Method for producing neural progenitor cells derived from induced pluripotent stem cells

Abstract

The present invention relates to a method for producing neural progenitor cells. The method for producing neural progenitor cells according to an aspect ensures reproducibility by setting criteria for each step, uniformly performs differentiation across production batches, and enables mass production of high-purity neural progenitor cells by removing unspecified types of cells other than neural progenitor cells using culture methods and culture conditions.

Description

Method for producing neural progenitor cells derived from induced pluripotent stem cells

[0001] The present invention relates to a method for producing neural progenitor cells.

[0002] This invention is a technology developed with the support of the Korean Fund for Regenerative Medicine (KFRM) supported by the Ministry of Science and ICT and the Ministry of Health and Welfare (RS-2022-00070674, 22C0625L1).

[0003] Induced pluripotent stem cells (IPS) are a cell line developed by Shinya Yamanaka in 2006, established by overexpressing specific genes (Yamanaka factors) in adult cells (Takahashi, Kazutoshi, and Shinya Yamanaka. "Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors." Cell 126.4 (2006): 663-676). Recently, human induced pluripotent stem cells have been actively studied as a starting material for cell therapy for various indications (Yoshida, Shinsuke, et al. "A clinical-grade HLA haplobank of human induced pluripotent stem cells matching approximately 40% of the Japanese population." Med 4.1 (2023): 51-66.).

[0004] The technology for establishing neural progenitor cells using human induced pluripotent stem cells is based on the technology established by Lorenz Studer in 2009, and mainly induces neural differentiation by treating two inhibitors (Noggin, SB431542) related to SMAD signaling (Chambers, Stuart M., et al. "Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling." Nature Biotechnology 27.3 (2009): 275-280). In 2012, Lorenz Studer successfully established neural progenitor cells using LDN193189 as an alternative to Noggin (Chambers, Stuart M., et al. "Combined small-molecule inhibition accelerates developmental timing and converts human pluripotent stem cells into nociceptors." Nature Biotechnology 30.7 (2012): 715-720.). However, the method described above has the disadvantage that the criteria for each differentiation stage are not clear, and the differentiation results vary from experiment to experiment or experimenter to experimenter due to various variables that may arise during the experimental process. Due to these disadvantages, the purity of the final product, neural progenitor cells, cannot be guaranteed when used as cell therapy. As a method to solve this problem, a method to selectively isolate neural progenitor cells using the MACS method, a separation method using surface antigens, has been developed (Kikuchi, Tetsuhiro, et al. "Human iPS cell-derived dopaminergic neurons function in a primate Parkinson's disease model." Nature 548.7669 (2017): 592-596., Lee, Dongjin R., et al. "PSA-NCAM-negative neural crest cells emerging during neural induction of pluripotent stem cells cause mesodermal tumors and unwanted grafts." Stem Cell Reports 4.5 (2015): 821-834.). In fact, the MACS method has improved problems caused by various variables of existing technologies (especially, improvement of purity), but it has the problem of excessive time and cost because it utilizes antibodies. Currently, other than the method using the MACS method, there is no known technology for isolating and long-term maintaining and culturing high-purity neural progenitor cells.

[0005] When using the above conventional techniques, there is a high possibility that other types of cells other than neural progenitor cells will also be generated as a result of differentiation during the differentiation process. Furthermore, the criteria for each step in the process of differentiating human induced pluripotent stem cells into neural progenitor cells are ambiguous, which increases the possibility of batch effects and makes it difficult to ensure reproducibility. The high-purity neural progenitor cell isolation method using the MACS method has improved the problems of the conventional technique, but its introduction requires a lot of time and money, and there are clear limitations in applying it to mass production. In other words, due to the above limitations, it is difficult to mass-produce high-purity neural progenitor cells from human induced pluripotent stem cells using the existing method, and it is also difficult to ensure reproducibility.

[0006] Accordingly, the present researchers have made efforts to overcome the above technical limitations, and have established a method for producing high-purity neural progenitor cells with mass production and reproducibility by discovering the optimal culture dish, culture method for each stage, and culture conditions, thereby completing the present invention.

[0007] One aspect provides a method for producing neural precursor cells, comprising the steps of: (a) culturing stem cells in three dimensions, forming embryoid bodies (EBs) from the stem cells, and differentiating the embryoid bodies into neural precursor cells; (b) selecting spheres containing the neural precursor cells in three dimensions; and (c) culturing the selected spheres in two dimensions to proliferate them.

[0008] One aspect provides a method for producing neural precursor cells, comprising the steps of: (a) culturing stem cells in three dimensions, forming embryoid bodies (EBs) from the stem cells, and differentiating the embryoid bodies into neural precursor cells; (b) selecting spheres containing the neural precursor cells in three dimensions; and (c) culturing the selected spheres in two dimensions to proliferate them.

[0009] The term “stem cell” as used herein refers to a cell that can differentiate into various cells that constitute biological tissues, and is a general term for undifferentiated cells in the pre-differentiation stage that can be obtained from each tissue of embryos, fetuses, and adults. It refers to a cell that has pluripotency that can differentiate into cells derived from endoderm, mesoderm, and ectoderm, or limited differentiation ability (multipotency) that can differentiate into cells closely related to tissue or function. Stem cells that can be used for the purpose of the present invention include without limitation, but are preferably pluripotent stem cells, and more preferably embryonic stem cells or induced pluripotent stem cells (induced pluripotent stem cells or induced pluripotent stem cells).

[0010] The above stem cells may be of animal origin, and the animals include not only humans and primates, but also livestock such as cows, pigs, sheep, horses, dogs, mice, rats, and cats. The above stem cells preferably refer to stem cells derived from humans.

