Method for producing neural cells

Abstract

To provide a method for producing neural cell which is capable of efficiently producing a neural cell in a short period without hurting the gene of a cell.SOLUTION: A method for producing a neural cell according to one embodiment of the present invention includes preparing a stem cell, introducing an inducing factor RNA into the stem cell, and differentiating the cell into a neural cell. A method for producing a neural cell according to another embodiment of the present invention includes preparing a cell, introducing a reprogramming factor RNA into the cell, and introducing an inducing factor RNA into the cell, into which the reprogramming factor RNA was introduced, to differentiate the cell into a neural cell.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to cell technology and to a method for producing nervous system cells. [Background technology]

[0002] Induced pluripotent stem cells (iPS cells) can transform into any cell that makes up the body. Because iPS cells can transform into various types of somatic cells and tissues, they are expected to be used in cell transplantation therapy and drug discovery research. For example, in 2014, retinal cells created from iPS cells were used in transplant therapy. Projects are underway not only in Japan but also in countries around the world to create brain cells and cells for various organs from iPS cells for use in transplant therapy.

[0003] There are many methods to change iPS cells into differentiated cells. However, in order to use iPS cells in transplantation therapy, it is important to establish an efficient method for inducing differentiation of iPS cells. Specifically, it is necessary to establish the technology used to induce iPS cells into differentiated cells, improve the efficiency and accuracy of differentiation induction, and ensure that the functionality of the differentiated cells produced can withstand transplantation therapy.

[0004] Conventionally, methods for inducing iPS cells or embryonic stem cells (ES cells) into differentiated cells have been performed by combining hormones, growth factors, and low molecular weight compounds that determine the properties of the cells, and changing their quantitative ratios and concentrations over time to mimic the developmental process. However, it is difficult and inefficient to completely mimic the developmental process in a test tube. In addition, compared to inducing somatic cell differentiation in mice, a very long differentiation induction period is required in humans; for example, it takes more than three months to create mature nerves.

[0005] Furthermore, there are problems such as the efficiency of differentiation induction differing greatly depending on the ES / iPS cell line, and the properties of the induced somatic cells being non-uniform. In fact, when chemical substances were added to multiple ES cell clones to produce various cells, it was shown that there were clones that were easily differentiated into pancreatic cells and clones that were easily differentiated into cardiac cells, and that there were differences in differentiation ability depending on the clones (see, for example, Non-Patent Document 1). In addition, when nerve cells were produced from several dozen types of iPS cells using a method called serum-free aggregate suspension culture method (SFEBq method), in which iPS cells are cultured in a medium that does not contain serum or chemical substances that inhibit nerve cell differentiation, it was proven that there are iPS / ES cell clones that are not easily transformed into nerve cells (see, for example, Non-Patent Document 2).

[0006] Specifically, it has been confirmed that cells induced to differentiate from human ES / iPS cells using methods that utilize hormones or chemicals are fetal-stage somatic cells at the initial stage. Inducing differentiation of mature human somatic cells is extremely difficult, and requires long-term culture over several months. However, in drug discovery and transplantation medicine, which target individuals that have completed development, it is important to create somatic cells that match the maturity of the individual.

[0007] In addition, there are various subtypes of nerve cells, but these subtypes cannot be uniformly induced to differentiate from ES / iPS cells using methods that use hormones or chemicals. This means that drug discovery screening specific to a particular nerve subtype is not possible, making drug discovery screening less efficient. In transplantation medicine, it is also not possible to concentrate and transplant only specific diseased cells.

[0008] In response to this, a method has been proposed in which genes that determine the properties of specific somatic cells are directly introduced into ES / iPS cells using viruses to produce the desired somatic cells. Compared to methods that use hormones or chemicals, methods that use viruses can specifically produce mature nerve cells in a very short period of time, such as two weeks. In addition, when nerve cells are produced by introducing a specific gene, it is possible to uniformly obtain only excitatory nerves, for example. This makes it possible to screen drugs specific to a specific nerve subtype, and it is thought that in transplantation medicine, it will be possible to concentrate and transplant only specific diseased cells.

[0009] However, in the method of inducing differentiation of stem cells into somatic cells using a virus to express a specific gene, the gene is inserted into the genome of the ES / iPS cell, damaging the endogenous gene. As a result, drug discovery screening cannot be performed properly, and there is a risk of cancer formation during transplantation (see, for example, Non-Patent Documents 3 and 4). [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Nature Biotechnol 26(3): 313-315, 2008. [Non-Patent Document 2] PNAS, 111:12426-12431, 2014 [Non-Patent Document 3] N Eng J Med, 346:1185-1193, 2002 [Non-Patent Document 4] Science 302: 415-419, 2003 Summary of the Invention [Problem to be solved by the invention]

[0011] An object of the present invention is to provide a method for producing nervous system cells, which can produce nervous system cells efficiently in a short period of time without damaging the genes of the cells. [Means for solving the problem]

[0012] According to an aspect of the present invention, there is provided a method for producing neural cells, comprising: preparing stem cells; and introducing an inducer RNA into the stem cells to differentiate them into neural cells.

[0013] In the above method for producing neural cells, the stem cells may be induced pluripotent stem cells. The neural cells may be any of nerve cells, neural stem cells, and neural progenitor cells. The neural cells may be any of inhibitory nerve cells, excitatory nerve cells, and dopamine-producing nerve cells. Alternatively, the neural cells may be motor nerve cells, oligodendrocyte precursor cells, oligodendrocytes, and the like.

[0014] In the above-mentioned method for producing neural cells, the inducer RNA may be introduced into the stem cells by lipofection.

[0015] In the above-mentioned method for producing neural cells, the inducer RNA may contain mRNA corresponding to a drug resistance gene.

[0016] The above-mentioned method for producing neural cells may include, after introducing an inducer RNA into stem cells, selecting cells exhibiting drug resistance.

[0017] According to an embodiment of the present invention, there is provided a method for producing nervous system cells, comprising: preparing cells; introducing reprogramming factor RNA into the cells; and introducing an inducer RNA into the cells into which the reprogramming factor RNA has been introduced, thereby differentiating the cells into nervous system cells. Conventionally, it took two months to establish stem cells and three months to induce nervous system cells. In contrast, according to the method for producing nervous system cells according to an embodiment of the present invention, it is possible to induce nervous system cells from cells in a short period of time.

[0018] In the above-mentioned method for producing neural cells, reprogramming factor RNA may be introduced into cells in the same culture vessel, and inducer RNA may be introduced into the cells into which the reprogramming factor RNA has been introduced.

[0019] In the above-mentioned method for producing neural cells, after introducing the reprogramming factor RNA into the cells, an inducer RNA may be introduced into the cells into which the reprogramming factor RNA has been introduced without detaching the cells into which the reprogramming factor RNA has been introduced from the culture vessel.

[0020] In the above-mentioned method for producing neural cells, after introducing the reprogramming factor RNA into the cells, the cells into which the reprogramming factor RNA has been introduced may be detached from the culture vessel, and the cells into which the reprogramming factor RNA has been introduced may be seeded into another culture vessel, after which an inducer RNA may be introduced into the cells into which the reprogramming factor RNA has been introduced.

[0021] In the above-mentioned method for producing neural cells, the cells into which the reprogramming factor RNA is introduced may be somatic cells such as human fibroblasts or blood cells.

[0022] In the above-mentioned method for producing a nervous system cell, the nervous system cell may be any one of a neuron, a neural stem cell, and a neural progenitor cell. The nervous cell may be any one of an inhibitory neuron, an excitatory neuron, and a dopamine-producing neuron.

[0023] In the above-mentioned method for producing neural cells, the inducer RNA may be introduced into the stem cells by lipofection.

[0024] In the above-mentioned method for producing neural cells, the inducer RNA may contain mRNA corresponding to a drug resistance gene.

[0025] The above-mentioned method for producing neural cells may include, after introducing an inducer RNA into stem cells, selecting cells exhibiting drug resistance.

[0026] Furthermore, according to an embodiment of the present invention, there is provided an RNA corresponding to the DNA of any one of SEQ ID NOs: 1 to 10.

[0027] Furthermore, according to an embodiment of the present invention, there is provided an inducer comprising RNA corresponding to the DNA of any one of SEQ ID NOs: 1 to 10. Effect of the Invention

[0028] According to the present invention, it is possible to provide a method for producing nervous system cells, which can produce nervous system cells efficiently in a short period of time without damaging the genes of the cells. [Brief description of the drawings]

[0029] [Figure 1] 1 is a photograph of a nerve cell according to Example 1 of the first embodiment. [Diagram 2] 1 is a photograph of a nerve cell according to Example 1 of the first embodiment. [Diagram 3] 1 is a photograph of a nerve cell according to Example 1 of the first embodiment. [Figure 4] 4 is a photograph of nerve cells according to Examples 2 to 4 of the first embodiment. [Diagram 5] 13 is a photograph of a nerve cell according to Example 3 of the first embodiment. [Figure 6] 13 is a photograph of a nerve cell according to Example 3 of the first embodiment. [Figure 7] 13 is a photograph of a nerve cell according to Example 5 of the first embodiment. [Figure 8] 13 is a photograph of a nerve cell according to Example 6 of the first embodiment. [Figure 9] 13 is a photograph of a nerve cell according to Example 7 of the first embodiment. [Figure 10] 13 is a photograph of a nerve cell according to Example 8 of the first embodiment. [Figure 11] 1 is a photograph of a nerve cell according to a comparative example of the first embodiment. [Figure 12] 1 is a table showing components of a master mix of reprogramming factor mRNA used in Example 1 of the second embodiment. [Figure 13] 1 is a table showing the contents of the kit used in Example 1 of the second embodiment. [Figure 14]13 is a photograph of a nerve cell according to Example 1 of the second embodiment. [Figure 15] 13 is a photograph of a nerve cell according to Example 2 of the second embodiment. [Figure 16] 13 is a photograph of a nerve cell according to Example 2 of the second embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] The following is a detailed description of the embodiments of the present invention. Note that the embodiments shown below are merely examples of devices and methods for embodying the technical idea of ​​the present invention, and the technical idea of ​​the present invention is not limited to the combination of components described below. The technical idea of ​​the present invention can be modified in various ways within the scope of the claims.

[0031] (First embodiment) The method for producing neural cells according to the first embodiment includes preparing stem cells, and introducing an inducer RNA into the stem cells to differentiate them into neural cells.

[0032] As stem cells, both induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells) can be used.

