COMPOSITION FOR PROMOTING DIFFERENTIATION OF NEURAL STEM CELLS INTO DOPAMINERGIC NEURONS

Abstract

Provided are a composition and a method for promoting differentiation of neural stem cells into dopaminergic neurons, the composition including fusaric acid, ascorbic acid, nicotinamide adenine dinucleotide, or a combination thereof. The composition and method according to an aspect may not only increase differentiation of neural stem cells isolated at an early stage of development into dopaminergic neurons, but also increase differentiation of subcultured neural stem cells into dopaminergic neurons, and thus, it is possible to secure more dopaminergic neurons, and increase therapeutic effects on Parkinson's disease.

Claims

1. A composition for promoting differentiation of neural stem cells into dopaminergic neurons, comprising fusaric acid, ascorbic acid, nicotinamide adenine dinucleotide (NAD+), or a combination thereof.

2. The composition of claim 1, comprising fusaric acid, ascorbic acid, and nicotinamide adenine dinucleotide.

3. The composition of claim 1, wherein the neural stem cells are subcultured 10 or more times and not more than 30 times.

4. The composition of claim 1, wherein the neural stem cells are subcultured 10 or more times and not more than 20 times.

5. The composition of claim 1, wherein the neural stem cells are embryonic stem cells, embryonic germ cells, embryonic carcinoma cells, induced pluripotent stem cells (iPSCs), or adult stem cells.

6. The composition of claim 1, wherein the dopaminergic neurons are dopaminergic neural progenitors, or dopaminergic neural precursor cells, or mature dopaminergic neurons.

7. The composition of claim 1, wherein the dopaminergic neurons are midbrain dopaminergic neurons.

8. A method of promoting differentiation of neural stem cells into dopaminergic cells, comprising: subculturing neural stem cells; and differentiating the subcultured neural stem cells in a medium comprising fusaric acid, ascorbic acid, nicotinamide adenine dinucleotide (NAD+), or a combination thereof.

9. The method of claim 8, wherein the subculturing is performed 10 or more times and not more than 30 times.

10. The method of claim 8, wherein the subculturing is performed 10 or more times and not more than 20 times.

11. The method of claim 8, wherein the medium comprises fusaric acid, ascorbic acid, and nicotinamide adenine dinucleotide.

12. Dopaminergic neurons differentiated by the method of claim 8.

13. A pharmaceutical composition for preventing or treating Parkinson's disease, comprising the dopaminergic neurons of claim 12 as active ingredients.

14. A method of preventing or treating Parkinson's disease, comprising: administering the pharmaceutical composition of claim 13 to a subject.

15. A use of the composition of claim 1, for preparation of a drug for preventing or treating Parkinson's disease.

Description

DESCRIPTION OF DRAWINGS

[0069] FIG. 1 is results of confirming expression of stem cell markers SOX2 and Nestin in neural stem cells isolated from the central nervous system of a 10-week-old fetus by immunostaining chemistry.

[0070] FIG. 2 is a diagram confirming the expression of TH, a marker of dopaminergic neurons, after differentiating FMD-NSPCs in a differentiation medium supplemented with FA alone, a combination of FA and AA, a combination of FA and NAD+, or a combination of FA, AA, and NAD+ (FA: 0.1 mM, AA: 0.2 μm, and NAD+: 1 mM).

[0071] FIG. 3 is a diagram confirming the expression of TH, a marker of dopaminergic neurons, and Tuj1, a marker of neural cells, after differentiating FMD-NSPCs in a differentiation medium supplemented with FA alone (FA: 0.1 mM).

[0072] FIG. 4 is a diagram confirming the expression of TH, a marker of dopaminergic neurons, and Tuj1, a marker of neural cells, after differentiating FMD-NSPCs obtained in subculture 9 in a differentiation medium supplemented with a combination of FA and AA (a), or a combination of FA and NAD+ (b).

[0073] FIG. 5 is a diagram confirming the expression of TH, a marker of dopaminergic neurons, after differentiating FMD-NSPCs obtained in subculture 12 in a differentiation medium supplemented with a combination of FA and AA (FA: 0.1 mM, AA: 0.2 mM).

[0074] FIG. 6 is a diagram confirming the expression of TH, a marker of dopaminergic neurons, after differentiating FMD-NSPCs obtained in subculture 14 (a) and subculture 15 (b) in a differentiation medium supplemented with a combination of FA and NAD+ (FA: 0.1 mM, NAD+ 0.5: 0.5 mM, NAD+ 1.0: 1 mM).

[0075] FIG. 7 is a diagram confirming the expression of TH, a marker of dopaminergic neurons, after differentiating FMD-NSPCs obtained in subculture 16 (a), subculture 17 (b), subculture 18 (c), and subculture 19 (d) in a differentiation medium supplemented with a combination of FA and AA (FA: 0.1 mM, AA: 0.2 mM).