[0011] In this specification, the term "induced pluripotent stem cells (iPSC)" refers to cells that have been induced to have pluripotent differentiation capacity through an artificial dedifferentiation process targeting differentiated cells. They have the ability to differentiate into all cells in the body except for the placenta, and in particular, have characteristics similar to embryonic stem cells (ESCs) derived from blastocysts. Embryonic stem cells (ESCs) are cells that have pluripotency that can differentiate into all cells of an animal, and are cultured in vitro by extracting the inner cell mass from an embryo in the blastocyst stage just before the fertilized egg is implanted in the mother's uterus. Specifically, induced pluripotent stem cells exhibit cell shape, gene, protein expression pattern, etc. similar to embryonic stem cells, have pluripotency in vitro and in vivo, form teratomas, form chimera mice when injected into mouse blastocysts, and are capable of germline transmission of genetic traits to the next generation. The artificial dedifferentiation process is performed by introducing virally mediated or non-viral vectors using retrovirus or lentivirus, non-virally mediated dedifferentiation factors using proteins and cell extracts, etc., or includes a dedifferentiation process using stem cell extracts, compounds, etc.

[0012] The somatic cells used to produce the above iPSCs may be derived from various animals, and preferably, they may be human-derived human-induced pluripotent stem cells (hiPSCs) produced using a dedifferentiation technique from human somatic cells. Furthermore, the above ESCs or iPSCs can be easily constructed by those skilled in the art using known methods.

[0013] The term "embryoid body" as used herein refers to a spherical stem cell-derived cell mass produced in a suspension culture state, which has the potential to differentiate into endoderm, mesoderm, and ectoderm, and is thus used as a precursor in most differentiation induction processes to secure tissue-specific differentiated cells. The present invention can dramatically improve the process of obtaining stem cell-derived differentiated cells by effectively enhancing the proliferation of the embryoid body itself, and can contribute to the development of high-value-added stem cell utilization technology.

[0014] In this specification, the term "subculture" refers to a method of continuously culturing cells in a healthy state for a long period of time by periodically transferring a portion of cells to a new culture vessel and then changing the culture medium to continue culturing the cells. As the number of cells increases in a culture vessel with a limited space, over a certain period of time, the growth nutrients are consumed or contaminants accumulate, causing the cells to die naturally. Therefore, it is used as a method to increase the number of healthy cells. Typically, replacing the medium (culture vessel) once or dividing a cell group and culturing it is called 1 passage. Currently, the traditional method of culturing embryoid bodies is mainly to cultivate until the utilization stage in the form of continuous culture without subculture.

[0015] As used herein, the term "precursor cells" refers to progenitor cells that do not express differentiation traits but possess the differentiation fate, when the cells corresponding to their progeny are found to express specific differentiation traits. For example, in the present invention, for neural cells (neurons), neuroblasts (neural stem cells) may correspond to neural precursor cells.

[0016] As used herein, the term “neural precursor cells (NPCs)” refers to cells at a stage before they fully acquire the form and function of neural cells. Neural precursor cells are precursor cells of the central nervous system (CNS) and are capable of differentiating into glial or neural cell types that constitute the CNS. Neural precursor cells may be cells that express one or more genes selected from the group consisting of Sox2, Nestin, and Musashi. These genes are genes uniquely expressed in neural precursor cells, and their expression indicates that the formed cells are neural precursor cells.

[0017]

[0018] Step (a) is described in detail. Step (a) refers to a step of culturing stem cells in three dimensions, forming embryoid bodies (EBs) from the stem cells, and differentiating them into neural precursor cells. In other words, the neural precursor cells obtained in step (a) may differentiate into neural precursor cells through embryoid bodies.

[0019] In one embodiment, step (a) may comprise differentiating the stem cells in a medium that does not contain bovine or other non-human animal-derived components. That is, step (a) may comprise differentiating the stem cells in a xeno-free medium.

[0020] As used herein, the term “xeno-free medium” refers to a xenobiotic-free medium that does not contain animal-derived components, and does not contain components derived from animals other than humans. For example, the medium may be commercially available CTS-SR (CTS Knock Out Serum Replacement). The medium according to the present invention excludes all animal-derived components, including serum, albumin, and other proteins as well as serum derived from xenobiotic animals, thereby preventing the introduction of potential mutations or contamination due to animal-derived components. By minimizing the risks associated with xenobiotic contamination, it has the advantage of enhancing safety in clinical applications or production fields, such as improving consistency in performance and quality, maintaining stem cell function, and enhancing the accuracy of experiments.

[0021] In one specific example, step (a) may include differentiating stem cells in a medium comprising at least one selected from the group consisting of DMEM / F12, CTS-SR (CTS Knock out serum replacement), NEAA (non-essential amino acid), 2-mercaptoethanol, LDN193189, and SB431542.

[0022] In one specific example, the concentration of the CTS-SR in the medium of step (a) may be 0.1 to 50% (v / v), 0.1 to 20% (v / v), 0.1 to 10% (v / v), 0.1 to 5% (v / v), 0.5 to 20% (v / v), 0.5 to 10% (v / v), 1 to 20% (v / v), 1 to 10% (v / v), 3 to 8% (v / v), or 4 to 6% (v / v) based on the total medium volume.

[0023] In this specification, “LDN193189” may be a compound having a structure represented by the following chemical formula 1.

[0024] [Chemical Formula 1]

[0025]

[0026] In this specification, “SB431542” may be a compound having a structure represented by the following chemical formula 2.