[0033] Examples of the induced neural cells include nerve cells, neural stem cells, and neural progenitor cells. Examples of the neural cells include inhibitory nerve cells, excitatory nerve cells, and dopamine-producing nerve cells. Alternatively, the neural cells may be motor nerve cells, oligodendrocyte precursor cells, oligodendrocytes, and the like.

[0034] Primate ES Cell Medium, mTeSR1, TeSR2, and TeSRE8 (STEMCELL Technologies), etc. may be used as the culture medium for culturing stem cells.

[0035] The medium for culturing stem cells may also contain a gel. The gel may contain at least one polymer compound selected from the group consisting of deacylated gellan gum, gellan gum, hyaluronic acid, rhamsan gum, diutan gum, xanthan gum, carrageenan, fucoidan, pectin, pectic acid, pectinic acid, heparan sulfate, heparin, heparitin sulfate, keratosulfate, chondroitin sulfate, deltamann sulfate, and rhamnan sulfate, and salts thereof. The gel medium may also contain methylcellulose. By containing methylcellulose, aggregation between cells is further suppressed.

[0036] The gel may be a temperature-sensitive gel. The temperature-sensitive gel may be at least one selected from poly(glycerol monomethacrylate) (PGMA), poly(2-hydroxypropyl methacrylate) (PHPMA), Poly(N-isopropylacrylamide) (PNIPAM), amine terminated, carboxylic acid terminated, maleimide terminated, N-hydroxysuccinimide (NHS) ester terminated, triethoxysilane terminated, Poly(N-isopropylacrylamide-co-acrylamide), Poly(N-isopropylacrylamide-co-acrylic acid), Poly(N-isopropylacrylamide-co-butylacrylate), Poly(N-isopropylacrylamide-co-methacrylic acid), Poly(N-isopropylacrylamide-co-methacrylic acid-co-octadecyl acrylate), and N-isopropylacrylamide.

[0037] The medium for culturing stem cells may contain at least one substance selected from the group consisting of cadherin, laminin, fibronectin, and vitronectin.

[0038] The inducer RNA introduced into the stem cell may contain at least one of, for example, ASCL1 (Achaete-Scute Homolog 1) mRNA, DLX2 (Distal-Less Homeobox 2) mRNA, MYT1L (Myelin Transcription Factor 1-Like) mRNA, and NGN2 (neurogenin 2) mRNA. Note that although gene symbols are written in human here, capitalization is not intended to limit the species. For example, even if all symbols are written in capital letters, it does not exclude the inclusion of mouse or rat genes. However, in the examples, gene symbols are written according to the species actually used.

[0039] The inducer RNA may contain mRNA corresponding to a drug resistance gene. The drug may be an antibiotic such as puromycin, neomycin, blasticidin, G418, hygromycin, or zeocin. The cells into which the inducer RNA has been introduced exhibit drug resistance.

[0040] The mRNAs contained in the inducer RNA were pseudouridine (Ψ), 5-methyluridine (5meU), N1-methylpseudouridine (me1Ψ), 5-methoxyuridine (5moU), 5-hydroxymethyluridine (5hmU), 5-formyluridine (5fU), 5-carboxymethylesteruridine (5camU), thienoguanosine (thG), N4-methylcytidine (me4C), 5-methylcytidine (m5C), 5-methyoxycytidine (5moC), and 5 and may be modified with at least one selected from the group consisting of 5-hydroxymethylcytidine (5hmC), 5-hydroxycytidine (5hoC), 5-formycytidine (5fC), 5-carboxycytidine (5caC), N6-methyl-2-aminoadenosine (m6DAP), diaminopurine (DAP), 5-methyluridine (m5U), 2'-O-methyluridine (Um or m2'-OU), 2-thiouridine (s2U), and N6-methyladenosine (m6A).

[0041] The mRNA may be polyadenylated. The mRNA may be prepared by polyadenylation of in vitro transcribed (IVT) RNA. The mRNA may be polyadenylated during IVT by using a DNA template that codes for poly(A) tail. The mRNA may be capped. To maximize the efficiency of expression in cells, most mRNA molecules may contain a cap.

[0042] The mRNA may have a 5'cap [m7G(5')ppp(5')G] structure. This sequence stabilizes the mRNA and promotes transcription. From mRNA that has 5'triphosphate, the 5'triphosphate may be removed by dephosphorylation treatment. The mRNA may have [3'O-Me-m7G(5')ppp(5')G] as an Anti-Reverse Cap Analog (ARCA). ARCA is a sequence inserted before the transcription start site, which doubles the efficiency of the transcribed mRNA. The mRNA may have a PolyA tail.

[0043] The inducer RNA includes, for example, NGN2-T2A-PURO mRNA (TriLink, RNA corresponding to the DNA set forth in SEQ ID NO: 1). Cells transfected with NGN2-T2A-PURO mRNA (Trilink) produce neurogenin2 (NGN2) and exhibit puromycin resistance. The mRNA may be capped with Anti-Reverse Cap Analog (ARCA), polyadenylated, and substituted with 5-methylcytidine and pseudouridine. 5-methylcytidine and pseudouridine reduce the recognition ability of the mRNA by antibodies. RNA corresponding to the DNA set forth in SEQ ID NO: 2 may also be used. The DNA set forth in SEQ ID NO: 2 is DNA obtained by removing the xba1 restriction site from the DNA set forth in SEQ ID NO: 1.

[0044] Alternatively, the inducer comprises NGN2-T2A-PURO mRNA (RNA corresponding to the DNA set forth in SEQ ID NO: 3). Cells transfected with RNA corresponding to the DNA set forth in SEQ ID NO: 3 produce NGN2 and exhibit puromycin resistance.

[0045] Alternatively, the inducer comprises ASCL1-T2A-PURO mRNA (RNA corresponding to the DNA set forth in SEQ ID NO: 4). Cells transfected with RNA corresponding to the DNA set forth in SEQ ID NO: 4 produce ASCL1 and exhibit puromycin resistance.

[0046] Alternatively, the inducer comprises DLX2-T2A-PURO mRNA (RNA corresponding to the DNA set forth in SEQ ID NO: 5). Cells transfected with RNA corresponding to the DNA set forth in SEQ ID NO: 5 produce DLX2 and exhibit puromycin resistance.

[0047] Alternatively, the inducer comprises DLX2-T2A-HYGRO mRNA (RNA corresponding to the DNA set forth in SEQ ID NO: 6). Cells transfected with RNA corresponding to the DNA set forth in SEQ ID NO: 6 produce DLX2 and exhibit hygromycin resistance.

[0048] Alternatively, the inducer comprises DLX2-T2A-BLAST mRNA (RNA corresponding to the DNA set forth in SEQ ID NO: 7). Cells transfected with RNA corresponding to the DNA set forth in SEQ ID NO: 7 produce DLX2 and exhibit blasticidin resistance.

[0049] Alternatively, the inducer comprises DLX2-IRES-HYGRO mRNA (RNA corresponding to the DNA set forth in SEQ ID NO: 8). Cells transfected with RNA corresponding to the DNA set forth in SEQ ID NO: 8 produce DLX2 and exhibit hygromycin resistance.

[0050] Alternatively, the inducer comprises DLX2-IRES-BLAST mRNA (RNA corresponding to the DNA set forth in SEQ ID NO: 9). Cells transfected with RNA corresponding to the DNA set forth in SEQ ID NO: 9 produce DLX2 and exhibit blasticidin resistance.

[0051] Alternatively, the inducer comprises ASCL1-T2A-PURO mRNA (RNA corresponding to the DNA set forth in SEQ ID NO: 10). Cells transfected with RNA corresponding to the DNA set forth in SEQ ID NO: 10 produce ASCL1 and exhibit puromycin resistance.

[0052] The inducer RNA is introduced into stem cells by a transfection method such as lipofection. The lipofection method is a method in which a complex of a negatively charged substance, a nucleic acid, and a positively charged lipid is formed by electrical interaction, and the complex is taken up into cells by endocytosis or membrane fusion. The lipofection method has the advantages of causing little damage to cells, being highly efficient in introduction, being easy to operate, and not taking much time.

[0053] For transfection of inducer RNA, for example, Lipofectamine Messenger Max (registered trademark) is used as a transfection reagent. Furthermore, examples of lipofection reagents that may be used include Lipofectamine® RNAiMAX (Thermo Fisher Scientific), Lipofectamin® 2000, Lipofectamin® 3000, NeonTransfection System (Thermo Fisher Scientific), Stemfect RNA transfection reagent (Stemfect), mRNA-In (registered trademark, Molecular Transfer, Inc.), NextFect® RNA Transfection Reagent (BiooSientific), Amaxa® Human T cell Nucleofector® kit (Lonza, VAPA-1002), Amaxa® Human CD34 cell Nucleofector® kit (Lonza, VAPA-1003), and ReproRNA® transfection reagent (STEMCELL Technologies).

[0054] Transfection of the inducer RNA may be carried out multiple times.

[0055] The medium used during transfection of the inducer RNA is, for example, a serum-free or low-serum medium such as Plurito Reprogramming Medium (STEMGENT) or Opti-MEM (registered trademark, Gibco). The medium used during, before, and after transfection of the inducer RNA may contain B18R protein. B18R protein alleviates the innate antiviral response of cells. B18R protein may be used to suppress cell death associated with immune responses that accompany the insertion of RNA into cells. However, the medium may not contain B18R protein, or may contain B18R protein at a low concentration such as 0.01% to 1%.

[0056] After transfection of the inducer RNA, or during multiple rounds of transfection of the inducer RNA, the medium is replaced with a medium suitable for neural cells.

[0057] If the inducer RNA contains an mRNA corresponding to a drug resistance gene, cells exhibiting drug resistance may be selected during or after transfection. For example, if the inducer RNA contains an mRNA corresponding to a puromycin resistance gene, cells after transfection can be exposed to puromycin to kill cells other than those into which the inducer RNA has been introduced, and cells into which the inducer RNA has been introduced can be selected. The inducer RNA may contain any one selected from neomycin, blasticidin, G418, hygromycin, zeocin, etc., as the mRNA corresponding to the drug resistance gene.

