JP7435942B2 - Method for differentiating neural stem cells or neural progenitor cells into dopaminergic neurons - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Act Molecular Therapy, September 2017; 25 (9): 2028-2037

The present invention relates to a method for differentiating neural stem cells or neural progenitor cells into dopaminergic neurons, and more specifically, the present invention relates to a method for differentiating neural stem cells or neural progenitor cells into dopaminergic neurons, and more specifically, to inject mRNA of a dopaminergic neuron-inducing transcription factor into neural stem cells or neural progenitor cells based on temporal control. It relates to a method for differentiating into dopaminergic neurons in which chromosomal stability is maintained by introducing the present invention.

Parkinson's disease (PD) is a central nervous system degenerative brain disease accompanied by movement disorders such as akinesia, rigidity, and tremors, and is caused by gradual loss of dopaminergic neurons in the substantia nigra of the midbrain. It is known that the cause of the disease is a decrease in the number of dopamine neurons that must reach the striatum due to death.

Current treatment for Parkinson's disease involves administering levo-DOPA (L-DOPA), a dopamine precursor, but this treatment has been shown to cause nausea, irritability, sleep disturbances, hypotension, stereotypic movements, and hallucinations. There are side effects such as delusions, and other methods have the effect of temporarily improving the disease. However, about 15 years ago, when brain tissue from a stillborn human fetus containing dopamine neurons was transplanted into Parkinson's disease patients, dopamine neurotransmission was restored and dopamine neurons were able to reach the striatum. As a result, improvement in akinesia symptoms was observed in some Parkinson's disease patients (NeuroRX, October 2004, Volume 1, Issue 4, pp. 382-393). As a result, the concept of cell therapy was introduced in which the lost cells are transplanted from the outside in hopes of improving symptoms. However, this also comes with ethical and technical issues, and in order to solve these issues, it is possible to produce cells that are the same as oneself through cell division, and differentiate into different specific cells using differentiation stimulation. Research on cell therapy methods using neural stem cells, which have the flexibility to achieve this goal, has been proposed. As a method for inducing dopaminergic neurons using existing neural stem cells, a method using a retrovirus is known. However, this method has problems such as the risk of genetic modification and the occurrence of mutations, and has the limitation that it is not suitable for clinical application. Therefore, gene transfer methods based on RNA and proteins, which have been used in research on reverse differentiated stem cells, have attracted attention as an appropriate method for generating DNA-free dopaminergic neurons. However, the protein delivery method requires the preparation and purification of a large amount of protein in order to express the desired gene, and the RNA delivery method uses RNA viruses, so it is necessary to prepare and purify a large amount of protein in order to express the desired gene. A limitation is that an additional screening step is required.

There is therefore a need to develop methods that can differentiate stable dopaminergic neurons from neural stem cells using new efficient gene expression techniques.

The present inventors solved the conventional problems described above and researched a method for efficiently and stably differentiating dopaminergic neurons from neural stem cells or neural progenitor cells. The present invention was completed based on the finding that functional dopaminergic neurons in which chromosome stability is maintained can be differentiated by introducing mRNA of a cell-inducing transcription factor.

Accordingly, an object of the present invention is to provide a method for differentiating neural stem cells or neural progenitor cells into dopamine neurons, and dopamine neurons produced by the differentiation method.

Another object of the present invention is to provide a cell therapeutic agent for treating Parkinson's disease containing the dopaminergic neurons.

However, the technical problems to be achieved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the following description.

To achieve the object of the present invention, the present invention provides a method for differentiating neural stem cells or neural progenitor cells into dopaminergic neurons, which comprises the step of introducing mRNA of a dopaminergic neuron-inducing transcription factor into neural stem cells or neural progenitor cells. provide a method.

In one embodiment of the present invention, the dopaminergic neuron-inducing transcription factor may be Nurr1 (Nuclear receptor related 1) or FoxA2 (Forkhead box protein A2).