[0027] [Chemical Formula 2]

[0028]

[0029] In one specific example, step (a) may be any culture method or culture medium capable of inducing intercellular aggregation of stem cells. Any culture method or culture medium capable of producing stem cell aggregates, i.e., single spherical neural progenitor cells, may be used, and is not limited thereto. For example, Aggrewell EB formation medium, Gibco Essential 6 Medium, CTS Essential 6 Medium, or TeSR-E6 may be used.

[0030] In one specific example, the culture in step (a) may be cultured for 5 to 30 days, 5 to 15 days, for example, 10 days.

[0031] In one specific example, the method for producing neural progenitor cells of the present invention may additionally include a step of proliferating stem cells by subculturing them prior to step (a).

[0032] The above subculture may include a step of culturing stem cells in a stem cell proliferation medium containing fibroblast growth factor and Y-27632.

[0033] The above subculture can be cultured for 1 to 10 days, for example, 5 days.

[0034] The above subculture may be performed repeatedly before step (a). Specifically, it may be repeated 1 to 100 times, 1 to 10 times, or 3 times.

[0035] In one specific example, the culture medium comprises 10 2 10 inland 5 10 cells, specifically 2 10 inland 5 Dog, 10 2 10 inland 4 Dog, 10 3 10 inland 5 Dog, 10 3 10 inland 4 Dog, 3×10 3 10 inland 4 Dog, 3×10 3 8×10 3 Dog, 3×10 3 6×10 3 Dog, 4×10 3 6×10 3 or 5×10 3 6×10 3 It may be made up of dog cells.

[0036] In one specific example, step (a) may include seeding stem cells into a well.

[0037] The concentration of the above seeded stem cells is 10 5 Cells / well up to 10 7 Cells / well, 3×10 5 Cells / well or 3×10 6 Cells / well, 3×10 5 Cells / well or 1.8×10 6 Cells / well, 6×10 5 Cells / well or 1.8×10 6 Cells / well, 9×10 5 Cells / well or 1.8×10 6 Cells / well, 1.2×10 6 Cells / well or 1.8×10 6 cells / well or 1.5×10 6 Cells / well or 1.8×10 6 It can be cell / well.

[0038]

[0039] (b) Step is described in detail. Step (b) refers to a step of three-dimensionally selecting a sphere containing the neural progenitor cells.

[0040] As used herein, the term “sphere” or “spheroid” means a cell aggregate forming a round spherical shape, which is also commonly expressed as a spheroid shape. As used herein, the spheroid cell aggregate may be a neurosphere.

[0041] In one specific embodiment, step (b) can be performed by mechanical separation or enzymatic separation.

[0042] The term "mechanical separation" as used herein refers to physically or mechanically separating a cell mass, and any method known in the art may be used without limitation. In one specific example, the mechanical separation may be separation using a blade, a tissue chopper, a needle, a pipetting device, and / or a scraper.

[0043] As used herein, the term "enzymatic separation" refers to the separation of cell masses through enzyme treatment, and any method known in the art may be used without limitation. In one specific example, the enzymatic separation may be separation through treatment with collagenase, including collagenase I, II, III, and IV, accutase, dispase, and / or trypsin.

[0044] As used herein, the term "Accutase" refers to a cell detachment solution, a proteolytic and collagenolytic enzyme solution. It is typically used to detach adherent cells from culture dishes, but it can also inhibit cell aggregation within cell masses, converting them into single cells. While reaction conditions and times may vary depending on the cell line, Accutase can be used without limitation on a wide range of cell lines. As a relatively mild cell detachment solution, Accutase maintains high cell viability even after use. Furthermore, Accutase is a cell detachment enzyme that does not require inhibitors.

[0045] In one specific example, step (b) may include a step of recovering the cells of step (a), dispensing the recovered cells into a medium containing Y-27632, and then three-dimensionally selecting spheroids containing neural progenitor cells.

[0046] The above medium may additionally contain at least one selected from the group consisting of DMEM / F12, NEAA, glutamine, sodium pyruvate, glucose, B27 supplement without vitamin A, 2-mercaptoethanol, and fibroblast growth factor (FGF).

[0047] The above medium may not contain an N2 supplement. Specifically, the medium may contain a B27 supplement without an N2 supplement. The medium may inhibit or reduce cell-to-cell adhesion during neurosphere formation compared to a medium containing an N2B27 supplement. This allows neurosphere formation and recovery without the need for an additional cell detachment process.

[0048] In one specific example, the concentration of the vitamin A-excluding B27 supplement in the medium of step (b) may be 0.01 to 10% (v / v), 0.01 to 5% (v / v), 0.01 to 2% (v / v), 0.1 to 10% (v / v), 0.1 to 5% (v / v), 0.1 to 2% (v / v), 1 to 10% (v / v), 1 to 5% (v / v), 1 to 3% (v / v), or 1 to 2% (v / v) based on the total medium volume.

[0049] In one specific example, step (b) may be performed repeatedly before step (c). Specifically, it may be repeated 1 to 20 times, 1 to 10 times, or 3 times.

[0050]

[0051] (c) Step is described in detail. Step (c) refers to the step of two-dimensionally culturing and proliferating the selected spheroids.

[0052] In one specific embodiment, step (c) may include recovering the cells selected in step (b) and proliferating the recovered cells in a medium containing at least one selected from the group consisting of DMEM / F12, Neurobasal medium, NEAA, N2 supplement, vitamin A-excluded B27 supplement, 2-mercaptoethanol, and fibroblast growth factor.

[0053] In one specific example, step (c) may be performed repeatedly. Specifically, it may be repeated at least once, three times, five times, specifically from 1 to 100 times, or from 1 to 10 times.

