METHOD FOR DIFFERENTIATING MOTOR NEURONS FROM TONSIL-DERIVED MESENCHYMAL STEM CELLS

Abstract

The present disclosure relates to a method for differentiating motor neurons from tonsil-derived mesenchymal stem cells, and a cell therapy agent using the same. The differentiation method of the present disclosure exhibits high differentiation potency into motor neurons, and thus enables a large quantity of motor neurons to be secured. Since the cells which are differentiated according to the present disclosure are obtained using discarded tonsillar tissues, there are fewer ethical issues. In addition, the cells are highly applicable as a cell therapy agent because they can be obtained easily in large quantities.

Claims

1. A differentiation medium composition for differentiating tonsil-derived mesenchymal stem cells or precursor cells differentiated therefrom into motor neurons, comprising DMEM (Dulbecco's modified Eagle's medium), FBS, N.sub.2 supplement, retinoic acid, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) and sonic hedgehog (SHH).

2. The differentiation medium composition according to claim 1, wherein the differentiation medium comprises low-glucose DMEM, 0.25-25% (w/v) FBS, 0.1-10% (w/v) N.sub.2 supplement, 0.1-10 μM retinoic acid, 1-100 ng/mL brain-derived neurotrophic factor (BDNF), 1-100 ng/mL nerve growth factor (NGF) and 0.01-1 ng/mL sonic hedgehog (SHH).

3. A method for differentiating into motor neurons, comprising a step of inducing differentiation into motor neurons by culturing tonsil-derived mesenchymal stem cells or precursor cells differentiated therefrom in the differentiation medium composition according to claim 1.

4. The differentiation method according to claim 3, wherein the culturing is performed for 2-4 weeks.

5. The differentiation method according to claim 3, wherein the differentiation method further comprises, before the step of inducing differentiation into motor neurons, a step of forming cell aggregates by culturing the tonsil-derived mesenchymal stem cells in a suspended state.

6. The differentiation method according to claim 5, wherein, in the step of forming cell aggregates, a proliferation medium comprising FBS, penicillin/streptomycin, β-mercaptoethanol and non-essential amino acids is used.

7. The differentiation method according to claim 6, wherein the proliferation medium of the step of forming cell aggregates is DMEM (Dulbecco's modified Eagle's medium) comprising 5-20% (w/v) FBS, 0.5-2% (w/v) penicillin/streptomycin, 0.05-0.2 mM β-mercaptoethanol and 0.5-2% (w/v) non-essential amino acids.

8. The differentiation method according to claim 5, wherein the cell aggregates are formed by culturing 5×10.sup.6 to 7×10.sup.6 cells per 10 mL of a medium on a polyethyleneimine-coated culture dish in a suspended state for 1-7 days.

9. The differentiation method according to claim 5, wherein, the differentiation method further comprises a step of differentiating the cell aggregates into neural precursor cells by subculturing up to 1-3 passages.

10. The differentiation method according to claim 1, wherein the precursor cells are neural precursor cells.

11. The differentiation method according to claim 3, wherein the tonsil-derived mesenchymal stem cells exhibit higher expression of the neural precursor cell marker vimentin as compared to mesenchymal stem cells derived from other tissues.

12. The differentiation method according to claim 3, wherein the precursor cells differentiated from the tonsil-derived mesenchymal stem cells exhibit higher expression of the neuron-specific marker Tuj1 as compared to precursor cells differentiated from mesenchymal stem cells derived from other tissues.

13. Motor neurons prepared by the differentiation method according to any of claims 3 to 12.

14. The motor neurons according to claim 13, wherein the motor neurons exhibit increased expression of ISL1 (insulin gene enhancer protein), HB9 (homeobox protein) or ChAT (choline acetyltransferase).

15. The motor neurons according to claim 13, wherein the motor neurons exhibit increased secretion of acetylcholine.

16. The motor neurons according to claim 13, wherein the motor neurons are capable of forming a neuromuscular junction.

17. The motor neurons according to claim 13, wherein the motor neurons can be subcultured up to 1-3 passages.

18. The motor neurons according to claim 13, wherein the motor neurons can be used by thawing after freezing.

19. A pharmaceutical composition for preventing or treating a neurological disorder, comprising the motor neurons according to claim 13 as an active ingredient.