In another embodiment of the present invention, the method may further include the step of inducing differentiation of neural stem cells or neural progenitor cells into dopaminergic neurons for 5 to 10 days before introducing the mRNA.

In yet another embodiment of the present invention, the mRNA may be repeatedly introduced into dopaminergic neurons of neural stem cells or neural progenitor cells at intervals of 1 to 2 days.

The present invention also provides dopaminergic neurons produced by the above differentiation method.

Further, the present invention provides a cell therapy agent for treating Parkinson's disease, which includes the dopamine neuron.

The method for differentiation into dopaminergic neurons according to the present invention synthesizes and introduces dopaminergic neuron-inducing transcription factors in the form of mRNA, which has no risk of genetic modification, unlike conventional methods using retroviral vectors. Furthermore, since functional chromosome-stable dopaminergic neurons can be produced, the present invention can be usefully used in the clinical field for the treatment of Parkinson's disease.

1 is a structure of plasmid DNA that is a template for synthesizing dopamine-induced transcription factor mRNA according to the present invention. FIG. 2 is a diagram illustrating a process in which the plasmid DNA is expressed into mRNA through in vitro transcription. FIG. 2 shows the verification of protein expression of mRNA introduced into HEK293 cells or rat neural stem cells (rat NPCs), and FIG. 2a shows the experimental method. These are the results of confirming the protein expression level using immunocytochemical staining after introducing Nurr1 or FoxA2 mRNA, each of which is a dopamine-induced transcription factor, into the cells, and the results showing an increase in the expression level due to cAMP. FIG. 3 shows how protein expression is maintained by mRNA introduced into cells, and FIG. 3a shows the experimental method. The results show that after introducing Nurr1 mRNA into rat neural stem cells (rat NPCs), the protein expression level decreases during the differentiation period (diff.0, 1, 2, and 3). FIG. 4 shows the results of verifying the differentiation of dopaminergic neurons by repeated introduction of mRNA into cells, and FIG. 4a shows the experimental method. Nurr1 mRNA was repeatedly introduced (N mRNA) into rat neural stem cells (rat NPCs), and differentiation into dopaminergic neurons was confirmed on day 3 (diff.3) and day 7 (diff.7) of differentiation. At this time, the results show that when FoxA2 is additionally expressed, the differentiation efficiency is significantly increased. FIG. 5 shows the effect of repeated introduction of mRNA into cells, and FIG. 5a shows the experimental method. These results show that repeated introduction of Nurr1 mRNA (N mRNA) into rat neural stem cells (rat NPCs) induces cell death of dopaminergic neurons from day 7 of differentiation (diff.7). FIG. 6 shows the results of verifying the efficiency of differentiation of dopaminergic neurons by repeatedly introducing mRNA based on temporal control, and FIG. 6a shows the experimental method. Nurr1 or FoxA2 mRNA was repeatedly introduced into rat neural stem cells (rat NPCs) from day 7 of differentiation, and observations were made during the differentiation period (diff. 10, 16, 22, and 28), indicating that protein expression was maintained and These results confirmed an increase in dopaminergic neuron differentiation efficiency. FIG. 7 shows the verification of the characteristics and functionality of dopaminergic neurons differentiated by the method shown in FIG. 6, and FIG. 7a shows the experimental method. These are the results of confirming the expression of specific markers (TH, AADC, DAT, VMAT2, and Lmx1A) of dopaminergic neurons. These are the results of confirming the expression of specific markers (TH, AADC, DAT, VMAT2, and Lmx1A) of dopaminergic neurons. This is the result of confirming dopamine secretion ability. This is the result of confirming active sodium current and action potential firing (Figure 7e). These results show the chromosomal stability of dopaminergic neurons differentiated by Nurr1 mRNA introduction according to the present invention compared to Nurr1 gene introduction using a retrovirus vector.

The present inventors have discovered that it is possible to differentiate into mature and functional chromosome-stable dopaminergic neurons by introducing mRNA of a dopaminergic neuron-inducing transcription factor into neural stem cells. completed.