[0076] FIG. 8 is a diagram confirming the expression of TH, a marker of dopaminergic neurons, after differentiating FMD-NSPCs obtained in subculture 21, a late stage, in a differentiation medium supplemented with a combination of FA and AA, or a combination of FA and NAD+.

[0077] FIG. 9 is a diagram confirming the expression of TH, a marker of dopaminergic neurons, and Tuj1, a marker of neural cells, after differentiating FMD-NSPCs obtained in subculture 10 (a), subculture 11 (b), and subculture 12 (c) in a differentiation medium supplemented with a combination of FA, AA, and NAD+ (FA: 0.1 mM, AA: 0.2 mM, NAD+: 1 mM).

[0078] FIG. 10 is a diagram confirming the expression of TH, a marker of dopaminergic neurons, and Tuj1, a marker of neural cells, after differentiating FMD-NSPCs obtained in subculture 17 and subculture 19 in a differentiation medium supplemented with FA alone, or a combination of FA, AA, and NAD+ (FA: 0.1 mM, AA: 0.2 mM, NAD+: 1 mM).

[0079] FIG. 11 is a diagram measuring fluorescence intensity by confirming the expression of TH, a marker of dopaminergic neurons, after differentiating FMD-NPCs in a differentiation medium supplemented with a combination of FA, AA, and NAD+.

MODE FOR INVENTION

[0080] Hereinafter, the present disclosure will be described in more detail through examples. However, these examples are intended only to illustrate at least one embodiment, but not to limit the scope of the present disclosure thereto.

Example 1. Isolation and Culture of Neural Stem Cells

[0081] Neural stem cells (FMD-NSPCs: fetal midbrain derived neural stem/progenitor cells) were isolated from the central nervous system of a 10-week-old fetus. Specifically, the human neural stem cells were isolated according to a method disclosed in Storch et al. 2001; Milosevic et al. in 2006, 2007 and the like. Ventral midbrain tissues from brain tissues of 10-week-old fetuses were isolated, and treated in a solution including 0.1 mg/ml of papain and 100 μg/ml of DNase at 37° C. for about 30 minutes, to separate into a single cell suspension. The suspension was washed with phosphate buffered saline (PBS) and incubated in 50 ug/ml of antipain at 37° C. for 30 minutes. The human neural stem cells (hNSPCs) obtained above were inoculated as a monolayer at a density of 30,000 cells/cm.sup.2 on a culture dish coated with 15 μg/ml of poly-L-ornithine and 4 μg/ml of fibronectin, and cultured.

[0082] Thereafter, expression of SOX2 and Nestin was confirmed in the isolated human neural stem cells, by using immunostaining chemistry. Specifically, the isolated hNSPCs were washed three times with PBS and fixed with PBS containing 4% paraformaldehyde for 10 minutes. After washing three times with PBS, blocking was performed by reacting with PBS containing 3% normal goat serum, 0.2% Triton X-100, and 1% BSA at room temperature for one hour. Anti-nestin (rabbit anti-nestin, COVANCE, CA, USA) and anti-Sox2 (rabbit-anti-Sox2, Abcam) primary antibodies were incubated overnight, washed three times with PBS, and the obtained cells were incubated with secondary antibodies of anti-mouse (Alexa Fluor™ 488), anti-mouse (Alexa Fluor™ 594), anti-rabbit (Alexa Fluor™ 488), anti-rabbit (Alexa Fluor™ 594) antibodies at room temperature for 60 minutes, and stained (counterstained)with 4′-6-diamidino-2-phenylindole (DAPI).

[0083] FIG. 1 is results of confirming expression of stem cell markers SOX2 and Nestin in neural stem cells isolated from the central nervous system of a 10-week-old fetus by immunostaining chemistry.

[0084] As a result, as shown in FIG.1, the isolated neural stem cells, unlike human neural progenitor cells (FMD-NPCs: fetal midbrain derived neural progenitor cells) isolated from a 14-week-old fetus, were confirmed to be cells obtained at an earlier stage than a developmental stage, through expression of stem cell markers SOX2 and Nestin.

Example 2. Confirmation of Effects of Fusaric Acid, NAD+, and Ascorbic Acid of Enhancing Differentiation Potency of Neural Stem Cells into Dopaminergic Neurons

[0085] In order to confirm effects of NAD+, ascorbic acid (AA), and fusaric acid (FA) on differentiation potency of neural stem cells, human neural stem cells were differentiated in a differentiation medium (50 ml of Neurobasal Media ; B27-CTS or B27 supplement (50×) 1×, L-glutamine (100×) 1×, 10 μM of forskolin, 100 μM of dibutyryl cAMP) further supplemented with fusaric acid; fusaric acid and NAD+; fusaric acid and ascorbic acid; or fusaric acid, NAD+, and ascorbic acid, and differentiation potencies into dopaminergic neurons were compared (D2: day 2 of differentiation, D4: day 4 of differentiation, and D6: day 6 of differentiation).