20. The pharmaceutical composition for preventing or treating a neurological disorder according to claim 19, wherein the neurological disorder is amyotrophic lateral sclerosis (ALS), myasthenia gravis (MG), spinal muscular atrophy (SMA) or Charcot-Marie-Tooth disease (CMT).

21. A cell therapy agent comprising the motor neurons according to claim 13.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0064] FIG. 1 schematically shows induction of tonsil-derived mesenchymal stem cells (T-MSC, A) to neural precursor cells (NP, B) and differentiation into motor neurons (MN, C) as well as the ingredients of a differentiation medium and the morphology of the cells.

[0065] FIG. 2 shows that motor neurons differentiated by a method according to the present disclosure can proliferate normally when subcultured for 2 and 3 passages.

[0066] MN2.5w: motor neurons differentiated for 2.5 weeks;

[0067] p2: subculturing for 2 weeks; and

[0068] p3: subculturing for 3 weeks (the same for FIG. 3).

[0069] FIG. 3 shows that motor neurons (MN) differentiated by a method according to the present disclosure can be used even after freezing and then thawing.

[0070] FIG. 4 shows a result of differentiating tonsil-derived mesenchymal stem cells (T-MSC) into motor neurons (MN) and confirming the increased expression of ISL1, HB9 and ChAT depending on differentiation period by real-time PCR.

[0071] MN2w: motor neurons differentiated for 2 weeks;

[0072] MN3w: motor neurons differentiated for 3 weeks; and

[0073] MN4w: motor neurons differentiated for 4 weeks.

[0074] FIG. 5a shows a result of differentiating tonsil-derived mesenchymal stem cells into motor neurons for 2 weeks and confirming the increased expression of ISL1 in the cells by immunofluorescence staining.

[0075] FIG. 5b shows a result of differentiating tonsil-derived mesenchymal stem cells into motor neurons for 2 weeks and confirming the increased expression of HB9 in the cells by immunofluorescence staining.

[0076] FIG. 5c shows a result of differentiating tonsil-derived mesenchymal stem cells into motor neurons for 2 weeks and confirming the increased expression of ChAT in the cells by immunofluorescence staining.

[0077] FIGS. 6a-6d show a result of differentiating tonsil-derived mesenchymal stem cells into motor neurons for 4 weeks and investigating the change in the expression of ISL1 (FIG. 6b), HB9 (FIG. 6c) and ChAT (FIG. 6d) in the motor neurons depending on differentiation period by western blotting.

[0078] FIG. 7 shows a result of differentiating tonsil-derived mesenchymal stem cells into motor neurons and statistically comparing the concentration of acetylcholine in supernatants depending on differentiation period as percentage with respect to a differentiation medium.

[0079] NPC: neural precursor cells;

[0080] MNC2w: motor neurons differentiated for 2 weeks;

[0081] MNC3w: motor neurons differentiated for 3 weeks; and

[0082] MNC4w: motor neurons differentiated for 4 weeks.

[0083] FIG. 8a shows the optical microscopic images of tonsil-derived mesenchymal stem cells before and after differentiating into motor neurons and motor neurons being co-cultured with muscle cells.

[0084] FIG. 8b shows a result of co-culturing T-MSCs that have been differentiated for 2 weeks into motor neurons according to the present disclosure with human skeletal muscle cells and confirming that a neuromuscular junction can be formed by fluorescence immunostaining and α-BTX treatment (hSKMC: human skeletal muscle cells only; T-MSC-MNC: motor neurons derived from tonsil-derived stem cells only; hSKMC & T-MSC-MNC: motor neurons derived from tonsil-derived stem cells co-cultured with human skeletal muscle cells).

[0085] FIG. 8c shows a result of co-culturing T-MSCs differentiated into motor neurons with human skeletal muscle cells as in FIG. 8b and confirming the formation of a neuromuscular junction and the morphology of the two cells by staining with different proteins. α-SMA (α-smooth muscle actin, blue) shows the morphology of the muscle cells, Tuj1 (beta III tubulin, green) shows the morphology of the neurons, and α-BTX (bungarotoxin, red) shows acetylcholine receptor clusters at the neuromuscular junction. The three images are merged in the rightmost image.

