KR101662792B1 - Method for proliferating neuronal progenitor cells and composition for treating nervous diseases comprising the neuronal progenitor cells - Google Patents

Abstract

The present invention relates to a method for proliferating neuronal progenitor cells and a composition for treating neuronal diseases including proliferated neuronal progenitor cells. It has been found that when the fetal neural progenitor cells are cultured under hypoxic conditions and / or in a medium containing tocopherol, tocopherol acetate, or a mixture thereof, the cell proliferation rate is improved. In addition, the effect of preventing the differentiation into neurons during proliferation can contribute to the mass production of neural stem cells, and the proliferated cells are expected to be used for the treatment of neurological diseases.

Description

TECHNICAL FIELD The present invention relates to a method for proliferating neuronal progenitor cells and proliferating neuronal progenitor cells,

The present invention relates to a method for proliferating neuronal progenitor cells and a composition for treating neuronal diseases including proliferated neuronal progenitor cells.

Neurological diseases are a wide range of diseases in which central nervous system or peripheral nervous system nerve cells die or malfunction. It is known that degenerative neurological diseases are caused by extinction of neuronal cells, synaptic formation between neurons, or functional problems with age.

Neural stem cells are cells that continue to proliferate in undifferentiated state and can differentiate into various types of neuronal cells. Neural stem cells are found in various anatomical regions in the fetal period to form the nervous system and are known to not regenerate once damaged. Neural stem cells are also present in the adult central nervous system of mammals, and they proliferate and differentiate throughout their life to produce new neurons. Neural stem cells can be cultured in vitro for a long time and can be integrated into the host system by engraftment, migration and differentiation during in vivo transplantation. As a result, there is growing interest in the possibility of treating neurogenesis using neural stem cells in neurological diseases.

For the clinical application of neural stem cells, identification of the proliferation, growth, and differentiation mechanism of neural stem cells enables safe cultivation and proliferation of a sufficient number of neural stem cells for cell transplantation, and induction of proper neural cell differentiation It is necessary to establish the technology. Since neural stem cells have the ability to differentiate into desired cell types, they can be usefully used for cell therapy by improving the proliferative capacity of neural stem cells.

One aspect provides a method of growing stem cells, comprising culturing the stem cells in a medium comprising tocopherol, tocopherol acetate, or a mixture thereof.

Another aspect provides a composition for treating a neurological disease comprising neural precursor cells proliferated by the above method.

Yet another aspect provides a medium composition for the undifferentiated growth of neural progenitor cells, comprising tocopherol, tocopherol acetate, or a mixture thereof.

One aspect provides a method of growing stem cells, comprising culturing the stem cells in a medium comprising tocopherol, tocopherol acetate, or a mixture thereof.

The stem cells may be cultured under a hypoxia condition. The hypoxic condition may be, for example, 0.1% to 10%, 1% to 10%, 1% to 8%, 1% to 5%, 2% to 5%, or 2% to 3% .

The proliferated stem cells can be used to treat or prevent various diseases. The disease may be, for example, a vascular or cardiovascular disease, atherosclerosis, diabetes, aplastic anemia, ischemia, spinal cord dysgenesis, myocardial infarction, fainting disorder, multiple sclerosis, stroke, stroke, hypotension, Alzheimer's disease, Parkinson's disease, Parkinson's Disease Dementia, Huntington's disease, Ray's disease, AIDS dementia, memory loss, amyotrophic lateral sclerosis (" amyotrophic lateral sclerosis, ALS), multiple system atrophy, ischemic heart disease, brain or spinal cord injury, neurological diseases caused by spinal cord injury, heart-lung bypass, glaucoma, retinal ischemia, Niemarm-Pick, Fabry's, Gaucher's , Hunter's, lysosomal accumulation diseases such as Hurler's syndrome, mucopolysaccharidosis, glycogenosis, congenital metabolic disorder, adrenoleukodystrophy, cystic fibrosis, glycogen accumulation Pseudomonas aeruginosa, Pseudomonas aeruginosa, Pseudomonas aeruginosa, Pseudomonas aeruginosa, Pseudomonas aeruginosa, Pseudomonas aeruginosa, Pseudomonas aeruginosa, Pseudomonas aeruginosa, Pseudomonas aeruginosa, , Cancer, tumors, and other pathological or oncological conditions.