Accordingly, the present invention provides a method for differentiating neural stem cells or neural progenitor cells into dopaminergic neurons, which comprises the step of introducing mRNA of a dopaminergic neuron-inducing transcription factor into neural stem cells or neural progenitor cells, and a method for producing dopaminergic neurons by the differentiation method. Provides dopaminergic neurons.

The term "differentiation" used in the present invention refers to a process in which stem cells in an undifferentiated state at an early stage acquire the characteristics of each tissue. This means that the progenitor cells come to have characteristics as dopaminergic neurons.

The term "neural stem cell" as used in the present invention means a cell that has the ability to differentiate into nerve cells and glial cells, and said cell has been differentiated more than 40 times, more preferably more than 50 times, most preferably Capable of unlimited cell division. The neural stem cells can be obtained from various sources, preferably from mammals such as humans, mice, or rats, and more preferably from embryonic bodies, adult tissues, fetal tissues, embryos. Derived from sexual stem cells (ES).

In addition, "neural progenitor cells" are cells that can produce progeny that are mature nerve cells, and may be used after being differentiated from embryonic stem cells, adult stem cells, or neural stem cells, or may be used by differentiation from embryonic stem cells, adult stem cells, or neural stem cells, or It can be used by directly separating it from the brain, cerebral cortex, or lateral ganglionic eminence (striatal primordium).

The term "dopamine neuron" as used in the present invention refers to a neuron that expresses tyrosine hydroxylase (TH). Dopaminergic neurons are specifically located in the substantia nigra of the midbrain and innervate the striatum, limbic system, and neocortex in vivo to modulate postural reflexes, locomotion, and compensation-related behaviors. In particular, in order to actually function as dopaminergic neurons in the body, they must exhibit midbrain characteristics.

In the present invention, the dopamine neuron-inducing transcription factor may be Nurr1 (Nuclear receptor related 1) or FoxA2 (Forkhead box protein A2).

Each nucleic acid encoding Nurr1 or FoxA2 according to the present invention can be used without restriction as long as it has a base sequence encoding Nurr1 or FoxA2 known in the art.

Nurr1 is a transcription factor that belongs to the steroid/thyroid hormone receptor expressed in midbrain dopaminergic neurons, and is expressed in dopaminergic neurons and plays a role in the development of midbrain dopaminergic neurons. It is considered. Alterations in Nurr1 are associated with disorders associated with dopaminergic dysfunction, including Parkinson's disease, schizophrenia, and manic depression, and dysregulated expression of this gene is known to be associated with rheumatoid arthritis. .

FoxA2 is a transcription factor expressed in the central nervous system, and although little is known about its relationship with midbrain dopaminergic neuron development, it is a transcription factor necessary for the development and maintenance of dopaminergic neurons. It is known that In an example of the present invention, it was revealed that when Nurr1 mRNA was introduced into neural progenitor cells alone, the efficiency of differentiation into dopaminergic neurons was significantly increased when FoxA2 was introduced together with the Nurr1 mRNA. (See Figure 4b).

In the present invention, the introduction of mRNA refers to a method of synthesizing a gene to be expressed using messenger NA and transmitting the gene. This is because when mRNA is introduced into cells, it contains modified 5-methylcytidine and pseudouridine instead of the existing base components CTP and UTP, so that it can respond to the innate antiviral immune response. However, such a gene expression method using mRNA introduction has the advantages of being simple, without the risk of genetic modification, and having excellent conversion efficiency due to high gene control.

The mRNA can be synthesized and manufactured through in vitro transcription of plasmid DNA designed to synthesize mRNA.

The present invention may further include a step of inducing differentiation of neural stem cells or neural progenitor cells into dopaminergic neurons for 5 to 10 days before introducing the dopaminergic neuron-inducing transcription factors Nurr1 and/or FoxA2 mRNA. By repeatedly introducing the mRNA into neural stem cells or neural progenitor cells at intervals of 1 to 2 days, preferably at intervals of 1 day, differentiation into dopaminergic neurons can be successfully induced.