[0086] FIG. 9 shows a result of differentiating tonsil-derived mesenchymal stem cells (T-MSC) into motor neurons (MNC) and confirming the increased expression of four neurotrophic factors in the cells by real-time PCR.

[0087] FIG. 10 shows a result of investigating the expression of vimentin in T-MSCs by immunofluorescence staining.

[0088] FIG. 11 shows a result of investigating the expression of Tuj1 in T-MSCs and neural precursor cells (NPCs) derived therefrom by immunofluorescence staining.

BEST MODE

[0089] Hereinafter, the present disclosure will be described in detail through examples. However, the following examples are for illustrative purposes only and the scope of the present disclosure is not limited by the examples.

Example 1: Differentiation of Tonsil-Derived Mesenchymal Stem Cells into Motor Neurons

Example 1-1: Culturing of Tonsil-Derived Mesenchymal Stem Cells

[0090] Tonsil-derived mesenchymal stem cells (TMSC) were obtained from the tonsillar tissues of patients who received tonsillectomy Department of Otorhinolaryngology-Head and Neck Surgery of Ewha Womans University Mokdong Hospital (tissues of young patients aged 4-20 years, approved by the Institutional Review Board: ECT 11-53-02). Stem cells were isolated and cultured in DMEM (Dulbecco's modified Eagle's medium, GIBCO) supplemented with 10% FBS (Hyclone), 1% penicillin/streptomycin (GIBCO), 0.1 mM β-mercaptoethanol (Sigma) and 1% non-essential amino acids (GIBCO).

Example 1-2: Differentiation of Tonsil-Derived Mesenchymal Stem Cells into Motor Neurons

[0091] The tonsil-derived mesenchymal stem cells were differentiated into motor neurons (MN) according to the following stages.

[0092] Spheres were formed as a first stage of inducing differentiation. The spheres were prepared by suspending 5,000,000-7,000,000 cells per 10 mL of the proliferation medium of Example 1 on a PEI-coated 100-mm Petri dish and inducing cell aggregation for 1-2 days. The formed spheres were replated onto a culture dish and differentiation into neural precursor cells (NPC) was induced by subculturing in a proliferation medium up to passage 1, 2 or 3.

[0093] The differentiated neural precursor cells were cultured additionally in a differentiation medium [low-glucose DMEM, 2.5% FBS, 1% N.sub.2 supplement, 1 μM retinoic acid, 10 ng/mL brain-derived neurotrophic factor (BDNF), 10 ng/mL nerve growth factor (NGF), 0.1 ng/mL sonic hedgehog (SHH)] for 2-4 weeks. Through this, motor neurons were prepared (FIG. 1).

Example 1-3: Subculturing of Differentiated Motor Neurons

[0094] As a result of subculturing the differentiated motor neurons for 2.5 weeks, it was confirmed that the motor neurons subcultured for 2 and 3 passages have normal proliferation ability. Accordingly, it was confirmed that the motor neurons differentiated according to the present disclosure can proliferate normally even when subcultured (FIG. 2).

Example 1-4: Use of Differentiated Motor Neurons after Freezing and Thawing

[0095] Motor neurons differentiated for 2.5 weeks according to the method described above were frozen on day 10 after culturing and then cell morphology was observed after thawing on day 14. No change in morphology was observed even after the freezing and thawing.

[0096] Accordingly, it was confirmed that the motor neurons differentiated according to the present disclosure can be used as normal motor neurons even after freezing and thawing (FIG. 3).

Example 3: Investigation of Differentiation Potency from Tonsil-Derived Mesenchymal Stem Cells into Motor Neurons by PCR

[0097] In order to investigate the differentiation potency from tonsil-derived mesenchymal stem cells into motor neurons, the expression level of ISL1 (insulin gene enhancer protein), HB9 and ChAT (choline acetyltransferase), which are representative markers of motor neurons, was analyzed by real-time PCR.