[0058] Whether or not the cells have differentiated into neural cells is confirmed by whether or not NGN2, β-IIITubulin, MAP2, PSA-NCAM, vGLUT, GAD67, TH (Tyrosine Hydroxylase), SOX1, SOX2, CD133, Nestin, HB9, ISL1, O4, PLP1, MOG, or MBP are positive. NGN2 is a switch protein required for neural cell differentiation. β-IIITubulin, MAP2, and PSA-NCAM are markers for neural cells. vGLUT is a marker for excitatory neurons. GAD67 is a marker for inhibitory neurons. TH is a marker for dopamine-producing neurons. SOX1, SOX2, CD133, and Nestin are markers for neural stem cells. HB9 and ISL1 are markers for motor neurons. O4, PLP1, MOG, and MBP are markers for oligodendrocyte precursors.

[0059] Additionally, GFAP and CD44 are available as markers for astrocyte precursors and astrocytes, and ChAT is available as a marker for cholinergic neurons.

[0060] According to the method of the first embodiment described above, by expressing RNA encoding a specific gene in stem cells, it is possible to efficiently produce nervous system cells without damaging the genes of the stem cells.

[0061] In the method of producing neural cells from stem cells using only hormones and chemicals, it takes a very long time to produce neural cells. In contrast, the method according to the first embodiment makes it possible to produce neural cells in a very short time.

[0062] In addition, in the method of producing neural cells from stem cells using hormones or chemical substances, only a portion of the stem cells become the target neural cells, whereas in the method according to the first embodiment, 90% or more of the cells into which the inducer RNA is introduced become the target neural cells.

[0063] Furthermore, in the method of producing neural cells from stem cells using hormones or chemical substances, even if the same protocol is followed, some clones will become the target neural cells and some will not, resulting in variation between clones. In contrast, the method according to the first embodiment makes it possible to obtain high differentiation induction efficiency for multiple clones.

[0064] Furthermore, when preparing transplant cells by inducing differentiation from an undifferentiated cell population using cytokines or the like, there is a possibility that undifferentiated cells remain among the transplant cells. These remaining undifferentiated cells undergo cell division and proliferation independently at the transplant site, and there is a risk of forming teratomas or the like. In contrast, according to the method of the first embodiment, a drug resistance gene can also be expressed at the same time, making it possible to select drugs for cells into which inducer RNA has been introduced. This makes it possible to avoid the risk of contamination with undifferentiated cells and the formation of teratomas, making it suitable for transplantation medicine.

[0065] (Example 1 of the first embodiment) Plates were coated with a solubilized basement membrane preparation (Matrigel, Corning). 1 × 10 cells were then added to 1 mL of human ES / iPS cell maintenance medium (mTeSR1, TEMCELL Technologies) containing 200 ng / mL B18R recombinant protein and ROCK inhibitor (Y-27632, Selleck). 5 pieces, 2×10 5 pcs or 4 x 10 5 Single-cell iPS cells were suspended, the suspension was added to a plate to seed the cells, and the plate was left to stand for one day.

[0066] The medium was replaced with 1 mL of xeno-free medium (Pluriton, STEMGENT) containing B18R recombinant protein at a concentration of 200 ng / mL and ROCK inhibitor.

[0067] In addition, a 1.5 mL microcentrifuge tube A and a 1.5 mL microcentrifuge tube B were prepared.

[0068] Tube A was charged with 62.5 μL of low serum medium (Opti-MEM (registered trademark), Gibco), to which was added 1.875 μL of an mRNA introduction reagent (Lipofectamine MessengerMax (registered trademark), Invitrogen), and mixed well to prepare a first reaction solution.

[0069] Tube B was charged with 62.5 μL of low serum medium (Opti-MEM®, Gibco), and 750 ng of ASCL1 mRNA, 250 ng of DLX2-T2A-BLAST mRNA, and 100 ng of GFP mRNA (Trilink) were added and mixed well to prepare the second reaction solution. ASCL1 is a protein that controls differentiation into neurons in general. DLX2 is a protein that controls differentiation into inhibitory neurons.

[0070] The second reaction solution was added to the first reaction solution in tube A to form a mixed reaction solution, and then tube A was tapped lightly at room temperature for 10 minutes to form liposomes. Next, the mixed reaction solution was added to the plate and left to stand at 37°C for 6 to 8 hours. This transfected the cells with mRNA (Day 0). Next, all the medium was removed from the plate, and 1 mL of xeno-free medium (Pluriton, STEMGENT) containing B18R recombinant protein and ROCK inhibitor at a concentration of 200 ng / mL was added to the plate and left to stand at 37°C overnight.

[0071] The cells were transfected with mRNA in the same manner as the previous day (Day 1). Next, all the medium was removed from the plate, and 1 mL of xeno-free medium (Pluriton, STEMGENT) containing B18R recombinant protein at a concentration of 200 ng / mL and the antibiotic blasticidin at a concentration of 20 ng / mL was added to the plate, and the plate was left to stand at 37°C overnight.

[0072] All media was removed from the plate, and 1 mL of xeno-free medium (Pluriton, STEMGENT) containing B18R recombinant protein at a concentration of 200 ng / mL and ROCK inhibitor was placed on the plate. Next, the cells were transfected with mRNA in the same manner as the previous day (Day 2). Then, all media was removed from the plate, and 1 mL of neuronal medium (DMEM / F12, 25 μg / mL insulin, 50 μg / mL human transferrin, 30 nmol / L sodium selenite, 20 nmol / L progesterone, and 100 nmol / L putrescine) containing B18R recombinant protein at a concentration of 200 ng / mL and blasticidin at a concentration of 20 ng / mL was placed on the plate, and the neuronal medium was called N3 medium. The plate was then left to stand overnight at 37°C.

[0073] All media was removed from the plate, and 1 mL of N3 medium containing B18R recombinant protein at a concentration of 200 ng / mL was added to the plate. Next, the cells were transfected with mRNA in the same manner as the previous day (Day 3). Then, all media was removed from the plate, and 1 mL of N3 medium containing B18R recombinant protein at a concentration of 200 ng / mL and blasticidin at a concentration of 20 μg / mL was added to the plate, and the plate was left standing at 37 ° C. overnight.

[0074] All media was removed from the plate, and 1 mL of N3 media containing B18R recombinant protein at a concentration of 200 ng / mL was added to the plate and left to stand overnight at 37°C. The cells were then cultured for 7 days. Selection with blasticidin was performed until Day 6.

[0075] As a result, as shown in Figure 1, it was confirmed from cell morphology that nerve cells were induced from iPS cells.

[0076] After that, the medium was removed from the plate, and the cells were washed with PBS. Next, 4% PFA was added to the plate, and the cells were fixed by reacting for 15 minutes at 4°C. Furthermore, after washing the cells twice with PBS, the primary antibody was diluted in PBS medium containing 5% CCS and 0.1% Triton, and added to the plate. The primary antibody used was a mouse monoclonal antibody IgG2a (MAB5406, Millepore) against GAD67, a marker for inhibitory neurons.

[0077] After one hour of reaction at room temperature, PBS was added to the plate, and the plate was thoroughly mixed with PBS, which was then discarded. PBS was added again, discarded, and a solution containing a fluorescently labeled donkey anti-mouse IgG (H+L) secondary antibody (Alexa Fluor, registered trademark, 555, Conjugate, Invitrogen) was added to the plate and reacted at room temperature for 30 minutes. The cells were then washed twice with PBS and observed under a fluorescent microscope. As a result, as shown in Figure 2 (fluorescence microscope image) and Figure 3 (merged image of phase contrast microscope image and fluorescence microscope image), it was confirmed that the neurons induced from iPS cells expressed GAD67, a marker for inhibitory neurons.

[0078] (Example 2 of the first embodiment) A plate was prepared by coating with a solubilized basement membrane preparation (Matrigel, Corning). Single-cell-dissociated iPS cells were suspended in 1 mL of human ES / iPS cell maintenance medium (mTeSR1, TEMCELL Technologies) containing 200 ng / mL of B18R recombinant protein and ROCK inhibitor, and the suspension was added to the plate to seed the cells, which were then left to stand for one day.

[0079] The medium was replaced with 1 mL of xeno-free medium (Pluriton, STEMGENT) containing B18R recombinant protein at a concentration of 200 ng / mL and ROCK inhibitor.

[0080] In addition, a 1.5 mL microcentrifuge tube A and a 1.5 mL microcentrifuge tube B were prepared.

[0081] Tube A was charged with 62.5 μL of low serum medium (Opti-MEM (registered trademark), Gibco), to which was added 1.875 μL of an mRNA introduction reagent (Lipofectamine MessengerMax (registered trademark), Invitrogen), and mixed well to prepare a first reaction solution.

[0082] Tube B contained 62.5 μL of low serum medium (Opti-MEM (registered trademark), Gibco), and 500 ng of ASCL1-PURO mRNA, 250 ng of DLX2-T2A-BLAST mRNA, and 100 ng of GFP mRNA (Trilink). The mixture was mixed well to prepare the second reaction solution.

[0083] The second reaction solution was added to the first reaction solution in tube A to form a mixed reaction solution, and then tube A was tapped lightly at room temperature for 10 minutes to form liposomes. Next, the mixed reaction solution was added to the plate and left to stand at 37°C for 6 to 8 hours. This transfected the cells with mRNA (Day 0). Next, all the medium was removed from the plate, and 1 mL of xeno-free medium (Pluriton, STEMGENT) containing B18R recombinant protein and ROCK inhibitor at a concentration of 200 ng / mL was added to the plate and left to stand at 37°C overnight.

[0084] The cells were transfected with mRNA in the same manner as the previous day (Day 1). Next, all the medium was removed from the plate, and 1 mL of xeno-free medium (Pluriton, STEMGENT) containing B18R recombinant protein at a concentration of 200 ng / mL, the antibiotic blasticidin at a concentration of 20 μg / mL, and puromycin and ROCK inhibitor at concentrations of 2 μg / mL was added to the plate, and the plate was left to stand at 37° C. overnight.

[0085] All media was removed from the plate, and 1 mL of xeno-free medium (Pluriton, STEMGENT) containing B18R recombinant protein at a concentration of 200 ng / mL and ROCK inhibitor was placed on the plate. Next, the cells were transfected with mRNA in the same manner as the previous day (Day 2). Then, all media was removed from the plate, and 1 mL of N3 medium containing B18R recombinant protein at a concentration of 200 ng / mL, blasticidin at a concentration of 20 μg / mL, and puromycin at a concentration of 2 μg / mL was placed on the plate, and the plate was left to stand overnight at 37 ° C.