In the present invention, the mRNA can be introduced into cells by a DNA-calcium precipitation method, a method using liposomes, a method using a polyamine system, an electroporation method, a method using a retrovirus, a method using an adenovirus, etc. Those skilled in the art can appropriately select and use techniques for introducing genes into cells known in the art, and in the present invention, preferably, a liposome-mediated method was used.

Through Examples, the present inventors experimentally confirmed the efficiency of differentiation of dopamine neurons and the functionality of differentiated dopamine neurons using the mRNA introduction method.

In one example of the present invention, it was confirmed that the protein was expressed as a result of introducing Nurr1 or FoxA2 mRNA into HEK293 cells and neural stem cells, respectively (see Example 2). In addition, as a result of verifying whether the above expression was maintained, we observed that the protein level decreased within 1 to 2 days of differentiation. Differentiated dopamine neurons were confirmed (see Example 3).

In another embodiment of the present invention, it was observed that the death of dopaminergic neurons was induced after 7 days as a result of promoting differentiation by the above method, and in order to solve this problem, a method based on temporal control was used. An mRNA introduction method was used. More specifically, as a result of repeatedly introducing mRNA of a dopamine neuron-inducing transcription factor into the cells 7 days after the start of differentiation of neural stem cells into dopamine neurons, the maintenance period of protein expression of the transcription factor and the number of dopamine neurons increased. This was confirmed (see Example 4).

In yet another embodiment of the present invention, as a result of verifying the characteristics and functionality of dopaminergic neurons produced by the differentiation method of the present invention, the expression of specific markers of dopaminergic neurons, dopamine secretion, and electrophysiological properties By confirming this, it was confirmed that differentiation into mature and functional dopaminergic neurons was successfully performed (see Example 5).

In still another embodiment of the present invention, differentiation into dopaminergic neurons was induced using the mRNA introduction method of the present invention and a conventional gene introduction method using retrovirus vectors, and as a result, unlike retrovirus vectors, It was confirmed that the chromosomal stability of the neurons was maintained when mRNA was introduced (see Example 6).

The above examples demonstrate that the differentiation method of the present invention can produce mature, functional dopaminergic neurons that maintain chromosome stability.

In another aspect of the present invention, the present invention provides a cell therapeutic agent for treating Parkinson's disease that includes dopaminergic neurons.

The cell therapy agent is a drug (as defined by the US FDA) that is used for the purpose of treatment, diagnosis, and prevention using cells and tissues that are isolated, cultured, and produced from humans through special mechanisms. Treatment, diagnosis and prevention through a range of actions such as growing living autologous, allogeneic, or xenogeneic cells in vitro, sorting them, or otherwise altering the biological properties of cells to restore function. refers to pharmaceuticals used for this purpose. Cell therapeutic agents are broadly classified into somatic cell therapeutic agents and stem cell therapeutic agents, depending on the degree of cell differentiation.

In the following, preferred embodiments are presented to aid understanding of the present invention. However, the following examples are merely provided for easier understanding of the present invention, and the content of the present invention is not limited by the following examples.

Example 1. Experiment preparation and experimental method 1-1. Isolation of Rat Neural Stem Cells (rat NPCs) Experimental animals were raised and treated according to the guidelines of the Laboratory Animal Management Committee (IACUC, 2016-0194A) of Hanyang University, Korea. Rat NPCs (Neural precursor cells) were obtained from the cerebral cortex of a 14.5-day-old fetus of a Sprague-Dawley (SD) rat (DaeHan BioLink). Thereafter, neural stem cells isolated from rat cerebral cortex tissue were treated with 15 mg/mL poly-L-ornithine (PLO, Sigma-Aldrich) and 1 mg/mL fibronectin at 37°C and 5% CO2. ; FN, Sigma-Aldrich) coated culture dishes.