[0098] Total RNA was extracted using an RNeasy mini kit (Qiagen Inc.) according to the manufacturer's instructions. cDNA was synthesized using Superscript II (Invitrogen) and an oligo-d(T)20 primer by conducting reaction at 42° C. for 1 hour and at 72° C. for 15 minutes. For the cDNA, quantitative real-time PCR was performed using SYBR® Premix Ex Taq™ kits (TaKaRa Bio Inc., Shiga, Japan) on an ABI 7500 fast real-time PCR system (Applied Biosystems/Thermo Fisher Scientific, Waltham, Mass., USA). The relative expression level of the ISL1, HB9 and ChAT genes was calculated using the comparative C.sub.t method (2.sup.−ΔΔCt), and all measurements were carried out in triplicate.

[0099] The result is shown in FIG. 4. As shown in FIG. 4, it was confirmed that, when tonsil-derived mesenchymal stem cells were differentiated into motor neurons, the expression of ISL1, HB9 and ChAT, which are markers of motor neurons, was increased from 2 weeks after the differentiation, which confirms the differentiation into motor neurons.

[0100] Specifically, ISL1 is a motor neuron-specific marker whose expression is increased during the early stage of differentiation into motor neurons. As shown in FIG. 4, the highest expression of ISL1 at 2 weeks after the differentiation means that the differentiation rate is the highest at 2 weeks after the differentiation, and the relatively decreased expression of ISL1 at 3 weeks as compared to 2 weeks after the differentiation means that differentiation into motor neurons has proceeded already. Statistically significant increased expression of ISL1 as compared to undifferentiated T-MSCs cells was observed at 2 weeks and 3 weeks. HB9 is also a motor neuron-specific marker whose expression is increased during the early stage of differentiation into motor neurons. Although the expression of HB9 was increased gradually with differentiation period, statistically significant expression as compared to undifferentiated T-MSCs cells was observed only at 2 weeks.

[0101] ChAT is a motor neuron marker whose expression is increased as differentiation proceeds, whereas the expression of ISL1 is increased during the early stage of differentiation, and is called an acetylcholinergic neuron marker. There exist two isoforms of ChAT: common type ChAT (cChAT) present in both the central nervous system and the peripheral nervous system; and peripheral type ChAT (pChAT) preferentially expressed in the peripheral nervous system. As shown in FIG. 4, the expression of exon 3 of ChAT was significantly expressed between 2 weeks and 4 weeks after the differentiation, showing the characteristics of both the central nervous system and the peripheral nervous system. The expression of exon 6 was increased at 2 weeks after the differentiation and then was decreased relatively from 3 weeks. This is due to skipping from exon 6 to exon 9 during post-transcriptional modification, suggesting that the characteristics of the peripheral nervous system become dominant as the differentiation proceeds. The above results for ChAT suggest that the differentiation into motor neurons begins at 2 weeks after the differentiation and proceeds until 3 weeks and 4 weeks.

[0102] Through these experimental results, it was confirmed that the cells differentiated from tonsil-derived mesenchymal stem cells exhibit the characteristics of motor neurons. Accordingly, it was confirmed that the differentiation medium of the present disclosure exhibits superior differentiation potency into motor neurons.

Example 4: Investigation of Differentiation Potency from Tonsil-Derived Mesenchymal Stem Cells into Motor Neurons by Immunofluorescence Assay

[0103] The differentiation potency into motor neurons was investigated by immunofluorescence staining. After differentiating tonsil-derived mesenchymal stem cells for 2 weeks, motor neurons were cultured on a cover slip. After the differentiation was finished, the cells were fixed in a 4% paraformaldehyde solution for 15 minutes at room temperature and then washed with PBS. The washed cells were treated in a PBS solution with 0.1% Tween-20 and 2% bovine serum albumin added for 1 hour and diluted with antibodies for detection of differentiation at a ratio designated by the producer. After addition to PBS, incubation was conducted at room temperature for 1 hour or overnight at low temperature. Subsequently, after washing again with PBS, the cells were treated with TRITC (tetrarhodamine isothiocyanate)- or FITC (fluorescein isothiocyanate)-conjugated secondary antibodies at room temperature or low temperature in the same manner as the primary antibodies. A mounting solution (Vectashield) with DAPI added was used for contrast staining of cell nuclei. After mounting, the cells were observed using a fluorescence microscope.