[0054] In one specific example, the concentration of the vitamin A-excluded B27 supplement in the medium of step (c) may be 0.01 to 5% (v / v), 0.01 to 2% (v / v), 0.01 to 1% (v / v), 0.1 to 5% (v / v), 0.1 to 2% (v / v), 0.1 to 1% (v / v), 0.5 to 5% (v / v), 0.5 to 3% (v / v), 0.5 to 2% (v / v), 0.5 to 1% (v / v), 1 to 5% (v / v), or 1 to 2% (v / v) based on the total medium volume.

[0055] In one specific example, the concentration of the N2 supplement in the medium of step (c) may be 0.001 to 5% (v / v), 0.001 to 3% (v / v), 0.001 to 1% (v / v), 0.001 to 0.5% (v / v), 0.1 to 5% (v / v), 0.1 to 3% (v / v), 0.1 to 1.5% (v / v), 0.1 to 1% (v / v), 0.1 to 0.5% (v / v), 0.3 to 1% (v / v), or 0.5 to 1% (v / v) based on the volume of the entire medium.

[0056] In one specific example, the concentration ratio of the vitamin A-excluded B27 supplement and the N2 supplement in the medium of step (c) may be about 10:1, about 5:1, about 4:1, about 3:1, about 2:1, about 3:2, or about 1:1.

[0057]

[0058] A method for producing neural progenitor cells according to one specific embodiment of the present invention can solve the problems of cell safety and mass production. The production method of the present invention induces differentiation of stem cells into neural progenitor cells under the specific culture method and culture environment described above, thereby uniformly progressing differentiation of stem cells to form mature embryoid bodies of uniform shape and size, thereby minimizing quality differences, and improving differentiation efficiency and consistency by eliminating unspecified types of cells other than neural progenitor cells. This manufacturing method stably maintains characteristics even after repeated long-term subculture, enables mass production of highly pure neural progenitor cells, and can contribute to the commercialization of neural progenitor cells differentiated from stem cells as cell therapy products.

[0059] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0060] The production method for neural progenitor cells, based on the specific conditions, can reduce the possibility of batch effects and ensure reproducibility by establishing standards for each stage (differentiation, selection, and proliferation). Specifically, it ensures that differentiation proceeds uniformly across manufacturing units, and by utilizing culture methods and conditions to eliminate non-specific cell types other than neural progenitor cells, it is possible to mass-produce high-purity neural progenitor cells.

[0061] Figure 1 is a schematic diagram of the entire process of creating neural progenitor cells from human induced pluripotent stem cells.

[0062] Figure 2 is a diagram showing the daily shape of an embryoid body in the differentiation step of a manufacturing method according to one specific example, confirmed using a phase contrast microscope.

[0063] Figure 3 is a diagram showing the shape of a neurosphere confirmed using a phase contrast microscope in a three-dimensional selection step of a manufacturing method according to one specific example.

[0064] Figure 4 is a diagram showing the presence or absence of attached cells according to the combination of supplements during neural sphere formation.

[0065] Figure 5 is a diagram showing the presence or absence of other types of cells other than neural progenitor cells using a phase contrast microscope depending on the presence or absence of a three-dimensional selection step in a manufacturing method according to one specific example.

[0066] Figure 6 is a diagram showing the results of confirming the purity of neural progenitor cells by flow cytometry analysis according to the number of passages when the three-dimensional cell selection process of the manufacturing method according to one specific example was not performed.

[0067] Figure 7 is a diagram showing the results of confirming the purity of neural progenitor cells by flow cytometry analysis according to the number of subcultures when a three-dimensional cell selection process of a manufacturing method according to one specific example was performed.

[0068] Figure 8 is a diagram showing the results of flow cytometry analysis to confirm the purity of high-purity neural progenitor cells when they are proliferated in two dimensions after a three-dimensional cell selection process of a manufacturing method according to one specific example, according to the number of subcultures.

[0069] Figure 9 is a diagram showing the shape and characteristics of neural progenitor cells established from a manufacturing method according to one specific example.

[0070] The following examples are provided for more detailed description. However, these examples are provided solely to illustrate one or more specific examples, and the scope of the present invention is not limited to these examples.

[0071]

[0072] Example. Method for producing neural progenitor cells

[0073] To manufacture high-purity neural progenitor cells with guaranteed mass production and reproducibility, neural progenitor cells were manufactured as follows. A schematic diagram of a method for producing neural progenitor cells according to one specific example is shown in Fig. 1, and the shape and characteristics of the established neural progenitor cells are shown in Fig. 8.

[0074]

[0075] 0. 시약, 자재, 기기 및 기구

[0076] 1) 시약

[0077] ● StemFit Basic03 (Cat. No. AJBASIC03, Ajinomoto)

[0078] ● Basic FGF (fibroblast growth factor) (Cat. No. SP-FGF2-G-001MG, Ajinomoto)

[0079] ● Y-27632 (Cat. No. TB-1254-GMP, Tocris)

[0080] ● CTS™ TrypLE™ Select Enzyme (Cat. No. A1285901, Thermo Fisher Scientific)

[0081] ● iMatrix-511 (Cat. No. 892012, Nippi)

[0082] ● DMEM / F12 (Cat. No. 11320033, Thermo Fisher Scientific)

[0083] ● Neurobasal (Cat. No. 21103049, Thermo Fisher Scientific)

[0084] ● Non-essential amino acids (NEAA; Cat. No. 11140050, Thermo Fisher Scientific)

[0085] ● 2-Mercaptoethanol (Cat. No. 21985-023, Thermo Fisher Scientific)