[0086] All media was removed from the plate, and 1 mL of N3 medium containing B18R recombinant protein at a concentration of 200 ng / mL was placed on the plate. Next, the cells were transfected with mRNA in the same manner as the previous day (Day 3). Then, all media was removed from the plate, and 1 mL of N3 medium containing B18R recombinant protein at a concentration of 200 ng / mL, blasticidin at a concentration of 20 μg / mL, and puromycin at a concentration of 2 μg / mL was placed on the plate, and the plate was left standing at 37 ° C. overnight. This was repeated until Day 6.

[0087] All media was removed from the plate, and 1 mL of N3 media containing B18R recombinant protein at a concentration of 200 ng / mL was added to the plate (Day 7). Then, the cells were cultured until Day 9.

[0088] When observed under a microscope, it was confirmed that nerve cells had been induced from the iPS cells, as shown in Figure 4.

[0089] (Example 3 of the first embodiment) A plate was prepared by coating with a solubilized basement membrane preparation (Matrigel, Corning). Single-cell-dissociated iPS cells were suspended in 1 mL of human ES / iPS cell maintenance medium (mTeSR1, TEMCELL Technologies) containing 200 ng / mL of B18R recombinant protein and ROCK inhibitor, and the suspension was added to the plate to seed the cells, which were then left to stand for one day.

[0090] The medium was replaced with 1 mL of xeno-free medium (Pluriton, STEMGENT) containing B18R recombinant protein at a concentration of 200 ng / mL and ROCK inhibitor.

[0091] In addition, a 1.5 mL microcentrifuge tube A and a 1.5 mL microcentrifuge tube B were prepared.

[0092] Tube A was charged with 62.5 μL of low serum medium (Opti-MEM (registered trademark), Gibco), to which was added 1.875 μL of an mRNA introduction reagent (Lipofectamine MessengerMax (registered trademark), Invitrogen), and mixed well to prepare a first reaction solution.

[0093] Tube B was charged with 62.5 μL of low serum medium (Opti-MEM®, Gibco), and 500 ng of ASCL1-PURO mRNA, 250 ng of DLX2-T2A-BLAST mRNA, 250 ng of MYT1L mRNA, and 100 ng of GFP mRNA (Trilink) were added and mixed well to prepare the second reaction solution. MYT1L is a protein that controls differentiation into neurons.

[0094] The second reaction solution was added to the first reaction solution in tube A to form a mixed reaction solution, and then tube A was tapped lightly at room temperature for 10 minutes to form liposomes. Next, the mixed reaction solution was added to the plate and left to stand at 37°C for 6 to 8 hours. This transfected the cells with mRNA (Day 0). Next, all the medium was removed from the plate, and 1 mL of xeno-free medium (Pluriton, STEMGENT) containing B18R recombinant protein and ROCK inhibitor at a concentration of 200 ng / mL was added to the plate and left to stand at 37°C overnight.

[0095] The cells were transfected with mRNA in the same manner as the previous day (Day 1). Next, all the medium was removed from the plate, and 1 mL of xeno-free medium (Pluriton, STEMGENT) containing B18R recombinant protein at a concentration of 200 ng / mL, the antibiotic blasticidin at a concentration of 20 μg / mL, and puromycin and ROCK inhibitor at concentrations of 2 μg / mL was added to the plate, and the plate was left to stand at 37° C. overnight.

[0096] All media was removed from the plate, and 1 mL of xeno-free medium (Pluriton, STEMGENT) containing B18R recombinant protein at a concentration of 200 ng / mL and ROCK inhibitor was placed on the plate. Next, the cells were transfected with mRNA in the same manner as the previous day (Day 2). Then, all media was removed from the plate, and 1 mL of N3 medium containing B18R recombinant protein at a concentration of 200 ng / mL, blasticidin at a concentration of 20 μg / mL, and puromycin at a concentration of 2 μg / mL was placed on the plate, and the plate was left to stand overnight at 37 ° C.

[0097] All media was removed from the plate, and 1 mL of N3 medium containing B18R recombinant protein at a concentration of 200 ng / mL was placed on the plate. Next, the cells were transfected with mRNA in the same manner as the previous day (Day 3). Then, all media was removed from the plate, and 1 mL of N3 medium containing B18R recombinant protein at a concentration of 200 ng / mL, blasticidin at a concentration of 20 μg / mL, and puromycin at a concentration of 2 μg / mL was placed on the plate, and the plate was left standing at 37 ° C. overnight. This was repeated until Day 6.

[0098] All media was removed from the plate, and 1 mL of N3 media containing B18R recombinant protein at a concentration of 200 ng / mL was added to the plate (Day 7). Then, the cells were cultured until Day 21.

[0099] On Day 9, when observed under a microscope, it was confirmed that nerve cells were induced from the iPS cells as shown in FIG. 4. Furthermore, on Day 21, similarly to Example 1 of the first embodiment, the cells were fluorescently immunostained using an antibody (MAB5406, Millepore) for GAD67, which is a marker for inhibitory nerve cells, and it was confirmed that the nerve cells induced from the iPS cells expressed GAD67, which is a marker for inhibitory nerve cells, as shown in FIG. 5 and FIG. 6. Furthermore, cells into which mRNA was not introduced were efficiently killed with blasticidin and puromycin, and cells into which mRNA was introduced were selectively allowed to survive. Furthermore, it was shown that nerve cells were more efficiently introduced by introducing MYT1L mRNA into cells. Furthermore, by performing selection with both blasticidin and puromycin, the number of transformed cells that were incompletely initialized and had the ability to proliferate was reduced.

[0100] (Example 4 of the first embodiment) A plate was prepared by coating with a solubilized basement membrane preparation (Matrigel, Corning). Single-cell-dissociated iPS cells were suspended in 1 mL of human ES / iPS cell maintenance medium (mTeSR1, TEMCELL Technologies) containing 200 ng / mL of B18R recombinant protein and ROCK inhibitor, and the suspension was added to the plate to seed the cells, which were then left to stand for one day.

[0101] The medium was replaced with 1 mL of xeno-free medium (Pluriton, STEMGENT) containing B18R recombinant protein at a concentration of 200 ng / mL and ROCK inhibitor.

[0102] In addition, a 1.5 mL microcentrifuge tube A and a 1.5 mL microcentrifuge tube B were prepared.

[0103] Tube A was charged with 62.5 μL of low serum medium (Opti-MEM (registered trademark), Gibco), to which was added 1.875 μL of an mRNA introduction reagent (Lipofectamine MessengerMax (registered trademark), Invitrogen), and mixed well to prepare a first reaction solution.

[0104] Tube B contained 62.5 μL of low serum medium (Opti-MEM®, Gibco), and 500 ng of ASCL1 mRNA, 250 ng of DLX2-T2A-BLAST mRNA, 250 ng of MYT1L mRNA, and 100 ng of GFP mRNA (Trilink). The mixture was mixed well to prepare the second reaction solution.

[0105] The second reaction solution was added to the first reaction solution in tube A to form a mixed reaction solution, and then tube A was tapped lightly at room temperature for 10 minutes to form liposomes. Next, the mixed reaction solution was added to the plate and left to stand at 37°C for 6 to 8 hours. This transfected the cells with mRNA (Day 0). Next, all the medium was removed from the plate, and 1 mL of xeno-free medium (Pluriton, STEMGENT) containing B18R recombinant protein and ROCK inhibitor at a concentration of 200 ng / mL was added to the plate and left to stand at 37°C overnight.

[0106] The cells were transfected with mRNA in the same manner as the previous day (Day 1). Next, all the medium was removed from the plate, and 1 mL of xeno-free medium (Pluriton, STEMGENT) containing B18R recombinant protein at a concentration of 200 ng / mL and antibiotics blasticidin and ROCK inhibitor at concentrations of 20 ng / mL was added to the plate, and the plate was left to stand at 37°C overnight.

[0107] All media was removed from the plate, and 1 mL of xeno-free medium (Pluriton, STEMGENT) containing B18R recombinant protein at a concentration of 200 ng / mL and ROCK inhibitor was placed on the plate. Next, the cells were transfected with mRNA in the same manner as the previous day (Day 2). Then, all media was removed from the plate, and 1 mL of N3 medium containing B18R recombinant protein at a concentration of 200 ng / mL and blasticidin at a concentration of 20 ng / mL was placed on the plate, and the plate was left to stand overnight at 37 ° C.

[0108] All media was removed from the plate, and 1 mL of N3 medium containing B18R recombinant protein at a concentration of 200 ng / mL was placed on the plate. Next, the cells were transfected with mRNA in the same manner as the previous day (Day 3). Then, all media was removed from the plate, and 1 mL of N3 medium containing B18R recombinant protein at a concentration of 200 ng / mL and blasticidin at a concentration of 20 ng / mL was placed on the plate, and the plate was left standing at 37°C overnight. This was repeated until Day 6.

[0109] All media was removed from the plate, and 1 mL of N3 media containing B18R recombinant protein at a concentration of 200 ng / mL was added to the plate (Day 7). Then, the cells were cultured until Day 21.

[0110] When observed under a microscope, it was confirmed that nerve cells had been induced from the iPS cells, as shown in Figure 4.

[0111] (Example 5 of the first embodiment) A plate was prepared by coating with a solubilized basement membrane preparation (Matrigel, Corning). In addition, 2 × 10 single cells were dissolved in 1 mL of human ES / iPS cell maintenance medium (mTeSR1, TEMCELL Technologies) containing B18R recombinant protein at a concentration of 200 ng / mL and ROCK inhibitor. 5 The iPS cells were suspended, the suspension was added to a plate to seed the cells, and the plate was left to stand for one day.

[0112] The medium was replaced with 1 mL of xeno-free medium (Pluriton, STEMGENT) containing 200 ng / mL of B18R recombinant protein, ROCK inhibitor, 500 nmol / L of A83-1, a selective inhibitor of ALK5, ALK4 and ALK7, 200 ng / mL of SHH (Sonic Hedgehog), 100 nmol / L of LDN, 100 ng / mL of FGF8, 3 μmol / L of CHIR99021, a GSK-3β inhibitor, and 2 μmol / L of palmorfamine, a differentiation-promoting reagent. This medium is called Pluri-NPC medium.

[0113] In addition, a 1.5 mL microcentrifuge tube A and a 1.5 mL microcentrifuge tube B were prepared.

[0114] 62.6 μL of low serum medium (Opti-MEM (registered trademark), Gibco) was placed in tube A, and 1.875 μL of an mRNA introduction reagent (Lipofectamine MessengerMax (registered trademark), Invitrogen) was added thereto and mixed well to prepare a first reaction solution.