Next, the rat-derived neural stem cells were grown in N2 medium supplemented with 20 ng/mL basic fibroblast growth factor (bFGF, R&D Systems), and 0.2 mM ascorbic acid (Sigma- Aldrich), 20 ng/mL brain-derived neurotrophic factor (BDNF, R&D systems), 20 ng/mL glial cell line-derived neurotrophic factor (BDNF, R&D systems), ic factor; GDNF, R&D Systems) and Differentiation was performed in N2 medium supplemented with 250 mg/mL dibutyryl-cAMP (db-cAMP, Sigma-Aldrich). In addition, rat-derived neural stem cells were treated with cAMP derivatives such as db-cAMP, 10 mM forskolin (Sigma-Aldrich), and 5 mM NKH477 (Sigma-Aldrich) before transfecting the synthesized mRNA. Thereafter, the synthesized mRNA was transfected and introduced into the cells, and then 200 ng/mL of B18R (interferon-gamma inhibitor, eBioscience) was added.

1-2. pcDNA/UTR120A, a vector for plasmid construct mRNA synthesis, was constructed using the pcDNA3.1(+) (Invitrogen) plasmid. More specifically, some restriction enzyme sites (896-930bp, 980-992bp) of pcDNA3.1+ were replaced with restriction enzymes NheI, BamHI, NotI and XbaI, and the synthesized 5'UTR, 5'UTR reverse direction , 3′UTR, 3′UTR reverse, 120pA and 120pA reverse oligomer (IDT) were annealed and inserted after the T7 promoter of pcDNA3.1(+). Furthermore, the eGFP, FLAG-conjugated Nurr1, and HA-conjugated FoxA2 genes were inserted between the 5'UTR and 3'UTR of pcDNA/UTR120A.

1-3. After linearizing the mRNA synthesis eGFP/UTR120A, Nurr1 (FLAG)/UTR120A, and FoxA2 (HA)/UTR120A constructs using EcoRV (Takara Bio) restriction enzymes, we used the MEGAscript T7 Kit (Ambion) as a template. We utilized this to proceed with mRNA synthesis. Thereafter, the transcription mixture was incubated in a test tube at 37° C. for 2 hours, and modified ribonucleotides 5'-methylcytidine and pseudouridine (Trilink Bio technologies) were added to produce modified mRNA. DNase was then treated for 15 min at 37°C and the synthesized mRNA was added with ScriptCap m7G capping system, 2'-O-methyltransferase and poly(A) polymerase (Epiccenter, currently available from CELL SCRIPT). , 5' capping and polyA tailing. Finally, mRNA was precipitated with 2.5 M ammonium acetate (Ambion), dissolved in RNA storage solution (Ambion), and then stored frozen at -70°C.

1-4. mRNA Transfection One day before transfection, rat cerebral cortex-derived neural stem cells (NPCs) (50 ,000 cells/φ12 mm). Thereafter, the synthesized mRNA and the transfection reagent Lipofectamine 2000 (Invitrogen) were each diluted with Opti-MEM medium (Invitrogen), left at room temperature for 5 minutes, and then the two solutions were mixed and incubated at room temperature for 20 minutes. Cultured. The cultured mixture was treated with the rat-derived neural stem cells, and 3 hours later, the mixture was replaced with a proliferation medium or a differentiation medium supplemented with recombinant protein B18R.

1-5. RT-PCR and real-time PCR
To synthesize cDNA, RNA was extracted from rat-derived neural stem cells using TRI REAGENT (Molecular Research Center), and then cDNA was synthesized using Superscript Kit (Invitrogen) from 5 ug of the extracted total RNA. did. Thereafter, the synthesized cDNA was used for amplification, and the identity of the amplicon was confirmed through 1.5% agarose gel electrophoresis.

Real-time PCR analysis was performed by conventional methods and performed on a CFX96 real-time system using iQ SYBR Green Supermix (Bio-Rad), and the real-time PCR reaction conditions were set to an annealing temperature of 60 °C and 45 cycle repetitions. . The primer sequences used in the RT-PCR and real-time PCR are shown in Tables 1 and 2 below, respectively.