[0086] ● CTS-Knockout Serum Replacement (CTS-SR; Cat. No. 12618012, Thermo Fisher Scientific)

[0087] ● LDN193189 (Cat. No. TB6053-GMP, Tocris)

[0088] ● SB431542 (Cat. No. TB1614-GMP, Tocris)

[0089] ● L-Glutamine (Cat. No. 25030-081, Thermo Fisher Scientific)

[0090] ● Glucose Solution (Cat. No. A2494001, Thermo Fisher Scientific)

[0091] ● Sodium Pyruvate (Cat. No. 11360-070, Thermo Fisher Scientific)

[0092] ● B-27 w / o Vit A (Cat. No. 12587010, Thermo Fisher Scientific)

[0093] ● N-2 (Cat. No. 17502048, Thermo Fisher Scientific)

[0094] ● Accutase (Cat. No. AT-104, STEMCELL)

[0095] ● Anti-Adherence Rinsing Solution (Cat. No. 07010, STEMCELL Technologies)

[0096] ● CTS DPBS (Cat. No. A1285601, Thermo Fisher Scientific)

[0097] ● Trypan blue (Cat. No. T10282, Thermo Fisher Scientific)

[0098]

[0099] 2) 자재

[0100] 15 mL tube (Cat. No.430766, Corning)

[0101] 50 mL tube (Cat. No. 430291, Corning)

[0102] Storage bottle (Cat. No. 550500, SPL)

[0103] 1.5 mL tube (Cat. No. MCT-150-C-S, Axygen)

[0104] 50 mL serological pipette (Cat. No. 4490, Corning)

[0105] 25 mL serological pipette (Cat. No. 4489, Corning)

[0106] 10 mL serological pipette (Cat. No. 4488, Corning)

[0107] 5 mL serological pipette (Cat. No. 4487, Corning)

[0108] Countess cell counting chamber slide (Cat. No. C10228, Thermo Fisher Scientific)

[0109] Parafilm (Cat. No. PM-996, Bemis)

[0110] EasYFlaskTM Cell Culture Flask (25 cm2) (T25 flask, Cat. No. 156367, Thermo Fisher Scientific)

[0111] EasYFlaskTM Cell Culture Flask (75 cm2) (T75 flask, Cat. No. 156499, Thermo Fisher Scientific)

[0112] AggreWell™ 800 (24 well) (Cat. No. 34811, STEMCELL Technologies)

[0113]

[0114] 3) Device

[0115] ● Cell counter Countess 3 (Cat. No., Thermo Fisher Scientific)

[0116] ● Centrifuge (Cat. No., LABOGENE)

[0117] ● Incubator (Cat. No., Thermo Fisher Scientific)

[0118]

[0119] 4) Device

[0120] ● Micropipette

[0121] -1000p pipette (Cat. No. 3120000062, Eppendorf)

[0122] -200p pipette (Cat. No. 3120000054, Eppendorf)

[0123] -20p pipette (Cat. No. 3120000038, Eppendorf)

[0124] ● Pipette aid (Cat. No. 4-000-101, Drummond)

[0125]

[0126] 1. Preparation of culture medium

[0127] 1.1 Preparation of iPSC, iPSC-Y, and iPSC-C media

[0128] StemFit Basic03 was dispensed entirely into solution B into solution A bottle according to the protocol provided by the manufacturer and stored at 4℃.

[0129] iPSC medium was prepared by dispensing 40 mL of StemFit Basic03 and 10.8 uL of Basic FGF (fibroblast growth factor) 0.3 mg / mL into a 50 mL tube.

[0130] iPSC-Y medium was prepared by adding 40 uL of Y-27632 10 mM to the above iPSC medium.

[0131] iPSC-C medium was prepared by adding 200 uL of iMatrix-511 0.5ug / uL to the above iPSC-Y medium.

[0132]

[0133] 1.2 Preparation of NIM badge

[0134] Item Reagent / Material Volume 1 DMEM / F12 37.6 mL 2 CTS-SR2 mL 3 NEAA (100X) 400 uL 4 2-Mercaptoethanol 40 uL

[0135]

[0136] The reagents or materials in Table 1 were dispensed into 50 mL tubes according to the prescriptions presented in Table 1 and stored at 4°C until use. Before use, 40 μL of LDN193189 (100 μM) and 40 μL of SB431542 (10 mM) were added to NIM (Neural Induction Medium). The final concentrations were LDN193189 (100 nM) and SB431542 (10 μM).

[0137]

[0138] 1.3 Preparation of NEM and NEM-Y media

[0139] Item Reagent / Material Capacity 1DMEM / F12370.9 mL2NEAA (100X)4 mL3L-glutamine (200mM)4 mL4Sodium pyruvate (100mM)4 mL5D-glucose (200g / L)1080 uL6B27 w / o Vit A (50X)8 mL72-Mercaptoethanol400 uL

[0140]

[0141] The reagents or materials in Table 2 were dispensed into 50 mL tubes (40 mL each) according to the prescriptions presented in Table 2 and stored at 4°C until use. NEM (Neural Expansion Medium) medium was used by adding 5.4 uL of Basic FGF (0.3 mg / mL) before use, and NEM-Y medium was used by adding 40 uL of Y-27632 (10 mM) to NEM medium.