The stem cell may be, for example, a neural progenitor cell. The term "neuronal progenitor cell or neuronal precursor cell" refers to a cell capable of differentiating into various types of neuronal cells. The neural progenitor cells may be used in combination with neural stem cells. In addition, the neural progenitor cells may be cells derived from neural stem cells. The neural progenitor cells may be derived, for example, from a mammalian fetus or adult. The neural progenitor cells may be derived, for example, from the brain tissue of the fetus. The neural progenitor cells may be derived, for example, from the midbrain tissue of the fetus.

The stem cell may be, for example, grown in an undifferentiated state. The term "proliferation" means an increase in the number of cells. The undifferentiated proliferation means proliferation to cells having the same properties as the original cells without being differentiated into specific cells.

The tocopherol or tocopherol acetate in the medium may be, for example, alpha-tocopherol or alpha-tocopherol acetate. The concentration of the tocopherol, tocopherol acetate or a mixture thereof may be, for example, 0.01 to 50 μg / ml, 0.01 μg / ml to 40 μg / ml, 0.01 μg / ml to 30 μg / To 20 μg / ml, from 0.01 μg / ml to 10 μg / ml, or from 1 μg / ml to 10 μg / ml. The mixing ratio of tocopherol and tocopherol acetate in the medium is 1: 10 to 10: 1, 1: 5 to 5: 1, 1: 4 to 4: 1, 1: , Or 1: 1.

The medium may be, for example, one containing tocopherol and / or tocopherol acetate in a medium for normal cell culture. The medium for cell culture is prepared by mixing DMEM (Dulbecco's Modified Eagle's Medium, GIBCO, USA), MEM (Minimal Essential Medium, GIBCO, USA), BME (Basal Medium Eagle, GIBCO, USA), RPMI 1640 USA), DMEM / F10 (Dulbecco's Modified Eagle's Medium: GIBCO, USA), DMEM / F12 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F- (GIBCO, USA), G-MEM (Glasgow's Minimal Essential Medium, GIBCO, USA), and IMDM (Isocove's Modified Dulbecco's Medium, GIBCO, USA). The medium may further comprise an antibiotic, a B27 supplement and / or a growth factor. The antibiotic may be, for example, penicillin, streptomycin, gentamicin, or primocin ( ^TM ). The growth factor may be, for example, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) and / or fibroblast growth factor 4 (FGF4). The concentration of bFGF, EGF or FGF4 in the medium is, for example, from 2 ng / ml to 100 ng / ml, from 4 ng / ml to 80 ng / ml, from 5 ng / ml to 50 ng / ml, To 30 ng / ml.

The method may be a method of growing neural progenitor cells in which neural progenitor cells are cultured under medium and hypoxic conditions comprising tocopherol, tocopherol acetate, or a mixture thereof. The hypoxic condition may be, for example, 0.1% to 10%, 1% to 10%, 1% to 8%, 1% to 5%, 2% to 4%, or 2% to 3% . The neural progenitor cells may be proliferated in an undifferentiated state. The tocopherol or tocopherol acetate in the medium may be, for example, alpha-tocopherol or alpha-tocopherol acetate. The concentration of tocopherol, tocopherol acetate, or a mixture thereof, and the mixing ratio of tocopherol and tocopherol acetate in the culture medium are as described above. The medium may be one containing tocopherol and / or tocopherol acetate in a medium for normal cell culture. The cell culture medium is as described above. The medium may further comprise, for example, an antibiotic, a B27 supplement and / or a growth factor. The antibiotic or the growth factor is as described above.

Another aspect provides a composition for treating neurological diseases comprising neural precursor cells proliferated in a medium comprising tocopherol, tocopherol acetate, or a mixture thereof.