1-6. Immunocytochemistry
After fixing the cultured cells with 4% formaldehyde (Sigma-Aldrich), the fixed cells were treated with 0.1% BSA/PBS, 10% normal goat serum (NGS, Pel-Freez), and 0.1% BSA/PBS. Treated with .03% Triton X-100 (Sigma-Aldrich) for 1 hour. Thereafter, the primary antibody was treated and incubated overnight at 4°C, followed by a biotin-conjugated secondary antibody (Vector Laboratories) or a fluorescent (DTAF, Rhodamine or Cy3) labeled secondary antibody (Jackson ImmunoResearch Laboratories). tories) were treated. The slides were then mounted with VECTASHIELD containing DAPI (Vector Laboratories) mounting medium, and the stained cells were visualized with epifluorescence or confocal microscopy. Additionally, the length of cell fibers expressing tyrosine hydroxylase (TH+) was measured using the Leica Application Suite (LAS) image analysis package.

1-7. Production of recombinant retroviruses We used the retroviral vector pCL, which was disclosed in a previous study. Retroviral constructs encoding HA-conjugated FoxA2 or vacant retroviral constructs were transfected into 293GPG packaging cells using Lipofectamine 2000. After 72 hours, the virus-containing supernatant was collected for 10 days, and 2 mg/mL of polybrene (hexadimethrine bromide, Sigma-Aldrich) was added to the virus supernatant and then stored at -70°C.

1-8. Analysis of DA secretion Analysis of dopamine (DA) secretion was performed using the Dopamine Research ELISA Kit (Labor Diagnostika Nord) according to the manufacturer's instructions. However, the supernatant in which DA was secreted was collected under two conditions: culture for 24 hours and stimulation with 56 mM KCl for 30 minutes. The DA level was calculated based on the standard curve generated with the standard control.

1-9. Electrophysiological analysis Whole-cell patch-clamp recordings were performed from rat-derived neural stem cells at room temperature (22±1°C) using an EPC10 USB amplifier (HEKA Elektronik). When filling with a solution consisting of 140mM K-gluconate, 5mM di-Tris-phosphocreatine, 5mM NaCl, 4mM MgATP, 0.4mM Na2GTP, 15mM HEPES, and 2.5mM Na-pyruvate. , the resistance of the pipette was 4-8 MU, and the pH was adjusted to 7.3 with KOH. The series resistance was compensated by 70%-80% and the current was low-pass filtered at 2kHz and sampled at 10kHz, giving a potential of 60mV. The aquarium solution was 124mM NaCl, 26mM NaHCO3, 3.2mM KCl, 2.5mM CaCl2, 1.3mM MgCl2, 1.25mM NaHPO4 and 10mM saturated with 95% O2 and 5% CO2. of glucose.

1-10. Cell Number Determination and Statistical Analysis Cell number measurements were performed by randomly selecting 10-15 microscopic areas per well for three wells per experimental condition. Each experiment was performed independently a minimum of three times.

All experimental results are expressed as mean±SE, and the paired sample t-test method was used for statistical analysis of the data.

Example 2. Confirmation of protein expression by intracellular introduction of synthesized dopamine-induced transcription factor mRNA The present inventors conducted the following steps in order to differentiate rat cerebral cortex-derived neural stem cells isolated by the method of Example 1-1 into dopaminergic neurons. An attempt was made to synthesize the mRNA of the dopamine-induced transcription factor using protein expression means and introduce it into the neural stem cells.

To this end, first, in order to synthesize mRNA that stably expresses the dopamine-induced transcription factor, plasmid DNAs having the structure shown in FIG. 1a were prepared. Specifically, the plasmid DNA contains a T7 promoter (pT7) for in vitro transcription, a UTR (5'UTR and 3'UTR) and a poly A tail (120pA) for mRNA stability, respectively. ), and the gene to be expressed (control group: eGFP, dopamine-induced transcription factor: Nurr1 or FoxA2). As shown in Figure 1b, the plasmid DNA having the above structure becomes linear by cutting it using a restriction enzyme (Cut DNA Template with Restriction enzyme), and undergoes the in vitro transcription process, capping, and polyA. Stable mRNA is synthesized through the tailing process.