[0142]

[0143] 1.4 Preparation of N2B27 and N2B27-C media

[0144] Item Reagent / Material Volume 1 DMEM / F 12 196 mL 2 Neurobasal 194 mL 3 NEAA 4 mL 4 N-22 mL 5 B27 w / o VitA 4 mL 6 2-Mercaptoethanol 400 uL

[0145]

[0146] The reagents or materials in Table 3 were dispensed into 50 mL tubes (40 mL each) according to the prescriptions presented in Table 3 and stored at 4°C until use. N2B27 (N-2(DMEM / F12) / B-27(Neurobasal)) medium was used by adding 5.4 uL of Basic FGF (0.3 mg / mL) before use, and N2B27-C medium was used by adding 100 uL of iMatrix-511 to N2B27 medium.

[0147]

[0148] 2. Preparation of microwells

[0149] After dispensing the anti-adherence rinse solution into the Aggrewell to be used in the experiment, centrifugation was performed (1300 x g, 5 min). Afterwards, it was checked under a microscope whether bubbles were formed in the microwells in each well, and additional centrifugation was performed until the bubbles disappeared. After removing the dispensed rinse solution, 2 mL of the iPSC-Y medium of Example 1.1 was dispensed and placed in an incubator set at 37°C and 5% CO2. It was left in an incubator.

[0150]

[0151] 3. Cell counting method

[0152] 10 uL of the cell suspension was dispensed onto Parafilm and mixed with 10 uL of trypan blue. 10 uL of the prepared cell suspension was dispensed onto a Countess cell counting chamber slide, and the cell number was counted using a Cell Counter Countess 3 device (unit: cells / mL). The counted cells were used to calculate cell volume.

[0153]

[0154] 4. Stem cell proliferation before differentiation

[0155] 4.1 Primary culture of stem cells

[0156] 2 mL of the iPSC-Y medium of Example 1.1 was dispensed into a 15 mL tube. After thawing the frozen cell suspension until at least 80% thawed, 1 mL of iPSC-Y medium was dispensed into the freezing vial. After confirming that the cell suspension was completely thawed, the entire mixture in the freezing vial was transferred to a 15 mL tube and centrifuged (1200 rpm, 5 minutes). The supernatant except for the cell pellet was removed from the centrifuged tube, and the cells were suspended in 1 mL of the iPSC-Y medium of Example 1.1 and isolated. 5 mL of the iPSC-C medium of Example 1.1 and 6,000 cells / cm of the cell suspension were added to a T25 flask. 2 Calculate the cell volume and dispense it into a T25 flask, and store it in an atmosphere set at 37℃ and 5% CO2. The culture medium in the T25 flask was removed and 5 mL of the iPSC medium of Example 1.1 was dispensed. After 48 hours, the culture medium in the T25 flask was removed and 5 mL of the iPSC medium of Example 1.1 was dispensed. After another 48 hours, the culture medium in the T25 flask was removed again and 5 mL of CTS DPBS was dispensed. After that, the CTS DPBS in the T25 flask was removed and 2.5 mL of TrypLE select was dispensed, and this was incubated in an incubator set at 37°C and 5% CO2. After incubation in an incubator, the T25 flask was removed and tapped to confirm that the cells had completely detached from the flask. 2.5 mL of the iPSC-Y medium from Example 1.1 was dispensed into the T25 flask. Finally, all cells in the T25 flask were collected in a 15 mL tube.

[0157]

[0158] 4.2 Secondary culture of stem cells

[0159] For the cells recovered in Example 4.1, the culture process of Example 4.1 was repeated in a T75 flask. However, after cell counting, the prescribed volume of the medium during the culture process was 15 mL for iPSC-C medium, iPSC medium, and CTS DPBS, and 7.5 mL for TrypLE select and iPSC-Y medium. Finally, all cells in the T75 flask were recovered in a 50 mL tube.

[0160]

[0161] 4.3 Tertiary culture of stem cells

[0162] For the cells recovered in Example 4.2, the culture process of Example 4.1 was repeated in T75 flasks (x4). However, the cell volume was 10,000 cells / cm in the cell suspension. 2 The amount of iPSC-C medium, iPSC medium, and CTS DPBS was calculated to be 15 mL, and the iPSC-Y medium was 7.5 mL. 7.5 mL of Accutase was dispensed instead of TrypLE select. Finally, all cells in the T75 flask were collected in a 50 mL tube. The collected solution was centrifuged (1200 rpm, 5 minutes), and the supernatant excluding the cell pellet was removed from the centrifuged tube.

[0163]

[0164] 5. Differentiation into neural progenitor cells

[0165] The cell pellet obtained in Example 4 was suspended in 2 mL of iPSC-Y medium. [Number of wells to be used in the experiment] x [1.8 x 10 6] was calculated and dispensed into a 15 mL tube and centrifuged (1200 rpm, 5 minutes). After removing the supernatant except for the cell pellet in the centrifuged tube, iPSC-Y medium (number of wells to be used in the experiment) x 1 mL was dispensed. The cell pellet was suspended by pipetting to prepare a cell suspension. Afterwards, the microwell plate of Example 2 was taken out, the mixed solution was removed, 1 mL of the iPSC-Y medium of Example 1.1 was dispensed to prepare, and 1 mL of the cell suspension was dispensed into each well of the plate. After pipetting to ensure that the cells were evenly suspended throughout the well, centrifugation was performed (100 x g, 3 minutes), and the centrifuged tube was placed in an incubator set at 37°C and 5% CO2. It was placed in an incubator. After 5 hours, it was checked whether the embryoid body (EB) was well formed.

[0166] The medium was replaced daily after 24 hours. Specifically, after 24 hours, 1 mL of the supernatant from each well was carefully removed with a micropipette, and 1.5 mL of the NIM medium of Example 1.2 was carefully dispensed using a micropipette. From then on (Day 2-9), 900 uL of the supernatant was removed on each day, and 950 uL of the NIM medium of Example 1.2 was dispensed. After the medium replacement, the culture was maintained in an incubator set at 37°C and 5% CO2. I left it in the incubator.