[0115] Tube B was charged with 62.5 μL of low serum medium (Opti-MEM®, Gibco), and 200 ng of NGN2-T2A-PURO mRNA, 200 ng of ASCL1-T2A-PURO mRNA, 200 ng of NURR1 mRNA, 200 ng of LMX1A mRNA, 200 ng of EN1 (Engrailed-1) mRNA, 200 ng of PITX3 mRNA, 200 ng of FOXA2 mRNA, and 100 ng of GFP mRNA (Trilink) were added, and the mixture was mixed well to prepare the second reaction solution.

[0116] The second reaction solution was added to the first reaction solution in tube A to form a mixed reaction solution, and then tube A was tapped gently at room temperature for 10 minutes to form liposomes. Next, the mixed reaction solution was added to the plate and left to stand at 37°C for 6 to 8 hours. This transfected the cells with mRNA (Day 0). Next, all the medium was removed from the plate, and Pluri-NPC medium was added to the plate and left to stand at 37°C overnight.

[0117] The cells were transfected with mRNA in the same manner as the previous day (Day 1). Next, all media were removed from the plate, and Pluri-NPC media containing 2 μg / mL of puromycin was added to the plate and incubated at 37° C. overnight.

[0118] 1 mL of N3 medium containing B18R recombinant protein at a concentration of 200 ng / mL, growth factor SHH at 200 ng / mL, growth factor LDN at 100 nmol / L, growth factor FGF8 at 100 ng / mL, CHIR at 3 μmol / L, and palmorfamine at 2 μmol / L was prepared. This medium is called N3-C medium. All media was removed from the plate, and Pluri-NPC medium was placed in the plate. Next, the cells were transfected with mRNA in the same manner as the previous day (Day 2). Then, all media was removed from the plate, and 1 mL of N3-C medium containing puromycin at a concentration of 2 μg / mL was placed in the plate, and the plate was left to stand overnight at 37 ° C.

[0119] All medium was removed from the plate, and fresh N3-C medium was added to the plate. Next, the cells were transfected with mRNA in the same manner as the previous day (Day 3). 1 mL of N3-C medium containing puromycin at a concentration of 2 μg / mL was added to the plate, and the plate was left standing at 37°C overnight. Similarly, the cells were transfected with mRNA on Day 4.

[0120] All the medium was removed from the plate, and 1 mL of N3-C medium without puromycin was added to the plate (Day 5).

[0121] Immunostaining of the cells was performed on Day 7. A buffer containing rabbit anti-TUJ1 antibody (Covance) at 1:1000 and sheep anti-TH antibody (Pel-Freez Biologicals) at 1:1000 was added to the plate and left to stand overnight at 4°C. Then, donkey anti-mouse IgG (H+L) secondary antibody Alexa Fluor® 555 conjugate (Thermofisher, A-21428) and donkey anti-rabbit IgG (H+L) secondary antibody Alexa Fluor® 647 conjugate (Thermofisher, A31573) were added to the plate, and the cells were observed under a microscope.

[0122] As a result, as shown in Figure 7, it was confirmed that the cells were positive for TH, a marker specific to dopamine-producing neurons, and TUJ1, a marker for neurons in general. On the other hand, GFP was hardly expressed. This indicates that the exogenous RNA was degraded and safe dopamine-producing neurons were obtained.

[0123] (Example 6 of the first embodiment) As in Example 5 of the first embodiment, 2 × 10 5 The iPS cells were seeded and left to stand for one day. The medium was then replaced with Pluri-NPC medium. As in Example 5 of the first embodiment, tube A containing the first reaction solution was prepared.

[0124] Tube B was charged with 62.5 μL of low serum medium (Opti-MEM®, Gibco), and 200 ng of ASCL1-T2A-PURO mRNA, 200 ng of NURR1 mRNA, 200 ng of LMX1A mRNA, 200 ng of EN1 mRNA, 200 ng of PITX3 mRNA, 200 ng of FOXA2 mRNA, and 100 ng of GFP mRNA (Trilink) were added and mixed well to prepare the second reaction solution.

[0125] The second reaction solution was added to the first reaction solution in tube A to form a mixed reaction solution, and then tube A was tapped gently at room temperature for 10 minutes to form liposomes. Next, the mixed reaction solution was added to the plate and left to stand at 37°C for 6 to 8 hours. This transfected the cells with mRNA (Day 0). Next, all the medium was removed from the plate, and Pluri-NPC medium was added to the plate and left to stand at 37°C overnight.

[0126] The cells were transfected with mRNA in the same manner as the previous day (Day 1). Next, all media were removed from the plate, and Pluri-NPC media containing 2 μg / mL of puromycin was added to the plate and incubated at 37° C. overnight.

[0127] All media was removed from the plate, and Pluri-NPC medium was added to the plate. Next, the cells were transfected with mRNA in the same manner as the previous day (Day 2). Then, all media was removed from the plate, and 1 mL of N3-C medium containing puromycin at a concentration of 2 μg / mL was added to the plate, and the plate was left standing at 37 °C overnight.

[0128] All medium was removed from the plate, and fresh N3-C medium was added to the plate. Next, the cells were transfected with mRNA in the same manner as the previous day (Day 3). 1 mL of N3-C medium containing puromycin at a concentration of 2 μg / mL was added to the plate, and the plate was left standing at 37°C overnight. Similarly, the cells were transfected with mRNA on Day 4.

[0129] All the medium was removed from the plate, and 1 mL of N3-C medium without puromycin was added to the plate (Day 5).

[0130] As in Example 5 of the first embodiment, the cells were immunostained on Day 7. As a result, as shown in Figure 8, it was confirmed that TH and TUJ1 were positive in the cells. On the other hand, GFP was hardly expressed.

[0131] (Example 7 of the first embodiment) The number of iPS cells was 4 × 10 5 The iPS cells were seeded on the plate and left to stand for one day in the same manner as in Example 5 of the first embodiment, except that the number of cells was 1. The medium was then replaced with Pluri-NPC medium. Tube A containing the first reaction solution was prepared in the same manner as in Example 5 of the first embodiment.

[0132] Tube B was charged with 62.5 μL of low serum medium (Opti-MEM®, Gibco), and 500 ng of ASCL1-T2A-PURO mRNA, 200 ng of NURR1 mRNA, 200 ng of LMX1A mRNA, 200 ng of EN1 mRNA, 200 ng of PITX3 mRNA, 200 ng of FOXA2 mRNA, and 100 ng of GFP mRNA (Trilink) were added and mixed well to prepare the second reaction solution.

[0133] The second reaction solution was added to the first reaction solution in tube A to form a mixed reaction solution, and then tube A was tapped gently at room temperature for 10 minutes to form liposomes. Next, the mixed reaction solution was added to the plate and left to stand at 37°C for 6 to 8 hours. This transfected the cells with mRNA (Day 0). Next, all the medium was removed from the plate, and Pluri-NPC medium was added to the plate and left to stand at 37°C overnight.

[0134] All media was removed from the plate, and the cells were transfected with mRNA in the same manner as the previous day (Day 1). Next, all media was removed from the plate, and Pluri-NPC medium containing 2 μg / mL of puromycin was added to the plate, and the plate was left standing at 37°C overnight.

[0135] All media was removed from the plate, and Pluri-NPC medium was added to the plate. Next, the cells were transfected with mRNA in the same manner as the previous day (Day 2). Then, all media was removed from the plate, and 1 mL of N3-C medium containing puromycin at a concentration of 2 μg / mL was added to the plate, and the plate was left standing at 37 °C overnight.

[0136] All media was removed from the plate, and fresh N3-C media was added to the plate. Next, the cells were transfected with mRNA in the same manner as the previous day (Day 3). 1 mL of N3-C media containing puromycin at a concentration of 2 μg / mL was added to the plate, and the plate was left standing at 37°C overnight. Similarly, the cells were transfected with mRNA on Days 4 and 5.

[0137] All the medium was removed from the plate, and 1 mL of N3-C medium without puromycin was added to the plate (Day 6).

[0138] As in Example 5 of the first embodiment, the cells were immunostained on Day 7. As a result, as shown in Figure 9, it was confirmed that the cells were positive for TH and TUJ1. On the other hand, GFP was hardly expressed.

[0139] (Example 8 of the first embodiment) The number of iPS cells was 2 × 10 5 pcs or 4 x 10 5 The iPS cells were seeded on the plate and left to stand for one day in the same manner as in Example 5 of the first embodiment, except that the number of cells was 1. The medium was then replaced with Pluri-NPC medium. Tube A containing the first reaction solution was prepared in the same manner as in Example 5 of the first embodiment.

[0140] Tube B contained 62.5 μL of low serum medium (Opti-MEM (registered trademark), Gibco), and 500 ng of NGN2-T2A-PURO mRNA and 100 ng of GFP mRNA (Trilink) were added and mixed well to prepare the second reaction solution.

[0141] The second reaction solution was added to the first reaction solution in tube A to form a mixed reaction solution, and then tube A was tapped gently at room temperature for 10 minutes to form liposomes. Next, the mixed reaction solution was added to the plate and left to stand at 37°C for 6 to 8 hours. This transfected the cells with mRNA (Day 0). Next, all the medium was removed from the plate, and Pluri-NPC medium was added to the plate and left to stand at 37°C overnight.

[0142] The cells were transfected with mRNA in the same manner as the previous day (Day 1). Next, all media were removed from the plate, and Pluri-NPC media containing 2 μg / mL of puromycin was added to the plate and incubated at 37° C. overnight.

[0143] All media was removed from the plate, and Pluri-NPC medium was added to the plate. Next, the cells were transfected with 200ng of NGN2-T2A-PURO mRNA, 200ng of ASCL1-T2A-PURO mRNA, 200ng of NURR1 mRNA, 200ng of LMX1A mRNA, 200ng of EN1 mRNA, 200ng of PITX3 mRNA, 200ng of FOXA2, and 100ng of GFP mRNA (Day 2). Then, all media was removed from the plate, and 1mL of N3-C medium containing puromycin at a concentration of 2μg / mL was added to the plate, and the plate was left to stand at 37℃ overnight.

[0144] All medium was removed from the plate, and fresh N3-C medium was added to the plate. Next, the cells were transfected with mRNA in the same manner as the previous day (Day 3). 1 mL of N3-C medium containing puromycin at a concentration of 2 μg / mL was added to the plate, and the plate was left standing at 37°C overnight. Similarly, the cells were transfected with mRNA on Day 4.