The neural progenitor cells may be cultured under hypoxic conditions, for example. The hypoxic condition may be, for example, 0.1% to 10%, 1% to 10%, 1% to 8%, 1% to 5%, 2% to 4%, or 2% to 3% . The tocopherol or tocopherol acetate in the medium may be, for example, alpha-tocopherol or alpha-tocopherol acetate. The concentration of tocopherol, tocopherol acetate or a mixture thereof in the medium may be, for example, 0.01 to 50 μg / ml, 0.01 to 40 μg / ml, 0.01 μg / ml to 30 μg / ml, / ml to 20 占 퐂 / ml, 0.01 占 퐂 / ml to 10 占 퐂 / ml, or 1 占 퐂 / ml to 10 占 퐂 / ml. The mixing ratio of tocopherol and tocopherol acetate in the medium is 1: 10 to 10: 1, 1: 5 to 5: 1, 1: 4 to 4: 1, 1: , Or 1: 1. The medium may be one containing tocopherol and / or tocopherol acetate in a medium for normal cell culture. The cell culture medium is as described above. The medium may further comprise, for example, an antibiotic, a B27 supplement and / or a growth factor. The antibiotic or the growth factor is as described above.

Wherein said neurological disease is selected from the group consisting of Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, polycystic dystrophy, Parkinson's dementia, stroke, ischemia and neurological disorders caused by spinal cord injury Lt; / RTI >

Yet another aspect provides a medium composition for the undifferentiated growth of neural progenitor cells, comprising tocopherol, tocopherol acetate, or a mixture thereof.

The tocopherol or tocopherol acetate in the medium composition may be, for example, alpha-tocopherol or alpha-tocopherol acetate. The concentration of tocopherol, tocopherol acetate or a mixture thereof in the medium composition may be, for example, 0.01 to 50 μg / ml, 0.01 to 40 μg / ml, 0.01 μg / ml to 30 μg / Ml to 20 μg / ml, 0.01 μg / ml to 10 μg / ml, or 1 μg / ml to 10 μg / ml. The mixing ratio of tocopherol and tocopherol acetate in the medium composition is 1: 10 to 10: 1, 1: 5 to 5: 1, 1: 4 to 4: 1, 1: 3 to 3: 1, or 1: 1. The above-mentioned culture medium composition may contain tocopherol and / or tocopherol acetate in a medium for conventional cell culture. The cell culture medium is as described above. The medium composition may further comprise, for example, an antibiotic, a B27 supplement and / or a growth factor. The antibiotic or the growth factor is as described above.

According to one aspect, the proliferation method of stem cells can contribute to the mass production of stem cells. Proliferating stem cells are expected to be useful in the treatment of various diseases.

FIG. 1 shows the result of confirming cell viability by MTT assay after culturing fetal neural progenitor cells in each medium for 7 days.
FIG. 2 shows the result (FIG. 2A) and the cell count result (FIG. 2B) of the fetal neural progenitor cells cultured in each medium for 7 days after microscopic examination.
FIGS. 3A to 3D show the results of comparing the expression rates of Ki67, Ki67 + Nestin, and Dapi markers by immunostaining after culturing fetal neural progenitor cells in each medium for 7 days.
4A to 4E show the results of comparing the expression rates of GFAP, Tuj1, GFAP + Tuj1, and Dapi markers by immunostaining after culturing fetal neural progenitor cells in each medium for 7 days.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  One. MTT  (3- (4,5-dimethylthiazol-2-yl) -2,5- Diphenyltetrazolium Bromide ) Assay for cell viability