[0167]

[0168] 6. Embryo (EB) isolation process

[0169] The plate of Example 5 was taken out, mild pipetting was performed throughout the well, and the cell suspension of each well was collected in a 15 mL tube. 1 mL of the NEM-Y medium of Example 1.3 was dispensed into each well, pipetting was performed again, and the cell suspension of each well was collected in a 15 mL tube. The 15 mL tube containing the recovered EBs was centrifuged (100 x g, 1 min). The supernatant excluding the cell pellet was removed from the centrifuged tube, and 1 mL of CTS DPBS was additionally dispensed and centrifuged (100 x g, 1 min). The supernatant excluding the cell pellet was removed again, and Accutase (the number of recovered wells) x 1 mL was dispensed into the tube and incubated at 37°C and 5% CO2. After 10 minutes in the incubator, the EB suspension was pipetted to separate single cells (single cell dissociation), and the NEM-Y medium of Example 1.3 (number of wells recovered) x 1 mL was additionally dispensed, followed by centrifugation (1200 rpm, 5 minutes). The supernatant excluding the cell pellet was removed again, and the cells were suspended in 1 mL of the NEM-Y medium of Example 1.3. 5 mL of the NEM-Y medium of Example 1.3 and 80,000 cells / cm of the cell suspension were placed in a T25 flask. 2 Calculate the cell volume and dispense it into a T25 flask, and store it in an atmosphere set at 37℃ and 5% CO2. It was left in an incubator.

[0170]

[0171] 7. 3D selection of neural progenitor cells

[0172] The cells obtained in Example 5 and 3 mL of the NEM medium of Example 1.3 were dispensed into a T25 flask after being allowed to stand for 48 hours. After 48 hours, 3 mL of the NEM medium of Example 1.3 was additionally dispensed, and after 24 hours, the entire supernatant of the T25 flask (~11 mL) was collected in a 15 mL tube and centrifuged (100 x g, 1 min). After removing the supernatant except for the cell pellet, 2 mL of Accutase was dispensed, and the cell pellet was suspended by pipetting, and incubated in an incubator set at 37°C and 5% CO2. It was placed in an incubator. After 10 minutes, the cells were separated by pipetting, and 2 mL of the NEM-Y medium of Example 1.3 was additionally dispensed and centrifuged (1200 rpm, 5 minutes). The supernatant excluding the cell pellet was removed again and suspended in 1 mL of the NEM-Y medium of Example 1.3. In a T25 flask, 5 mL of the NEM-Y medium of Example 1.3 and 80,000 cells / cm of the cell suspension were added. 2 Calculate the cell volume and dispense it, and store it in a culture medium set at 37℃ and 5% CO2. It was left in an incubator.

[0173] The dispensing and centrifugation process of NEM medium, Accutase, and NEM-Y medium as described above was performed twice more. The supernatant except for the cell pellet in the centrifuged tube was removed and suspended in 1 mL of N2B27 medium of Example 1.4. In a T25 flask, 5 mL of N2B27-C medium of Example 1.4 and 20,000 cells / cm of the cell suspension were added. 2 Calculate the cell volume and dispense it, and store it in a culture medium set at 37℃ and 5% CO2. It was left in an incubator.

[0174]

[0175] 8. Two-dimensional proliferation of neural progenitor cells

[0176] 5 mL of N2B27-C medium of Example 1.4 and 20,000 cells / cm of cell suspension from which NPC selection was performed three times in Example 7 were added to a T25 flask. 2 The cell volume was calculated and dispensed.

[0177] After 48 hours, the medium in the T25 flask was removed, and 5 mL of the N2B27 medium of Example 1.4 was dispensed. After 48 hours, the culture medium in the T25 flask was removed, and 5 mL of the N2B27 medium of Example 1.4 was dispensed. After the next 24 hours, the medium in the T25 flask was removed, and 5 mL of CTS DPBS was dispensed. After that, the CTS DPBS in the T25 flask was removed, and 2.5 mL of Accutase was dispensed, and then it was placed in an incubator set at 37°C and 5% CO2. It was left in the incubator for 5 minutes. After taking out the T25 flask and tapping it, it was confirmed that the cells were completely detached from the flask, and 2.5 mL of the N2B27 medium of Example 1.4 was dispensed into the T25 flask. Finally, all the cells in the T25 flask were collected in a 15 mL tube. The collected solution was centrifuged (1200 rpm, 5 minutes), and the supernatant except for the cell pellet was removed from the centrifuged tube. The dispensing and centrifugation process of the N2B27 medium, Accutase, and N2B27-C medium described above was performed 5 more times.

[0178]

[0179] Experimental Example 1. Confirmation of Embryoid Body (EB) Formation During Differentiation into Neural Progenitor Cells

[0180] To confirm whether differentiation into neural progenitor cells was performed well according to Example 5, daily culture photos were taken, and these are shown in Figure 2.

[0181] As shown in Fig. 2, it was observed that the culture was well formed and maintained during the differentiation process.

[0182]

[0183] Experimental Example 2. Confirmation of Neuroblastoma Cell Formation

[0184] In order to confirm whether neurosphere cells were well formed according to Example 7, the cultures that completed the differentiation process were treated with Accutase enzyme, and then three-dimensionally cultured for 5 days, and then confirmed using a phase contrast microscope, which is shown in Fig. 3. In addition, the presence or absence of attached cells according to the combination of supplements was confirmed, which is shown in Fig. 4.