[0145] All the medium was removed from the plate, and fresh N3-C medium was added to the plate. Next, the cells were transfected with mRNA in the same manner as the previous day (Day 5). 1 mL of N3-C medium containing puromycin at a concentration of 2 μg / mL and B18R recombinant protein (eBioscience) was added to the plate, and the plate was left standing at 37° C. overnight.

[0146] All the medium was removed from the plate, and 1 mL of N3-C medium without puromycin was added to the plate (Day 6).

[0147] Immunostaining of the cells was performed on Day 7 in the same manner as in Example 5 of the first embodiment. As a result, as shown in FIG. 10, it was confirmed that the cells were positive for TH and TUJ1. On the other hand, GFP was hardly expressed. This indicates that the exogenous RNA was degraded and safe dopamine-producing nerve cells were obtained.

[0148] (Comparative Example of the First Embodiment) Pluri-NPC medium and N3-C medium are media used when inducing dopamine-producing neurons from iPS cells using only hormones. Here, the medium was replaced and the cells were cultured in the same manner as in Examples 5 to 8 of the first embodiment, except that the inducer RNA was not introduced into the iPS cells, and immunostaining of the cells was performed on Day 7. As a result, as shown in FIG. 11, it was confirmed that the cells were negative for TH and TUJ1. Furthermore, the morphology was not that of neurons.

[0149] Second embodiment The method for producing neural cells according to the second embodiment includes preparing cells, introducing reprogramming factor RNA into the cells, and introducing inducer RNA into the cells introduced with the reprogramming factor RNA to differentiate them into neural cells.

[0150] In the method for producing neural cells according to the second embodiment, reprogramming factor RNA may be introduced into cells in the same culture vessel, and inducer RNA may be introduced into the cells into which the reprogramming factor RNA has been introduced. For example, after the reprogramming factor RNA is introduced into the cells, the inducer RNA may be introduced into the cells into which the reprogramming factor RNA has been introduced without detaching the cells into which the reprogramming factor RNA has been introduced from the culture vessel. Alternatively, the inducer RNA may be introduced into the cells on the day after the reprogramming factor RNA is introduced into the cells.

[0151] Alternatively, in the method for producing neural cells according to the second embodiment, after introducing reprogramming factor RNA into cells, the cells into which the reprogramming factor RNA has been introduced may be detached from the culture vessel, and the cells into which the reprogramming factor RNA has been introduced may be seeded into another culture vessel, after which an inducer RNA may be introduced into the cells into which the reprogramming factor RNA has been introduced.

[0152] Examples of cells into which the reprogramming factors are introduced include differentiated cells (somatic cells) such as fibroblasts, blood cells, dental pulp stem cells, keratinocytes, hair papilla cells, oral epithelial cells, and somatic stem progenitor cells.

[0153] The blood cells are separated from blood, such as, but not limited to, peripheral blood and umbilical cord blood. The blood may be collected from an adult or a minor. During collection, an anticoagulant such as ethylenediaminetetraacetic acid (EDTA), heparin, and biologic standard blood preservation solution A (ACD-A) solution is used.

[0154] Blood cells are nucleated cells such as monocytes, neutrophils, eosinophils, basophils, and lymphocytes, and do not include red blood cells, granulocytes, and platelets. Blood cells may be, for example, endothelial progenitor cells, blood stem / progenitor cells, T cells, or B cells. T cells are, for example, αβ T cells.

[0155] Mononuclear cells are separated from blood using a blood cell separation medium and a centrifuge, etc. When Ficoll (GE Healthcare) is used as a blood cell separation medium, the method for separating mononuclear cells is as follows.

[0156] Since the accuracy of mononuclear cell separation tends to deteriorate at low temperatures, set the centrifuge to 4°C to 42°C, preferably 18°C. 10 μL to 50 mL of blood is collected from an adult or minor, and a chelating agent containing EDTA is added to the blood to prevent blood clotting, and the blood is gently mixed. In addition, 5 mL of human lymphocyte separation medium (Ficoll-Paque PREMIUM, GE Healthcare Japan) is dispensed into two 15 mL tubes. 5 mL of blood is diluted with 5 mL of PBS, and 5 mL is layered on top of the human lymphocyte separation medium in the tube. At this time, the diluted blood is slowly added onto the medium by running it down the tube wall so as not to disturb the interface.

[0157] The solution in the tube is centrifuged at 10×g to 1000×g, preferably 400×g, at 4°C to 42°C, preferably 18°C, for 5 minutes to 2 hours, preferably 30 minutes. After centrifugation, a cloudy white intermediate layer appears in the tube. This cloudy white intermediate layer contains mononuclear cells. Slowly collect the cloudy white intermediate layer in the tube with a pipette and transfer it to a new 15 mL tube. At this time, be careful not to suck up the bottom layer. About 1 mL of the cloudy white intermediate layer can be collected from one tube. Transfer the intermediate layers from two tubes together into one tube.

[0158] 1 mL to 48 mL, preferably 12 mL, of PBS is added to the collected mononuclear cells, and the solution is further centrifuged at 10×g to 1000×g, preferably 200×g, at 4° C. to 42° C., preferably 18° C., for 1 to 60 minutes, preferably 10 minutes. The supernatant of the solution is then removed by aspirating using an aspirator, and 1 mL to 12 mL, preferably 3 mL, of a chemically defined serum-free hematopoietic cell medium (X-VIVO® 10, Lonza) is added and suspended to obtain a mononuclear cell suspension. 10 μL of the mononuclear cell suspension is stained with trypan blue and counted using a hemocytometer.

[0159] When Vacutainer (registered trademark, BD) is used as a blood collection tube, the method for isolating mononuclear cells is as follows.

[0160] Since the accuracy of mononuclear cell separation tends to deteriorate at low temperatures, the centrifuge is set to 4°C to 42°C, preferably 18°C. 8 mL of blood is collected from an adult or minor using a blood collection tube (Vacutainer (registered trademark, BD) and mixed with an anticoagulant by inversion. Then, the balance is adjusted and the solution is centrifuged in a swing rotor at 4°C to 42°C, preferably 18°C, 100 x g to 3000 x g, preferably 1500 x g to 1800 x g for 1 minute to 60 minutes, preferably 20 minutes. After centrifugation, the upper layer, which is the plasma layer, is removed and the mononuclear cell layer and blood cells adhering to the gel are suspended by pipetting to obtain a suspension. The resulting suspension is transferred to another 15 mL tube.

[0161] Add 1 mL to 14 mL, preferably 12 mL, of PBS to the suspension in the 15 mL tube, and centrifuge the suspension at 4°C to 42°C, preferably 18°C, at 100 x g to 3000 x g, preferably 200 x g, for 1 to 60 minutes, preferably 5 minutes. After centrifugation, remove the supernatant with an aspirator. Also, dilute the hemolytic agent (PharmLyse (registered trademark), 10x concentration, BD) to 1x concentration with sterile water. Loosen the pellet in the 15 mL tube by tapping, and add 1 mL to 14 mL, preferably 1 mL, of hemolytic agent. Then, leave the solution at room temperature, protected from light, for 1 to 60 minutes, preferably 1 minute.

[0162] Next, 1 mL to 14 mL, preferably 12 mL, of PBS is added to a 15 mL tube, and the tube is centrifuged at 100×g to 3000×g, preferably 200×g, for 1 to 60 minutes, for 5 minutes, at 4° C. to 42° C., preferably at room temperature. After centrifugation, the supernatant is removed with an aspirator, and 1 mL to 15 mL, preferably 3 mL, of a chemically defined serum-free hematopoietic cell medium (X-VIVO® 10, Lonza) is added and suspended to obtain a mononuclear cell suspension. 10 μL of the mononuclear cell suspension is stained with trypan blue and counted using a hemocytometer.

[0163] The method for separating mononuclear cells from blood is not limited to the above-mentioned method, and for example, mononuclear cells may be separated from blood using a dialysis membrane. Filters such as PureCell Select System (registered trademark, PALL) for concentrating mononuclear cells from whole blood, a purifier for removing blood cells (CellSorba E, registered trademark, Asahi Kasei), and a leukocyte removal filter for platelet preparations (SepaCell PL, registered trademark, PLX-5B-SCD, Asahi Kasei) can also be used.

[0164] Mononuclear cells may be separated using an erythrocyte separator capable of separating nucleated cells by gravity sedimentation or centrifugation of erythrocytes. Examples of erythrocyte separators include HetaSep (registered trademark, STEMCELL Technologies) and HES40 (NIPRO).

[0165] Furthermore, as mononuclear cells, CTL-UP1 available from Cellular Technology Limited, PBMC-001 available from Sanguine Biosciences, and the like may be used.

[0166] Alternatively, blood cells that have been cryopreserved using a cell cryopreservation solution such as Cell Banker 1, Stem Cell Banker GMP Grade, and Stem Cell Banker DMSO-Free GMP Grade (Zenoac) may be thawed and used.

[0167] When thawing mononuclear cells, first, 1 mL to 15 mL, preferably 8 mL, of chemically defined serum-free hematopoietic cell medium (X-VIVO® 10, Lonza) is placed in a 15 mL tube, and the tube containing the frozen mononuclear cells is placed in a warm bath at 4°C to 42°C, preferably 37°C, to start thawing the mononuclear cells. Thereafter, with some ice remaining, the tube containing the mononuclear cells is removed from the warm bath, and the mononuclear cells are transferred to a tube containing chemically defined serum-free hematopoietic cell medium. 10 μL of the mononuclear cell suspension is stained with trypan blue and counted using a hemocytometer.

[0168] Blood cells may be separated based on cell surface markers. Blood stem and progenitor cells are positive for CD34. T cells are positive for any of CD3, CD4, and CD8. B cells are positive for any of CD10, CD19, and CD20. Blood stem and progenitor cells, T cells, or B cells are separated from blood cells using, for example, an automated magnetic cell separator and immunomagnetic beads. Alternatively, pre-separated mononuclear cells may be prepared. However, blood cells that have not been separated based on cell surface markers may also be used.

[0169] CD34 positive cells are stem / progenitor cells and tend to be easily reprogrammed. In addition, when iPS cells are generated using CD3 positive T cells, the T cell-derived iPS cells retain the TCR recombination type, and therefore tend to be efficiently induced to differentiate into T cells.

[0170] The method for isolating CD34 positive cells is as follows.