In order to verify whether the mRNA synthesized through the above process is actually expressed as protein within cells, the present inventors used HEK293 cells and HEK293 cells, which have a high intracellular introduction efficiency, by the method of Examples 1-4 above. Rat-derived neural stem cells (rat NPCs or rNPCs) were transfected with each of the synthesized mRNAs, and the expression level of each protein was observed using the immunocytochemical staining method described in Examples 1-6.

As a result of the experiment according to the process shown in FIG. 2a, as shown in FIG. 2b, it was observed that NURR1 and FOXA2 proteins, which are dopamine-induced transcription factors, were expressed by just one introduction of mRNA. Furthermore, in order to increase the stability and half-life of intracellular mRNA, it was confirmed that the protein expression level was significantly increased when cyclic AMP (cAMP) was added.

Example 3. Verification of maintenance of intracellular protein expression by introduction of synthesized mRNA In addition to the results of Example 2, in order to verify whether protein expression by mRNA introduced into cells is maintained, After introducing Nurr1 mRNA, a dopamine-induced transcription factor, into rat neural stem cells using the same method, the protein expression level was observed during the differentiation period (diff.1 to diff.3), as shown in FIG. 3a.

As a result, as shown in Figure 3b, when observed up to the third day of differentiation (diff.3), the expression of NURR1 protein, a dopamine-induced transcription factor, decreased for 1 to 2 days due to rapid mRNA degradation within the cells. It was confirmed that only a certain degree was maintained.

To solve these problems, the present inventors repeatedly transfected Nurr1 mRNA at 1-day intervals to maintain intracellular protein expression, as shown in Figure 4a. On the third day (diff.3) and the seventh day (diff.7) of differentiation, the differentiation into dopaminergic neurons was observed using immunocytochemical staining.

As a result, as shown in Figure 4b, as a result of repeatedly introducing Nurr1 mRNA into cells (N mRNA TF), dopamine neurons expressing tyrosine hydroxylase (TH), a specific marker for dopamine neurons, When it was confirmed that the cells differentiated into neurons, and the coactivator FoxA2 RNA was additionally introduced into the cells using a retrovirus (N mRNA + F mRNA), differentiation into dopaminergic neurons significantly increased. It was confirmed.

Example 4. Repeated introduction of intracellularly synthesized mRNA based on temporal control and verification of its effects The present inventors found that when dopamine-induced transcription factor mRNA is repeatedly introduced into rat neural stem cells as in Example 3, In order to verify the effect, Nurr1 mRNA was repeatedly transfected and observed until differentiation day 10 (diff.10), as shown in FIG. 5a.

As a result, as shown in Figure 5b, due to the repeated introduction of mRNA, cell death of dopamine neurons was induced after 7 days of dopamine neuron differentiation, and after 9 to 10 days, most of the cells were observed. We have confirmed that this is not the case.

In order to solve these problems, the present inventors confirmed that Nurr1 expression has a delayed expression pattern in the midbrain where dopamine neurons exist, and that Nurr1 and FoxA2, which are dopamine-induced transcription factors, have a delayed expression pattern. We attempted delayed onset for temporal control. More specifically, as shown in Figure 6a, dopamine-induced transcription factor mRNA was repeatedly introduced into the cells starting 7 days after the start of differentiation of rat neural stem cells into dopamine neurons, and 10 days after differentiation (diff. 10). ), the degree of differentiation into dopaminergic neurons was observed on day 16 (diff.16), day 22 (diff.22), and day 28 (diff.28).

As a result, as shown in Figure 6b, by measuring the expression level of NURR1 protein, it was confirmed that the maintenance period of the protein expression was further increased, and the number of TH-expressing cells (TH+) and TH fiber By measuring the number and length of neurons, it was found that the number of mature dopamine neurons increased.