Neuronal progenitor cells were isolated from fetuses at 14 weeks of age. Prior to obtaining the fetal samples, they received informed consent in advance and were also approved by the Cha Hospital for Institutional Research Ethics Committee for their collection and use for research purposes. Storch et al. 2001; Milosevic et al. Ventral midbrain tissue in 14-week-old fetal brain tissue was isolated and cultured in a solution containing 0.1 mg / ml of papain and 100 ug / ml of DNase at 37 < 0 > C Treated for about 30 minutes and separated into single cell suspensions. This was washed with phosphate buffered saline (PBS) and incubated at 50 / / ml antipain at 37 캜 for 30 minutes. The obtained fetal neural progenitor cells were inoculated in a single layer at a density of 30,000 cells / cm ² in a culture dish coated with 15 ㎍ / ml of poly-L-ornithine and 4 / / ml of fibronectin. Cultured fetal neural progenitor cells were inoculated in 96-well plates at 1 × 10 ⁴ cells / well, cultured for 7 days, and the cell viability was analyzed by measuring the absorbance (570 nm) after treatment with MTT reagent.

FIG. 1 shows the result of confirming cell viability by MTT assay after culturing fetal neural progenitor cells in each medium for 7 days. Antibiotics 20 ng / ml of bFGF and EGF (Peprotech, R & D system) and 10 ml of B27 supplement (GIBCO) were added to a mixed medium of 500 ml DMEM / F12 (1: 1) supplemented with 0.5 ml of primocin, To this was added 1 μg / ml of DL-alpha-tocopherol acetate (M3) or 1 μg / ml of alpha-tocopherol ((±) -alpha-tocopherol) ), Or both (M5), and these were compared with the general culture conditions. The culture medium containing 20 μg / ml of each of bFGF and EGF and α supplement (2.5 mg insulin, 25 mg transferrin, 0.03 μM sodium selenite, 0.2 mg / ml) was added to the mixed medium of 500 ml of DMEM / F12 (M1), and B27 supplement was further added to the cells (M2). The cells were cultured by adding L-glutamine (36.5 mg, 0.75 g L-glucose)

As a result, the cell viability was statistically significantly higher (P <0.02) in the medium containing alpha-tocopherol and / or DIA-alpha-tocopherol acetate compared to the normal culture conditions. In addition, the cell viability was statistically significantly higher (M3: P <0.009, M4: P <0.01, M5: P <0.001) in hypoxia of 0.1% to 10% than normoxia.

Therefore, it has been confirmed that the treatment of DIA-alpha-tocopherol acetate and / or alpha-tocopherol increases the survival rate of fetal neural progenitor cells, and has a synergistic effect especially in hypoxic culture conditions.

Example  2. Cell Count

Fetal neural progenitor cells were inoculated into 6-well plates at 2 × 10 ⁵ cells / well, cultured for 7 days, and then the number of cells was measured.

FIG. 2 shows the result of microscopic examination (FIG. 2A) and cell count result (FIG. 2B) after culturing fetal neural progenitor cells in each medium for 7 days. The conditions M1 to M5 for each of the culture conditions are as described in Example 1. Fetal neural progenitor cells showed a statistically significant increase in the number of cells (Ps <0.0001) in medium containing 1 μg / ml of alpha-tocopherol and / or 1 μg / ml of DIA-alpha-tocopherol acetate compared to the normal culture conditions . (M4 vs. M5: P <0.003, M3 vs. M5: P <0.001), whereas the number of cells was significantly increased in the hypoxic condition when both of them were added compared to the case of adding only DIA-alpha-tocopherol acetate or alpha-tocopherol P < 0.0006). Compared with the addition of only diel-alpha-tocopherol acetate, the number of cells increased statistically (M3 vs. M5: P <0.009).

As a result, the largest number of cells could be identified in the experimental group treated with DIA-alpha-tocopherol acetate and alpha-tocopherol.

Example  3: Immunocytochemical analysis 1

Fetal neural progenitor cells were inoculated on 24-well plates at 1 × 10 ⁵ cells / well for 7 days and immunocytochemistry was performed using Ki67, Nestin, and DAPI markers.

FIGS. 3A to 3D show the results of comparing the expression rates of Ki67, Ki67 + Nestin, and Dapi markers by immunostaining after culturing fetal neural progenitor cells in each medium for 7 days.