[0171] Prepare blood stem and progenitor cell medium by adding 10 μL of IL-6 (100 μg / mL), 10 μL of SCF (300 μg / mL), 10 μL of TPO (300 μg / mL), 10 μL of FLT3 ligand (300 μg / mL), and 10 μL of IL-3 (10 μg / mL) to 10 mL of serum-free medium (StemSpan H3000, STEMCELL Technologies).

[0172] Add 1 mL to 6 mL, preferably 2 mL, of hemocyte medium to one well of a 6-well plate. Also add 1 mL to 6 mL, preferably 2 mL, of PBS to each of the other 5 wells to prevent evaporation of the medium. Then, place the 6-well plate in a 4 °C to 42 °C, preferably 37 °C, incubator to warm it up.

[0173] A column buffer is prepared by adding 10 μL to 1 mL, preferably 80 μL, of EDTA (500 mmol / L) and 10 μL to 1 mL, preferably 200 μL, of FBS to 20 mL of PBS. 4 From 1×10 9 pcs, preferably 2 x 10 7 The mononuclear cell suspension containing 10 mononuclear cells is dispensed into a 15 mL tube, and the mononuclear cell suspension is centrifuged at 100×g to 3000×g, preferably 300×g, for 10 minutes at 4° C. to 42° C., preferably 4° C. After centrifugation, the supernatant is removed, and the mononuclear cells are suspended in 100 μL to 1 mL, preferably 300 μL, of the column buffer.

[0174] Add 10 μL to 1 mL, preferably 100 μL, of FcR blocking reagent (Miltenyi Biotec) and 10 μL to 1 mL, preferably 100 μL, of CD34 microbead kit (Miltenyi Biotec) to the mononuclear cell suspension in a 15 mL tube. The FcR blocking reagent is used to enhance the specificity of microbead labeling. Then, mix the mononuclear cell suspension and leave it at 4°C to 42°C, preferably 4°C, for 1 minute to 2 hours, preferably 30 minutes.

[0175] Next, add 1 mL to 15 mL, preferably 10 mL, of column buffer to the mononuclear cell suspension in the 15 mL tube to dilute it, and centrifuge it at 100×g to 1000×g, preferably 300×g, for 1 minute to 2 hours, preferably for 10 minutes, at 4° C. to 42° C., preferably 4° C. After centrifugation, remove the supernatant in the 15 mL tube with an aspirator, and add 10 μL to 10 mL, preferably 500 μL, of column buffer to resuspend it.

[0176] Attach a column for the automatic magnetic cell separator (MS column, Miltenyi Biotec) to the automatic magnetic cell separator (MiniMACS Separation Unit, Miltenyi Biotec), and wash the column with 10 μL to 10 mL, preferably 500 μL of column buffer. Next, place the mononuclear cells in the column. Furthermore, add 10 μL to 10 mL, preferably 500 μL of column buffer to the column and wash the column 1 to 10 times, preferably 3 times. Then, remove the column from the automatic magnetic cell separator and place it in a 15 mL tube. Next, add 10 μL to 10 mL, preferably 1000 μL of column buffer to the column, and quickly push the syringe to let the CD34 positive cells flow out into the 15 mL tube.

[0177] 10 μL of the CD34 positive cell suspension is stained with trypan blue, and the number of cells is counted using a hemocytometer. The CD34 positive cell suspension in the 15 mL tube is centrifuged at 4° C. to 42° C., preferably at 4° C., 100×g to 1000×g, preferably at 300×g, for 1 to 2 hours, preferably for 10 minutes. After centrifugation, the supernatant is removed with an aspirator. Furthermore, the CD34 positive cells are resuspended in a warmed blood cell medium, and the CD34 positive cells are plated on a culture plate. Then, the CD34 positive cells are cultured for 6 days at 4° C. to 42° C., preferably at 37° C., and 1% to 20%, preferably 5% CO2. During this time, the medium does not need to be replaced.

[0178] The method for isolating cells using a marker other than CD34 is similar to the method for isolating CD34-positive cells.

[0179] Examples of the reprogramming factor RNA introduced into the cells include OCT3 / 4 mRNA, SOX2 mRNA, KLF4 mRNA, and c-MYC mRNA. As the reprogramming factor RNA, M3O, which is an improved version of OCT3 / 4, may be used. The reprogramming factor RNA may further include mRNA of at least one factor selected from the group consisting of LIN28A, LIN28B, GLIS1, p53-dominant negative, p53-P275S, L-MYC, NANOG, DPPA2, DPPA4, DPPA5, ZIC3, BCL-2, E-RAS, TPT1, SALL2, NAC1, DAX1, TERT, ZNF206, FOXD3, REX1, UTF1, KLF2, KLF5, ESRRB, miR-291-3p, miR-294, miR-295, NR5A1, NR5A2, TBX3, MBD3sh, TH2A, and TH2B. These mRNAs are available from TriLink.

[0180] The mRNA was synthesized from pseudouridine (Ψ), 5-methyluridine (5meU), N1-methylpseudouridine (me1Ψ), 5-methoxyuridine (5moU), 5-hydroxymethyluridine (5hmU), 5-formyluridine (5fU), 5-carboxymethylesteruridine (5camU), thienoguanosine (thG), N4-methylcytidine (me4C), 5-methylcytidine (m5C), 5-methyoxycytidine (5moC), 5-hydroxymethylesteruridine (5camU), 5-methylcytidine (5meC), 5-methyl ... It may be modified with at least one selected from the group consisting of 5-methylcytidine (5hmC), 5-hydroxycytidine (5hoC), 5-formycytidine (5fC), 5-carboxycytidine (5caC), N6-methyl-2-aminoadenosine (m6DAP), diaminopurine (DAP), 5-methyluridine (m5U), 2'-O-methyluridine (Um or m2'-OU), 2-thiouridine (s2U), and N6-methyladenosine (m6A).

[0181] The mRNA may be polyadenylated.

[0182] The mRNA may be prepared by polyadenylation of in vitro transcribed (IVT) RNA. The mRNA may be polyadenylated during IVT by using a DNA template encoding a poly(A) tail. The mRNA may be capped. To maximize the efficiency of expression in cells, it is preferable that the majority of mRNA molecules contain a cap. The mRNA may have a 5'cap[m7G(5')ppp(5')G] structure. This sequence stabilizes the mRNA and promotes transcription. From mRNAs that have a 5'triphosphate, the 5'triphosphate may be removed by dephosphorylation treatment. The mRNA may have [3'O-Me-m7G(5')ppp(5')G] as an Anti-Reverse Cap Analog (ARCA). ARCA is a sequence inserted before the transcription start site, doubling the efficiency of transcribed mRNA. The mRNA may have a PolyA tail.

[0183] Furthermore, the mRNA may be a replicative RNA having a self-replicating ability. Replicative RNA is an RNA having a self-replicating ability, and unlike normal RNA, it also has the ability to express proteins necessary for RNA replication. Replicative RNA is derived from the Venezuelan equine encephalitis (VEE) virus, which is a type of alphavirus. When replicative RNA is transfected into cells, it is possible to cause the cells to express RNA that continues to produce reprogramming factors, making it possible to omit the need to introduce reprogramming factor RNA into cells multiple times.

[0184] The replicative RNA sequence may include a sequence obtained from an alphavirus selected from the group consisting of alphavirus replicon RNA, Eastern equine encephalitis virus (EEE), Venezuelan equine encephalitis virus (VEE), Everglades virus, Mucambo virus, Pixuna virus, and Western equine encephalitis virus (WEE).

[0185] The replicative RNA may also comprise sequences derived from an alphavirus selected from the group consisting of Sindbis virus, Semliki Forest virus, Middelburg virus, Chikungunya virus, O'nyong-nyong virus, Ross River virus, Barmah Forest virus, Getah virus, Sagiyama virus, Bebaru virus, Mayaro virus, Una virus, Aura virus, Whataroa virus, Babanki virus, Kyzylagach virus, Highlands J virus, Fort Morgan virus, Ndumu virus, and Buggy Creek virus.

[0186] The replicative RNA contains, for example, from 5' to 3', (VEE RNA replicase)-(promoter)-(RF1)-(self-cleaving peptide)-(RF2)-(self-cleaving peptide)-(RF3)-(IRES or core promoter)-(RF4)-(IRES or any promoter)-(optional selectable marker)-(VEE 3'UTR and polyA tail)-(optional selectable marker)-promoter. The above RF1-4 are factors that induce dedifferentiation of cells into pluripotent cells. The above RF2-3, RF3-4, and RF4 are optional. The RF1-4 may be selected from the group consisting of OCT3 / 4, KLF4, SOX-2, c-MYC, LIN28A, LIN28B, GLIS1, FOXH1, p53-dominant negative, p53-P275S, L-MYC, NANOG, DPPA2, DPPA4, DPPA5, ZIC3, BCL-2, E-RAS, TPT1, SALL2, NAC1, DAX1, TERT, ZNF206, FOXD3, REX1, UTF1, KLF2, KLF5, ESRRB, miR-291-3p, miR-294, miR-295, NR5A1, NR5A2, TBX3, MBD3sh, TH2A, and TH2B.

[0187] As a medium for culturing cells into which reprogramming factor RNA is introduced, for example, a stem cell medium such as human ES / iPS medium such as PluriQ (MTI-GlobalStem) and Primate ES Cell Medium (ReproCELL) can be used.

[0188] However, the stem cell medium is not limited thereto, and various stem cell media can be used. For example, Primate ES Cell Medium, Reprostem, ReproFF, ReproFF2, ReproXF (Reprocell), mTeSR1, TeSR2, TeSRE8, ReproTeSR (STEMCELL Technologies), PluriSTEM (registered trademark) Human ES / iPS Medium (Merck), NutriStem (registered trademark) XF / FF Culture Medium for Human iPS and ES Cells, Pluriton reprogramming medium (Stemgent), PluriSTEM (registered trademark), Stemfit AK02N, Stemfit AK03 (Ajinomoto), ESC-Sure (registered trademark) serum and feeder free medium for hESC / iPS (Applied StemCell), and L7 (registered trademark) hPSC Culture System (LONZA) may be used. The stem cell medium is placed in, for example, a dish, a well, or a tube.

[0189] The reprogramming factor RNA is introduced into cells using an RNA transfection reagent, such as mRNA-In (registered trademark, Molecular Transfer, Inc.).