Example 5. Verification of the characteristics and functionality of dopaminergic neurons differentiated by introduction of dopamine neuron-inducing transcription factor mRNA Based on the results of Example 4, the present inventors investigated the characteristics and functionality of dopamine neurons differentiated by introducing transcription factor mRNA. We attempted to observe the characteristics and functionality of dopaminergic neurons produced by introducing dopamine-induced transcription factor mRNA through controlled regulation.

To this end, after proceeding with the experiment according to the process shown in Figure 7a, first, on the 14th day of differentiation (diff.14), the expression of dopamine neuron markers (TH, AADC, DAT, VMAT2, Lmx1A) was determined by RT- Observations were made by PCR and quantitative real-time PCR. As a result, as can be seen from Figures 7b and 7c, the differentiated dopaminergic neurons (Nr(L)Fr(L)) were able to express all of the markers compared to the control group cells (NC). revealed.

Next, in order to confirm the dopamine secretion ability of the differentiated dopamine neurons, on the 15th day after the start of differentiation, early dopamine neurons (Nr + Fr) and late dopamine neurons (Nr (L) + Fr (L)) were isolated for 24 hours each. After incubation or stimulation with 56mM KCl for 30 minutes, culture supernatants were collected and dopamine levels were measured. As a result, as shown in Figure 7d, the dopamine secretion amount was significantly highest in the case of Nr(L) + Fr(L), and there was a noticeable difference when cultured for 24 hours compared to when stimulated with KCl. showed that. In addition, as a result of performing an electrophysiological experiment on the 22nd day after the start of differentiation, active sodium current and action potential firing were confirmed as shown in FIG. 7e.

From the above results, it was found that the differentiated dopaminergic neurons are mature cells that have all their properties and functionality.

Example 6. Verification of maintenance of chromosome stability in dopamine neurons The present inventors attempted to compare the chromosome stability of dopamine neurons differentiated through mRNA introduction according to the present invention and dopamine neurons differentiated using other conventional gene introduction methods. did. For this purpose, each gene was introduced into dopamine neurons (NURR1 retrovirus) that had been induced to differentiate by introducing Nurr1 gene using a retroviral vector and dopamine neurons (NURR1 mRNA) that had been induced to differentiate by introducing Nurr1 mRNA. Afterwards, genomic DNA was extracted and the residual state of the gene was measured.

As a result, as shown in Figure 8, when the viral vector is used, the retrovirus randomly inserts and remains in the chromosomes of nerve cells, causing chromosomal deformation, whereas mRNA It was confirmed that when chromosomal cells were introduced, chromosomal stability was maintained because there was no gene insertion into the chromosomes. As a result, unlike the existing method of inducing dopamine neurons using viral vectors, dopamine neurons produced using mRNA are chromosomally stable cells and are highly effective and effective for clinical applications. We found that it is possible to demonstrate safety and safety.

The foregoing description of the present invention is for illustrative purposes only, and those with ordinary knowledge in the technical field to which the present invention pertains will be able to provide other specific examples without changing the technical idea or essential features of the present invention. It can be understood that it can be easily transformed into a typical form. Therefore, the embodiments described above are illustrative in all respects and should be understood as limiting.

Claims (2)

a) culturing neural stem cells or neural progenitor cells for 5 days to induce differentiation; and b) after culturing for 5 days, administering a dopaminergic neuron-inducing transcription factor to the neural stem cells or neural progenitor cells. introducing the mRNA;
The dopamine neuron-inducing transcription factor is Nurr1 (Nuclear receptor related 1) or FoxA2 (Forkhead box protein A2),
A method for differentiating neural stem cells or neural progenitor cells into dopaminergic neurons. 2. The differentiation method according to claim 1, wherein the mRNA is repeatedly introduced into neural stem cells or neural progenitor cells at intervals of 1 to 2 days.