[0104] As can be seen from FIG. 5a, whereas T-MSCs showed no red fluorescence signal of ISL1 at all, the differentiated motor neurons (T-MSC-MNC) showed strong red fluorescence signal of ISL1 (b and e of FIG. 5a). In addition, it was confirmed that the expression of class III beta-tubulin (Tuj1), which is a neuron-specific protein, is increased as the differentiation proceeds (a and d of FIG. 5a). In addition, in order to verify the adequacy of the method for differentiating into motor neurons by immunofluorescence assay, induced pluripotent stem cell-derived motor neurons (iXCell™ human iPSC-derived motor neurons, iPSC-MNC) were purchased and the expression of ISL1 and Tuj1 was observed. As a result, the expression pattern of the two markers (ISL1 and Tuj1) was identical although T-MSC-MNCs and iPSC-MNCs showed slightly different cell morphologies (h, i, j and k of FIG. 5a).

[0105] As seen from FIG. 5b, whereas T-MSCs showed no red fluorescence signal of HB9 at all, the differentiated motor neurons (T-MSC-MNC) showed strong red fluorescence signal of HB9 (b and e of FIG. 5b). In addition, it was confirmed that the expression of Tuj1 is increased as the differentiation proceeds (a and d of FIG. 5b). In addition, the expression of HB9 and Tuj1 in iPSC-MNCs was observed. As a result, the expression pattern of the two markers (HB9 and Tuj1) was identical although T-MSC-MNCs and iPSC-MNCs showed slightly different cell morphologies (h, i, j and k of FIG. 5b).

[0106] As seen from FIG. 5c, whereas T-MSCs showed no red fluorescence signal of ChAT at all, the differentiated motor neurons (T-MSC-MNC) showed strong red fluorescence signal of ChAT (b and e of FIG. 5c). In addition, it was confirmed that the expression of Tuj1 is increased as the differentiation proceeds (a and d of FIG. 5c). In addition, the expression of ChAT and Tuj1 in iPSC-MNCs was observed. As a result, the expression pattern of the two markers (ChAT and Tuj1) was identical although T-MSC-MNCs and iPSC-MNCs showed slightly different cell morphologies (h, i, j and k of FIG. 5c).

[0107] Through these experimental results, it was confirmed that the cells differentiated from tonsil-derived mesenchymal stem cells have the characteristics of motor neurons. Accordingly, it was confirmed that the differentiation medium of the present disclosure exhibits superior differentiation potency into motor neurons.

Example 5: Investigation of Differentiation Potency from Tonsil-Derived Mesenchymal Stem Cells into Motor Neurons by Western Blotting

[0108] The differentiation from tonsil-derived mesenchymal stem cells into motor neurons was investigated by western blotting.

[0109] Tonsil-derived mesenchymal stem cells and cells in different stages of differentiation (undifferentiated tonsil-derived mesenchymal stem cells, neural precursor cells, and motor neurons differentiated for 2 to 4 weeks) were lysed by adding to a lysis buffer containing a protease inhibitor (Roche). Total proteins (10-30 μg) were immunoblotted with primary antibodies (ISL1, HB9, ChAT), and GAPDH (Abcam) was used as an internal control. Band intensity was quantified using LAS-3000 (Fuji Film) and normalized to the intensity of GAPDH.

[0110] The result is shown in FIGS. 6a-6d. The band intensities of FIG. 6a are plotted in FIG. 6b (ISL1), FIG. 6c (HB9) and FIG. 6d (ChAT). As can be seen from FIGS. 6a-6d, although the ISL1 protein was expressed slightly in T-MSCs, the expression was increased as the cells were differentiated into neural precursor cells (NPC) and reached maximum at 2 weeks after the differentiation (FIG. 6b). The HB9 protein was hardly expressed in T-MSCs and NPCs, and the expression was increased at 2 weeks and 3 weeks after the differentiation (FIG. 6c). The isotype 2 protein of ChAT showed two bands at 2 weeks and 3 weeks after the differentiation, confirming differentiation into motor neurons (FIG. 6d).