[0086] Specifically, NSPCs derived from 14-week-old embryos and fetuses were cultured, allowed to settle, and differentiated for 6 days in a differentiation medium supplemented with FA alone, a combination of FA and AA, a combination of FA and NAD+, or a combination of FA, AA, and NAD+. The culture was continuously subcultured under a condition of oxygen partial pressure of 3% by changing the culture medium once every two days. The differentiated neural stem cells, that is, dopaminergic neurons were washed with a buffer solution after removing culture solution from the culture vessel, and treated with Accutase (PAA) for 30 minutes to separate the cells from the culture dish, and then again washed with a buffer solution. The obtained cells were centrifuged at 1,000 rpm for about 5 minutes to remove the supernatant, and the differentiated dopaminergic neurons were harvested. In order to compare differentiation potencies of neural stem cells into dopamine neurons, tyrosine hydroxylase (TH) antibodies (rabbit-anti-TH, Pelfreez), which is a marker of dopaminergic neurons, and Tuj1 antibodies (mouse anti-Tuj1 Millipore, CA. USA), a marker of neural cells were used to perform immunochemical staining.

[0087] As shown in FIG. 2, after differentiating FMD-NSPCs in a differentiation medium supplemented with FA alone, a combination of FA and AA, a combination of FA and NAD+, or a combination of FA, AA, and NAD+ (FA: 0.1 mM, AA: 0.2 μM, NAD+: 1 mM), expression of TH, a marker of dopaminergic neurons was confirmed, and as a result, differentiation into dopaminergic neurons was more clearly observed when a combination of FA and AA, a combination of FA and NAD+, or a combination of FA, AA, and NAD+ were added to the differentiation medium than when TH was added alone. In particular, when the neural cells were differentiated in a differentiation medium supplemented with a combination of FA, AA, and NAD+, differentiation into dopaminergic neurons was most prominently observed.

[0088] As shown in FIG. 3, after differentiating FMD-NSPCs in a differentiation medium (FA: 0.1 mM) supplemented with FA alone, expression of TH, a marker of dopaminergic neurons, and Tuj1, a marker of neural cells was confirmed, and as a result, as Tuj1 expression increased over time, stem cell characteristics decreased as differentiation progressed, and mature neurons increased due to gradual differentiation of proliferating cells. On the other hand, expression of TH decreased over time, confirming that differentiation into dopaminergic neurons was reduced.

[0089] As shown in FIG. 4, after differentiating FMD-NSPCs obtained in subculture 9 in a differentiation medium supplemented with a combination of FA and AA (a), or a combination of FA and NAD+ (b) (FA: 0.1 mM, AA: 0.2 mM, and NAD+: 1 mM), expression of TH, a marker of dopaminergic neurons and Tuj1, a marker of neural cells was confirmed, and as a result of comparing with a case in which FA was added alone (compared with FIG. 3), it was confirmed that Tuj1 expression increased to a similar degree, but TH expression was maintained at a constant level without decreasing. That is, when neural stem cells were differentiated in a differentiation medium supplemented with a combination of FA and AA, or a combination of FA and NAD+, compared to a differentiation medium supplemented with FA alone, it was confirmed that differentiation of neural stem cells into dopaminergic neurons was increased.

[0090] As shown in FIG. 5, as a result of comparing expressions of TH, a marker of dopaminergic neurons, after differentiating FMD-NSPCs obtained in subculture 12 in a differentiation medium supplemented with FA alone or a combination of FA and AA, (FA: 0.1 mM, and AA: 0.2 mM), when the neural stem cells isolated from middle stage subcultures were differentiated in a medium supplemented with a combination of FA and AA, than in a medium supplemented with FA alone, differentiation efficiency of neural stem cells into dopaminergic neurons was confirmed to be slightly increased.

[0091] As shown in FIG. 6, as a result of comparing expressions of TH, a marker of dopaminergic neurons, after differentiating FMD-NSPCs obtained in subculture 14 (a) and subculture 15 (b) in a differentiation medium supplemented with FA alone or a combination of FA and NAD+ (FA: 0.1 mM, NAD+ 0.5: 0.5 mM, NAD+ 1.0: 1 mM), when the neural stem cells were differentiated in a medium supplemented with a combination of FA and NAD+, than in a medium supplemented with FA alone, differentiation efficiency of neural stem cells into dopaminergic neurons was confirmed to be slightly increased.