[0190] Alternatively, for transfection of reprogramming factor RNA, for example, Lipofectamine Messenger Max (registered trademark) is used as a lipofection reagent. In addition, as a lipofection reagent, Lipofectamine (registered trademark) RNAiMAX (Thermo Fisher Scientific), Lipofectamin (registered trademark) 2000, Lipofectamin (registered trademark) 3000, NeonTransfection System (Thermo Fisher scientific), Stemfect RNA transfection reagent (Stemfect), NextFect (registered trademark) RNA Transfection Reagent (BiooSientific), Amaxa (registered trademark) Human T cell Nucleofector (registered trademark) kit (Lonza, VAPA-1002), Amaxa (registered trademark) Human CD34 cell Nucleofector (registered trademark) kit (Lonza, VAPA-1003), and ReproRNA (registered trademark) transfection reagent (STEMCELL Technologies) and the like may be used.

[0191] The introduction of the reprogramming factor RNA into the cell may be carried out multiple times. The introduction of the reprogramming factor RNA into the cell may be carried out, for example, once every two days, or once a day, for 5 to 15 days, for 7 to 13 days, or for 10 days. However, when the mRNA is a replicative RNA, the introduction of the reprogramming factor RNA into the cell may be carried out once.

[0192] Thereafter, by the same method as in the first embodiment, an inducer RNA is introduced into the cells into which the reprogramming factor RNA has been introduced.

[0193] According to the method of the second embodiment, it is possible to produce neural cells from differentiated cells in a short period of time.

[0194] (Example 1 of the second embodiment) Add 0.5 μg / cm of iMatrix-511 (nippi) to each well of the well plate in 1.5 mL of PBS. 2 The well plate was then placed in an incubator at 37°C for at least 1 hour. Next, the basement membrane matrix was removed from each well of the well plate, and approximately 1 x 10 fibroblasts suspended in 10% FBS medium were placed in each well of the well plate. 5 The fibroblasts were then plated and cultured as adherent cells.

[0195] A master mix of reprogramming factor mRNA shown in FIG. 12 was prepared. In addition, the medium in each well of the well plate was replaced with 2 mL of PluriQ (registered trademark, MTI-GlobalStem). In addition, tubes A and B containing the contents shown in FIG. 13 were prepared. Next, the contents of tubes A and B were mixed to prepare a mixture of mRNA-In (registered trademark, Molecular Transfer, Inc.), an RNA transfection reagent, and the mRNA master mix, and the mixture was left to stand for 10 minutes. Thereafter, the mixture of RNA-In and the mRNA master mix was added to each well, and the well plate was shaken to diffuse the mixture of RNA-In and the mRNA master mix in the medium. Next, the fibroblasts were incubated overnight at 37°C and 5% CO2 to transfect the reprogramming factor RNA into the cells (Day 0).

[0196] From the next day onwards, the cells were transfected with the reprogramming factor RNA in the same manner as above for 9 consecutive days, so that the cells were transfected with the reprogramming factor RNA a total of 10 times.

[0197] A transfection medium was prepared by mixing 1.25 mL of xeno-free medium (Pluriton, STEMGENT), 0.5 μL of Pluriton Supplement (STEMGENT), and 2 μL of a solution containing B18R recombinant protein at a concentration of 100 ng / μL (eBioscience). Before transfection of the inducer RNA, the medium in each well was replaced with the transfection medium, and the cells transfected with the reprogramming factor RNA were cultured at 37°C for 2 hours.

[0198] NGN2-T2A-PURO mRNA (Trilink) and green fluorescent protein (GFP) mRNA (Trilink) were prepared as inducer RNA. The mRNA was capped with Anti-Reverse Cap Analog (ARCA), polyadenylated, and substituted with 5-methylcytidine and pseudouridine. The mRNA was purified with a silica membrane and mixed with an mRNA introduction reagent (Lipofectamine MessengerMax (registered trademark), Invitrogen) in a solution containing 1 mmol / L sodium citrate at pH 6. Furthermore, 1.5 mL microcentrifuge tubes A and 1.5 mL microcentrifuge tubes B were prepared in the same number as the number of wells.

[0199] 62.5 μL of low serum medium (Opti-MEM®, Gibco) was placed in tube A, and 1.875 μL of mRNA introduction reagent (Lipofectamine MessengerMax®, Invitrogen) was added thereto and mixed well to prepare the first reaction solution. Then, tube A was gently tapped at room temperature for 10 minutes to mix the first reaction solution.

[0200] Tube B contained 62.5 μL of low serum medium (Opti-MEM®, Gibco), to which 500 ng of NGN2-T2A-PURO mRNA (Trilink) and 100 ng of GFP mRNA (Trilink) were added, and the mixture was mixed well to prepare the second reaction solution.

[0201] The second reaction solution was added to the first reaction solution in tube A to form a mixed reaction solution, and then tube A was tapped lightly at room temperature for 5 minutes to form liposomes. Next, the mixed reaction solution was added to each well and left to stand at 37°C for 6 to 8 hours. As a result, 500ng of NGN2mRNA and 100ng of GFPmRNA were added to each well, and inducer RNA was introduced into the cells transfected with the reprogramming factor RNA (Day 10). Note that the day when the inducer RNA was first transfected into the cells was the day after the day when the reprogramming factor RNA was last transfected into the cells. Then, all the medium was removed from each well, and xeno-free medium (Pluriton, STEMGENT) containing B18R recombinant protein at a concentration of 200ng / mL was placed in each well and left to stand overnight at 37°C.

[0202] All medium was removed from each well, and xeno-free medium (Pluriton, STEMGENT) containing B18R recombinant protein at a concentration of 200ng / mL was added to each well. Next, the cells were transfected with inducer mRNA in the same manner as the previous day (Day 11). Then, all medium was removed from the plate, and xeno-free medium (Pluriton, STEMGENT) containing B18R recombinant protein at a concentration of 200ng / mL and puromycin at a concentration of 2μg / mL was added to each well, and the plate was left to stand overnight at 37℃.

[0203] All media was removed from each well, and xeno-free media (Pluriton, STEMGENT) containing B18R recombinant protein at a concentration of 200ng / mL was added to each well. Next, the cells were transfected with inducer mRNA in the same manner as the previous day (Day 12). Then, all media was removed from the plate, and N3 media containing B18R recombinant protein at a concentration of 200ng / mL and puromycin at a concentration of 2μg / mL was added to each well, and the plate was left to stand overnight at 37℃.

[0204] All media was removed from each well, and N3 media containing B18R recombinant protein at a concentration of 200ng / mL was added to each well. Next, the cells were transfected with inducer mRNA in the same manner as the previous day (Day 13). Then, all media was removed from the plate, and N3 media containing B18R recombinant protein at a concentration of 200ng / mL and puromycin at a concentration of 2μg / mL was added to each well, and the plate was left standing overnight at 37℃.

[0205] All media were removed from the plate, and N3 media containing B18R recombinant protein at a concentration of 200 ng / mL and puromycin at a concentration of 2 μg / mL was added to each well, followed by incubation at 37° C. overnight (Day 14).

[0206] All media were removed from the plate, and N3 medium containing B18R recombinant protein at a concentration of 200 ng / mL but not containing puromycin was added to each well, and the cells were cultured at 37°C until Day 20.

[0207] After that, the medium was removed from the plate, and the cells were washed with PBS. Next, 4% PFA was added to the plate, and the cells were fixed by reacting for 15 minutes at 4°C. Furthermore, after washing the cells twice with PBS, the primary antibody was diluted in PBS medium containing 5% CCS and 0.1% Triton and added to the plate. The primary antibodies used were a mouse monoclonal antibody (Sigma) against MAP2, a marker for neurons, and a rabbit polyclonal antibody (Synaptic Systems) against vGLUT, a marker for excitatory neurons.

[0208] After one hour of reaction at room temperature, PBS was added to the plate, and the plate was thoroughly mixed with PBS, which was then discarded. PBS was added again, discarded, and a solution containing fluorescently labeled donkey anti-mouse IgG (H+L) secondary antibody (Alexa Fluor, registered trademark, 555, conjugate, ThermoFisher) and rabbit anti-mouse IgG (H+L) secondary antibody (Alexa Fluor, registered trademark, 647, conjugate, ThermoFisher) was added to the plate and reacted at room temperature for 30 minutes. The cells were then washed twice with PBS and observed under a fluorescent microscope. As a result, as shown in FIG. 14, it was confirmed that the induced neurons expressed MAP2, a marker for neurons, and vGLUT, a marker for excitatory neurons.

[0209] (Example 2 of the second embodiment) Fibroblasts were prepared in the same manner as in Example 1 of the second embodiment, and the reprogramming factor RNA was transfected into the cells a total of 10 times. Then, the cells were cultured in PluriQ (registered trademark, MTI-GlobalStem) containing TGF-β at a concentration of 2 ng / mL for 5 days.

[0210] The cells were detached from the well plate, suspended in a xeno-free medium (Pluriton, STEMGENT), and seeded into each well of a new well plate. The cells were then transfected with the inducer RNA in the same manner as in Example 1 of the second embodiment.

[0211] As a result, it was confirmed that nerve cells were induced, as shown in Fig. 15. Furthermore, when the cells were immunostained in the same manner as in Example 1 of the second embodiment, it was confirmed that the induced nerve cells expressed MAP2, which is a nerve cell marker, and vGLUT, which is a marker for excitatory nerve cells, as shown in Fig. 16.

Claims

1. Providing stem cells, seeding the stem cells, including single cells, in a medium; introducing an inducer RNA into the stem cells to differentiate them into dopamine-producing nerve cells; Including, The inducer comprises EN1 mRNA, ASCL1 mRNA, NURR1 mRNA, LMX1A mRNA, PITX3 mRNA, and FOXA2 mRNA; A method for producing dopamine-producing neurons.

2. The method for producing dopamine-producing neurons according to claim 1 , wherein the dopamine-producing neurons express TH.

3. the inducer RNA comprises an mRNA corresponding to a drug resistance gene; The drug is at least one selected from blasticidin, puromycin, hygromycin, neomycin, G418, and zeocin; A method for producing dopamine-producing neurons according to claim 1 or 2.

4. The method for producing dopamine-producing neurons according to claim 1 , wherein the medium contains B18R protein.

5. Repeating the introduction of the inducer RNA into the stem cells multiple times; In the first stage of introducing the inducer RNA into the stem cell, the medium contains an ALK inhibitor; In the latter stage of introducing the inducer RNA into the stem cells, the medium does not contain an ALK inhibitor. A method for producing dopamine-producing neurons according to any one of claims 1 to 4.

6. A method for producing dopamine-producing neurons described in any one of claims 1 to 5, wherein the inducer further contains NGN2 mRNA.

Images