cells were cultured using the differentiation protocol of Example 1.1 to obtain iPSC-derived motor neurons expressing SNAP-25. The morphology of the cells was observed on days 12, 28, 32, and 40 of culture. In addition, the expression of SNAP-25 and TRA-1-60 in the cells according to each culture period was analyzed. The results of the morphological and expression analysis of the cells are shown in Figure 3.

[0138] As shown in Fig. 3, cells cultured with the differentiation protocol of Example 1.1 were confirmed to express SNAP-25 by more than 78% from day 12 of differentiation, and by more than 99% from day 40 of differentiation.

[0139] Additionally, to confirm the purity of differentiated cells, the undifferentiated marker TRA-1-60 was additionally analyzed, and it was confirmed that cells cultured using the differentiation protocol of Example 1.1 expressed TRA-1-60 at less than 0.1%. This means that there were no undifferentiated cells when cultured using the culture protocol of Example 1.1.

[0140] From the above results, it was determined that differentiation of motor neurons expressing SNAP-25 is suitable when cultured for approximately 40 days or more under 3D culture conditions, and a differentiation protocol was established under those conditions.

[0141]

[0142] Example 2. Characterization of iPSC-derived motor neurons expressing SNAP-25.

[0143] 2.1 Characterization of iPSC-derived motor neurons expressing SNAP-25

[0144] The expression of SNAP-25, an undifferentiated marker (TRA-1-60), neuronal markers (TuJ1, MAP2), and a motor neuron-specific marker (ChAT) in motor neurons produced by the differentiation method established in Example 1 was measured by the method of Experimental Example 1. The results of measuring the expression of related markers in the produced iPSC-derived motor neurons expressing SNAP-25 are shown in Fig. 4.

[0145] As shown in Fig. 4, all iPSC-derived motor neurons expressing SNAP-25 manufactured by the method of Example 1 were confirmed to have a consistent type of differentiated cell morphology that formed opaque (solid) spheroids without transparent (cystic) parts in a 3D-type differentiation state.

[0146] In addition, it was confirmed that the motor neurons produced by the method of Example 1 expressed the neuronal markers TuJ1 and MAP2 by more than 90%, and the motor neuron-specific marker ChAT by more than 90%. In addition, it was confirmed that the motor neurons produced by the method of Example 1 expressed the undifferentiated marker TRA-1-60 by less than 0.1%.

[0147] Through the above results, it was confirmed that all motor nerve stem cells manufactured by the method of Example 1 were successfully differentiated into motor nerve cells, and that their purity was also very high.

[0148]

[0149] 2.2 Verification of SNAP-25 expression in iPSC-derived motor neurons expressing SNAP-25

[0150] The expression of SNAP-25 in motor neurons produced using the differentiation method established in Example 1 was verified using the method of Experimental Example 1 above through flow cytometry. SiMa cells, a cell line currently used in actual cell-based botulinum toxin titer tests, served as the control group in this experiment. The results of the flow cytometry analysis are shown in Fig. 5.

[0151] As shown in Fig. 5, all motor neurons produced using the differentiation method established in Example 1 were confirmed to have 99% SNAP-25 expression. This was equivalent to the control SiMa cells.

[0152]

[0153] 2.3 Verification of SNAP-25 protein expression in iPSC-derived motor neurons expressing SNAP-25

[0154] The expression of SNAP-25 protein in motor neurons produced using the differentiation method established in Example 1 was verified using the method of Experimental Example 2. The control group in this experiment used SiMa cells, a cell line currently used in actual cell-based botulinum toxin titer testing. The Western blot results are shown in Figure 6.

[0155] As shown in Fig. 6, it was confirmed that all motor neurons produced by the differentiation method established in Example 1 expressed SNAP-25 at the protein level.

[0156]

[0157] 2.4 Quantitative analysis of SNAP-25 expression in iPSC-derived motor neurons expressing SNAP-25

[0158] SNAP-25 expression in hiPSC-derived motor neurons was quantified using an Enzyme Linked ImmunoSorbent Assay (ELISA) using the method described in Experimental Example 3. The control group in this experiment used SiMa cells, a cell line currently used in cell-based botulinum toxin titer testing. The ELISA results are shown in Figure 7.

[0159] As shown in Fig. 7, it was confirmed that all motor neurons produced by the differentiation method established in Example 1 had higher SNAP-25 expression than SiMa cells.

[0160]

[0161] Example 3. Botulinum toxin efficacy analysis

[0162] 3.1 Coating and cell seeding for attachment of iPSC-derived motor neurons expressing SNAP-25

[0163] While botulinum toxin can be applied directly to cells in three-dimensional configurations, neurite outgrowth through three-dimensional cell attachment is necessary to more accurately assess its efficacy in inhibiting neurotransmission. First, we explored the optimal coating conditions for motor neuron attachment and neurite outgrowth.

[0164] Culture vessel coating was compared with the method of Experimental Example 4 above, using (i) a mixed coating method in which a mixture containing poly-L-ornithine and laminin is coated, which is widely used; and (ii) a sequential coating method, which is a proprietary coating method in which ECM is layered by performing a second laminin coating after a first poly-L-ornithine coating.

[0165] The specific coating method is as follows.

[0166]

[0167] (i) Mixed coating method: A simple mixture of 50 μg / mL of poly-L-ornithine and 25 μg / mL of laminin was prepared. The prepared mixture was added to a culture vessel, and the mixture was reacted and coated at 37°C for 1 hour. The coated culture vessel was washed 2-3 times with DPBS, and then 3D cells were seeded.