[0111] Similarly to Example 3, the increased expression of isotype 2 in motor neurons 2 weeks after the differentiation means that the differentiated motor neurons exhibit the characteristics of peripheral nerves.

[0112] Through these experimental results, it was confirmed that the cells differentiated from tonsil-derived mesenchymal stem cells have the characteristics of motor neurons. Accordingly, it was confirmed that the differentiation medium of the present disclosure exhibits superior differentiation potency into motor neurons.

Example 6: Confirmation of Differentiation Potency into Motor Neurons from increase in acetylcholine

[0113] For a supernatant (or conditioned medium) taken from a culture dish in which tonsil-derived mesenchymal stem cells were being differentiated into motor neurons for 4 weeks and a differentiation medium, the increase in acetylcholine with respect to the differentiation medium was calculated as percentage using an acetylcholine assay kit (Fluorometric; Cell Biolabs, INC. Calif., USA).

[0114] The result is shown in FIG. 7. As can be seen from FIG. 7, when T-MSCs were differentiated into T-MSC-MNCs, the secretion of acetylcholine began to increase from 1 week after the differentiation and reached maximum at 2 weeks. This result was statistically significant when repeated three times. This means that, when tonsil-derived mesenchymal stem cells are differentiated into motor neurons, the highest differentiation rate is achieved at 2 weeks after the differentiation.

[0115] Acetylcholine is a neurotransmitter of the neuromuscular junction secreted at the axon terminal. The increased secretion of acetylcholine in the motor neurons prepared according to the present disclosure means that they can function as normal motor neurons.

[0116] Through this, it was confirmed that tonsil-derived mesenchymal stem cells are differentiated into motor neurons when cultured using the differentiation medium of the present disclosure.

Example 7: Neuromuscular Junction Forming-Ability of Differentiated Motor Neurons

[0117] It was investigated whether a neuromuscular junction is formed in order to investigate whether the motor neurons differentiated according to the present disclosure actually exhibit the characteristics of motor neurons.

[0118] Specifically, motor neurons differentiated from tonsil-derived mesenchymal stem cells for 2 weeks were co-cultured with human skeletal muscle cells (hSKMC) and fixed 4-5 days later. Then, it was investigated whether the cells are neurons by staining with Tui1 (green) by fluorescence immunostaining, and the presence of acetylcholine receptors was investigated by treating with Alexa 555-conjugated α-BTX to confirm the formation of the neuromuscular junction.

[0119] The result is shown in FIG. 8. First, the morphological change of T-MSC-MNCs was observed before investigating the formation of the neuromuscular junction (FIG. 8a). Compared with T-MSCs, T-MSC-MNCs became multipolar and the length of the cell body was increased like typical motor neurons (arrows in FIG. 8a). In addition, the change in the cell morphology of hSKMCs being co-cultured could be observed as well as the cellular characteristics of hSKMCs and T-MSC-MNCs being co-cultured.

[0120] As seen from FIG. 8b, when T-MSCs or hSKMCs were cultured alone, no red fluorescence was observed at all and the expression of Tuj1 was low. In contrast, when the motor neurons differentiated according to the present disclosure were co-cultured with skeletal muscle cells, red fluorescence was observed and the expression of Tuj1 was increased. In order to investigate the formation of the neuromuscular junction in more detail, triple staining was performed with the muscle-specific marker α-smooth muscle actin (α-SMA) and the neuron-specific markers Tuj1 and α-BTX (FIG. 8c). As a result, the presence of red acetylcholine receptors (arrows) was clearly observed when the two cells were co-cultured.

[0121] This result suggests that the cells differentiated from tonsil-derived mesenchymal stem cells for 2 weeks have the possibility of signaling through the junction with skeletal muscle cells, which is the most important function of motor neurons.

[0122] The red fluorescence indicates the presence of acetylcholine receptors in the motor neurons co-cultured with the skeletal muscle cells. A normal nerve signal transmission system mediated by acetylcholine can be established based on this experimental result because the motor neurons differentiated according to the present disclosure are capable of forming the neuromuscular junction.