[0092] As shown in FIG. 7, as a result of comparing expressions of TH, a marker of dopaminergic neurons, after differentiating FMD-NSPCs obtained in subculture 16 (a), subculture 17 (b), subculture 18 (c), and subculture 19 (d) in a differentiation medium supplemented with FA alone, or a combination of FA and AA (FA: 0.1 mM, and AA: 0.2 mM), FMD-NSPCs isolated from late stage subcultures did not show significant differences in their ability to differentiate into dopaminergic neurons when differentiated in a differentiation medium supplemented with FA alone, or in a differentiation medium supplemented with a combination of FA and NAD+.

[0093] As shown in FIG. 8, as a result of comparing expressions of TH, a marker of dopaminergic neurons, after differentiating FMD-NSPCs obtained in subculture 21 in a differentiation medium supplemented with FA alone, a combination of FA and AA, or a combination of FA and NAD+, it was confirmed that even when AA or NAD+ was further added, respectively, than when FA was added alone, there was no significant effect on an ability of the neural stem cells isolated from a late stage subculture to differentiate into dopaminergic neurons.

[0094] As shown in FIG. 9, as a result of comparing expressions of TH, a marker of dopaminergic neurons, and Tju1, a marker of neural stem cells, after differentiating FMD-NSPCs obtained in subculture 10 (a), subculture 11 (b), and subculture 12 (c), in a differentiation medium supplemented with a combination of FA, AA, and NAD+, it was confirmed that as Tuj1 expression increased, neural stem cells gradually differentiated and mature neurons increased, and since TH expression increased even until day 6 of differentiation, differentiation into dopaminergic neurons was increased.

[0095] As shown in FIG. 10, as a result of comparing expressions of TH, a marker of dopaminergic neurons, and Tju1, a marker of neural stem cells, after differentiating FMD-NSPCs obtained in subculture 17, and subculture 19, in a differentiation medium supplemented with FA alone, or a combination of FA, AA, and NAD+ (FA:0.1 mM, AA: 0.2 mM, and NAD+: 1 mM), even when differentiating neural stem cells isolated from late stage subcultures, when the neural stem cells were differentiated in a differentiation medium supplemented with FA, AA, and NAD+, than in a medium supplemented with FA alone, not only expression of TH was stronger, but also the differentiated cells showed a shape of a more mature neural cell in the development of the axon of a neural cell, and interconnection with the surrounding neural cells.

[0096] As shown in FIG. 11, after differentiating FMD-NPCs in a differentiation medium supplemented with FA alone, or a combination of FA, AA, and NAD+, expression of TH, a marker of dopaminergic neurons, was confirmed by measuring fluorescence intensity. For each group, neural stem cells (A) subcultured less than 10 times, (B) subcultured 10 or more times and 20 or less times, (C) subcultured 20 or more times were used to compare average values, to compare and analyze differentiation rates according to a subcultured number.

[0097] As a result, in cells treated with FA alone, a rate of differentiation into TH gradually decreased as the cells were subcultured.

[0098] In contrast, among the cells differentiated in the differentiation medium supplemented with a combination of FA, AA, and NAD+, there was no difference of differentiation rates in cells subcultured less than 10 times when using differentiation media supplemented with FA, and FA, AA, and NAD+.

[0099] In addition, as a result of differentiating cells subcultured 10 or more times and 20 or less times, it was confirmed that the differentiation rate was improved by about 2 times in the differentiation medium supplemented with FA, AA, and NAD+ than in the differentiation medium supplemented with FA alone, and about 1.3-fold improvement in the differentiation rate was confirmed in the cells subcultured 20 or more times.

[0100] From the above results, it was found that the differentiation of neural stem cells subcultured less than 10 times was less affected. In addition, neural stem cells subcultured 10 or more times and less than 20 times were most affected by the differentiation medium supplemented with a combination of FA, AA, and NAD+, and it was also confirmed that the differentiation rate increased in the cells subcultured 20 or more times in a differentiation medium supplemented with FA, AA, and NAD+. Therefore, it was confirmed that a differentiation rate of neural stem cells into dopaminergic cells, which gradually decreases when subcultured numbers increase, may be maintained or enhanced by culturing the neural stem cells in a differentiation medium supplemented with a combination of FA, AA, and NAD+.

[0101] Moreover, under the differentiation condition including a combination of FA, AA and NAD+, compared to the differentiation condition including FA alone, expression of NAD+ dependent histone deacetylases (SIRT1), which are known to inhibit intracellular protein entanglement and apoptosis caused by reactive oxygen stress, a pathogenesis of Parkinson's disease, may be maintained for a longer period of time. Therefore, it may be seen that the enhancement of an ability to differentiate into dopaminergic neurons by the combination of FA, AA, and NAD+ was achieved through increased SIRT1 activity.