[0168] (ii) Sequential coating method: The first coating was performed by reacting 50 μg / mL of poly-L-ornithine at 37°C for 1 hour. The first-coated culture vessel was washed with DPBS, and 25 μg / mL of laminin was added and incubated at 37°C for 1 hour for the second coating. The coated culture vessel was washed 2-3 times with DPBS, and then 3D cells were seeded.

[0169]

[0170] The results comparing the mixed coating method and the sequential coating method are shown in Fig. 8.

[0171] As shown in Fig. 8, it was confirmed that 3D cells adhered well in culture vessels coated with the mixed coating method and the sequential coating method. In addition, when 3D cells were cultured in culture vessels coated with the above two methods, it was confirmed that neuronal cell-specific markers such as MAP2 and TuJ1 were expressed in neurites and that neural axons extended. In particular, it was confirmed that neural axons extended better in 3D cells cultured in culture vessels coated with the sequential coating method than in the mixed coating method.

[0172]

[0173] 3.2 Measurement of SNAP-25 expression in botulinum toxin-treated motor neurons

[0174] iPSC-derived motor neurons expressing SNAP-25, manufactured by the method of Example 1, were treated with 1 unit of wonderTox, a commercially available botulinum toxin, and cultured for 48 hours. SNAP-25 expression was then analyzed using Western blotting. An antibody capable of detecting the 25 kD, uncleaved form of SNAP-25, a presynaptic membrane protein, was used for the Western blotting. The Western blotting results are shown in Figure 9.

[0175] As shown in Fig. 9, it was confirmed that all motor neurons manufactured by the method of Example 1 showed a decrease in the expression of SNAP-25 upon treatment with botulinum toxin. In particular, it was confirmed that the expression level of SNAP-25 was reduced to less than 50% upon treatment with botulinum toxin.

[0176] The above results show that the cell-based botulinum toxin potency test of iPSC-derived motor neurons expressing SNAP-25 showed a potency similar to the mouse-measured LD50 assay of 1 Unit of botulinum toxin, confirming that the iPSC-derived motor neurons expressing SNAP-25 of the present invention are cells applicable to the cell-based potency test of botulinum toxin. In particular, unlike SiMa cells, motor neurons differentiated from hiPSCs can reproduce the characteristics and mechanisms of actual motor neurons, and thus can represent the responsiveness of actual neurons to botulinum toxin administration, suggesting that they are more suitable cells for the study of botulinum toxin potency.

[0177]

[0178] In summary, the present inventors have developed a method for differentiating iPSC-derived motor neurons expressing SNAP-25. Using the method of the present invention, it was confirmed that motor neurons stably and highly express SNAP-25, neuronal markers, and motor neuron markers, and that undifferentiated markers are expressed at less than 0.1%. Furthermore, when the motor neurons produced by the method of the present invention are subjected to a botulinum toxin titer test, it was confirmed that they exhibited titers similar to those observed in the mouse LD50 assay. Furthermore, unlike conventional botulinum toxin titer testing methods, the present invention utilizes the neurons for analysis, which has the advantage of representing the responses of actual neurons. Therefore, the method of the present invention and the motor neurons produced by the method can be utilized in various fields of botulinum toxin titer research.

[0179]

[0180] While specific aspects of the present invention have been described in detail, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. (a) A step of culturing stem cells in a culture medium containing at least one selected from the group consisting of Y27632, CHIR99021, SB431542, and DMH1; (b) a step of culturing the cells of step (a) with a culture medium containing at least one selected from the group consisting of DMH1, SB431542, retinoic acid (RA), and purmorphamine (Pur); and (c) A method for producing motor neurons, comprising a step of culturing the cells of step (b) in a medium containing retinoic acid or permorphamine.

2. A method for producing motor neurons in the first paragraph, wherein the stem cells are pluripotent stem cells, induced pluripotent stem cells, or embryonic stem cells.

3. In the first paragraph, step (a) is culturing stem cells in a culture medium containing at least one selected from the group consisting of Y27632, CHIR99021, SB431542, and DMH1, A method for producing motor neurons, comprising: culturing cultured stem cells in a culture medium containing at least one selected from the group consisting of CHIR99021, SB431542, and DMH1.

4. A method for producing motor neurons in the first paragraph, wherein the culture in steps (a) to (c) is 3D culture.

5. A method for producing motor neurons in accordance with claim 4, wherein the 3D culture is performed in a non-adhesive culture vessel.

6. A method for producing motor neurons in the first paragraph, wherein step (a) is culturing for 1 to 7 days.

7. A method for producing motor neurons, wherein step (b) is performed by culturing for 1 to 3 days in the first paragraph.

8. A method for producing motor neurons in the first paragraph, wherein step (c) is culturing for 20 to 60 days.

9. Motor neurons manufactured by the method of paragraph 1. 10.(a) A step of culturing the motor neurons of Article 9 after treating them with botulinum neurotoxin; and (b) A method for measuring botulinum neurotoxin potency, comprising the step of measuring the expression of SNAP-25 of the cultured cells of (a).

11. A method for measuring botulinum neurotoxin titer in claim 10, wherein the culturing in step (a) is performed in (i) a culture vessel coated with a mixture containing poly-L-ornithine and laminin; or (ii) a culture vessel sequentially coated with poly-L-ornithine and laminin.

12. A composition for measuring the potency of botulinum neurotoxin containing motor neurons of Article 9.

13. A kit for measuring the potency of botulinum neurotoxin containing motor neurons of Article 9.

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