Example 8: Investigation of Increase of Neurotropic Factors in Motor Neurons Differentiated from Tonsil-Derived Mesenchymal Stem Cells by PCR

[0123] In order to investigate characterization of the motor neurons differentiated from tonsil-derived mesenchymal stem cells, the change in the expression of neurotrophic factors such as brain derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), nerve growth factor (NGF) and heregulin (HRG), which promote initial growth and development of neurons in the central nervous system and the peripheral nervous system, was analyzed by real-time PCR. Total RNA was extracted using an RNeasy mini kit (Qiagen Inc.) according to the manufacturer's instructions. cDNA was synthesized using Superscript II (Invitrogen) and an oligo-d(T)20 primer by conducting reaction at 42° C. for 1 hour and at 72° C. for 15 minutes. For the cDNA, quantitative real-time PCR was performed using SYBR® Premix Ex Taq™ kits (TaKaRa Bio Inc., Shiga, Japan) on an ABI 7500 fast real-time PCR system (Applied Biosystems/Thermo Fisher Scientific, Waltham, Mass., USA). The relative expression level of the BDNF, GDNF, NGF and HRG genes was calculated using the comparative C.sub.t method (2.sup.−ΔΔCt), and all measurements were carried out in triplicate.

[0124] As shown in FIG. 9, the expression of the four neurotrophic factors was increased statically significantly after differentiation into T-MSC-MNCs. In particular, it is to be noted that the expression of BDNF, GDNF and HRG, which are nerve growth factors not added to the differentiation medium, was increased significantly.

Example 10: Comparison with AdMSCS, BMMSCs AND WJ-MSCs

[0125] FIG. 10 shows a result of investigating the expression of vimentin in T-MSCs by immunofluorescence staining. Vimentin is a protein often used as a neural precursor cell marker. From FIG. 10, it can be seen that the T-MSCs have remarkably higher differentiation potency into motor neurons as compared to other MSCs (AdMACs, BM-MSCs and WJ-MSCs).

[0126] FIG. 11 shows a result of investigating the expression of Tuj1 in T-MSCs and neural precursor cells (NPCs) derived therefrom by immunofluorescence staining. From FIG. 11, it can be estimated that the neural precursor cells differentiated from the T-MSCs have remarkably higher differentiation potency into motor neurons as compared to the NPCs derived from other MSCs (AdMSCs and BM-MSCs) because the expression level of the neuron-specific marker Tuj1 is very high.

[0127] In the present specification, detailed description of the contents that can be fully recognized and inferred by those of ordinary skill in the art to which the present disclosure belongs was omitted. More various modifications can be made to the specific exemplary embodiments described in the present disclosure within the scope not changing the technical idea or essential constitution of the present disclosure. Accordingly, the present disclosure can be carried out in a way different from those described and exemplified specifically in the present disclosure, and this will be understood by those of ordinary skill in the art to which the present disclosure belongs.

[0128] [National R&D Program Supporting Invention]

[0129] [Project ID] 2017R1D1A1A02018634

[0130] [Ministry in charge] Ministry of Education

[0131] [Research management] National Research Foundation of Korea

[0132] [Research project title] Basic research project (Research promotion)-Basic research program in science and engineering-Basic research (SGER)

[0133] [Research title] Development of tonsil-derived mesenchymal stem cells for treatment of peripheral nerve disease

[0134] [Contribution rate] 70/100

[0135] [Research institute] Ewha University-Industry Collaboration Foundation

[0136] [Research period] 2017 Jun. 1 to 2020 May 31

[0137] [National R&D Program Supporting Invention]

[0138] [Project ID] HI12C0135010017

[0139] [Ministry in charge] Ministry of Health and Welfare

[0140] [Research management] Korea Health Industry Development Institute

[0141] [Research project title] Health technology R&D project-Rare disease project

[0142] [Research title] Development of novel biomarker and customized therapeutic technology for Charcot-Marie-Tooth disease

[0143] [Contribution rate] 30/100

[0144] [Research institute] Ewha University-Industry Collaboration Foundation

[0145] [Research period] 2017 Apr. 1 to 2018 Mar. 31