[0185] As shown in Figure 3, neural progenitor cells formed neurospheres during 3D culture, whereas other types of cells other than neural progenitor cells were confirmed to have died because they lacked the ability to form neurospheres and undergo 3D culture.

[0186] As shown in Figure 4, under the N2B27 condition, some cells were observed to adhere during neurosphere formation (black arrow area), but under the B27 only condition, no adherent cells were observed. Since additional cell detachment is required when cell attachment occurs, the above results imply that the process can be minimized at the same efficiency when using the B2 only supplement combination.

[0187]

[0188]

[0189] Experimental Example 3. Confirmation of neural progenitor cell proliferation according to the presence or absence of a 3D selection process.

[0190] The presence of other types of cells other than neural progenitor cells was observed using a phase contrast microscope, and this is shown in Figure 5.

[0191] As shown in Figure 5, when the 3D selection process was not performed, proliferation of unspecified types of cells other than neural progenitor cells was confirmed, whereas when the 3D selection process was performed, proliferation of unspecified types of cells other than neural progenitor cells was not confirmed.

[0192]

[0193] Experimental Example 4. Confirmation of neural progenitor cell expression markers according to the presence or absence of a 3D selection process.

[0194] The purity of the neural progenitor cells obtained in Example 8 was confirmed by flow cytometry (FACS) analysis according to the number of passages.

[0195] First, in the absence of a 3D selection process, only a 2D proliferation process was performed, so the analysis conditions were kept the same to control for variables in flow cytometry that may arise from differences in cell morphology between 3D and 2D cultures. Specifically, in the presence of a 3D selection process, a 2D proliferation process was performed once after the 3D selection process. The expression of PSA-NCAM(+), used as a marker of neuroectoderm, and CD271(+) / HNK-1(+), used as a marker of neural crest cells, was confirmed, and the results are shown in Figures 6 to 8.

[0196] As shown in Fig. 6, when the 2D proliferation process was performed without performing the 3D selection process, the ratio of PSA-NCAM(+) was maintained at 95% or higher, and the ratio of CD271(+) / HNK-1(+) was confirmed to increase from 2.37% (passage 1) to 24.4% (passage 6).

[0197] As shown in Fig. 7, when the 3D selection process was performed, the ratio of PSA-NCAM(+) was maintained at 95% or higher, and the ratio of CD271(+) / HNK-1(+) was confirmed to decrease from 0.78% (passage 1) to 0.24% (passage 6).

[0198] As shown in Fig. 8, when the 3D selection process was performed 3 times and the 2D expansion process was performed 3 times, it was confirmed that the ratio of CD271(+) / HNK-1(+) decreased from 11.4% to 1.43%, and when the 2D expansion process was performed 3 times for high-purity neural progenitor cells with a CD271(+) / HNK-1(+) ratio of 1.43%, it was confirmed that the ratio of CD271(+) / HNK-1(+) did not increase and high purity was maintained through expansion 1 time (0.83%), 2 times (1.39%), and 3 times (1.21%).

[0199]

[0200] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. (a) A step of culturing stem cells in three dimensions, forming embryoid bodies (EBs) from the stem cells, and differentiating them into neural precursor cells; (b) a step of three-dimensionally selecting a sphere containing the neural progenitor cells; and (c) A method for producing neural progenitor cells, including a step of two-dimensionally culturing and proliferating the selected spheroids.

2. A method for producing neural progenitor cells according to claim 1, wherein step (a) comprises a step of differentiating stem cells in a medium that does not contain a component derived from a xenogeneic animal.

3. A method for producing neural progenitor cells according to claim 2, wherein the medium further comprises at least one selected from the group consisting of DMEM / F12, NEAA (non-essential amino acid), 2-mercaptoethanol, LDN193189, and SB431542.

4. In claim 1, the culture medium is 10 2 10 inland 5 A method for producing neural progenitor cells comprising cells / culture bodies of a dog.

5. A method for producing neural precursor cells according to claim 1, wherein step (b) is performed by mechanical separation or enzymatic separation.

6. A method for producing neural progenitor cells according to claim 5, wherein the mechanical separation is performed using a blade, a tissue grinder, a needle, a pipette, a scraper, or a combination thereof.

7. A method for producing neural progenitor cells according to claim 5, wherein the enzymatic separation is performed using collagenase, accutase, dispase, hyaluronidase, DNase, papain, trypsin, or a combination thereof.

8. A method for producing neural progenitor cells according to claim 1, wherein step (b) comprises recovering the cells of step (a), distributing the recovered cells in a medium containing Y-27632, and then three-dimensionally selecting spheroids containing neural progenitor cells.

9. A method for producing neural precursor cells according to claim 8, wherein the medium further comprises at least one selected from the group consisting of DMEM / F12, VNEAA, glutamine, sodium pyruvate, glucose, B27 supplement without vitamin A, 2-mercaptoethanol, and fibroblast growth factor (FGF).

10. A method for producing neural progenitor cells according to claim 1, wherein the stem cells are induced pluripotent stem cells or embryonic stem cells.

11. A method for producing neural precursor cells according to claim 1, wherein step (b) is repeated at least once.

12. A method for producing neural precursor cells according to claim 1, wherein step (c) is repeated at least 5 times.

13. A method for producing neural progenitor cells, wherein the method further comprises a step of proliferating stem cells by subculturing them prior to step (a).

14. A method for producing neural progenitor cells according to claim 13, wherein the subculture comprises a step of culturing stem cells in a stem cell proliferation medium containing fibroblast growth factor and Y-27632.

15. A method for producing neural progenitor cells according to claim 13, wherein the subculture is repeated at least three times.

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