In FIG. 3B, fetal neural progenitor cells were statistically significantly increased when DiEL-alpha-tocopherol acetate (M3), alpha-tocopherol (M4), or both (M5) were added compared to the general culture conditions M1 and M2 Ki67 expression was increased (Ps <0.0001). In the hypoxic condition, Ki67 expression was higher (M4 vs. M5: P = 0.05), but not statistically significant when both were added compared to the case of adding only alpha-tocopherol. When B27 supplement was added under normal culture conditions, Ki67 expression was increased (P <0.03) statistically compared with (M1) when B27 supplement was not added (M2).

In FIG. 3c, the frequency of expression of Ki67 and Nestin was significantly higher in the fetal neural progenitor cells than in the normal culture conditions when DiEL-alpha-tocopherol acetate, alpha-tocopherol, or both were added Ps? 0.0001). When hypoxic cultures were added, both Ki67 and Nestin were expressed more frequently (M4 vs. M5, P <0.02) when both of them were added than when α-tocopherol alone was added.

In Figure 3D, fetal neural progenitor cells were found to have higher expression of Dapi water (F _{4, 48} = 57.91, P <0.0001) under hypoxic conditions compared to general oxygen conditions. In addition, fetal neural progenitor cells exhibited a significantly higher Dapi water expression ratio (Ps ≤ 0.0001) when added with diel-alpha-tocopherol acetate, alpha-tocopherol, or both, compared to normal culture conditions. In the hypoxic condition, the Dapi water expression was statistically significantly higher in the case of addition of both of Dl-alpha-tocopherol acetate and alpha-tocopherol (M3 vs. M5, P < 0.0001, M4. M5, P < 0.0002). Fetal neural progenitor cells exhibited significantly higher Dapi water expression in hypoxic conditions compared to normal oxygen conditions when alpha-tocopherol alone or both of DIA-alpha-tocopherol acetate and alpha-tocopherol were added ( P <0.006 for M4 and P <0.0001 for M5, respectively, under hypoxic conditions compared to normal oxygen conditions).

Therefore, in the experimental group treated with DIA-alpha-tocopherol acetate and alpha-tocopherol together, the expression of Dapi water, the proportion of cells showing Ki67 positive as a cell proliferation marker, or the proportion of cells showing Ki67 positive and Nestin positive neural progenitor cell markers (Fig. 3B to Fig. 3D).

Example  4: Immunocytochemical analysis 2

Fetal neural progenitor cells were inoculated into 24 well plates at 1 × 10 ⁵ cells / well for 7 days and immunocytochemistry was performed using GFAP, Tuj1, and DAPI markers.

4A to 4E show the results of comparing the expression rates of GFAP, Tuj1, GFAP + Tuj1, and Dapi markers by immunostaining after culturing fetal neural progenitor cells in each medium for 7 days.

In FIG. 4B, fetal neural progenitor cells were statistically significantly increased when DiEL-alpha-tocopherol acetate (M3), alpha-tocopherol (M4), or both (M5) were added compared to the general culture conditions M1 and M2 GFAP expression was decreased (Ps <0.04). In addition, GFAP expression was lower in the case of B27 supplement (M2) than in the absence of B27 supplement (M1) in the normal culture conditions. (Hypoxic condition: P <0.006, normal oxygen condition: P <0.007).

In Fig. 4C, fetal neural progenitor cells were statistically significantly increased when DiEL-alpha-tocopherol acetate (M3), alpha-tocopherol (M4), or both (M5) were added compared to the general culture conditions M1 and M2 Tuj1 expression was decreased (Ps <0.02). In addition, the expression of Tuj1 was lower in the case of B27 supplement (M2) than in the absence of B27 supplement (M1) under normal culture conditions. (Hypoxic condition: P < 0.008).

In FIG. 4d, the frequency of expression of GFAP and Tuj1 was lower in the fetal neural progenitor cells than in the normal culture conditions, when Diel-alpha-tocopherol acetate, alpha-tocopherol, or both were added Ps < 0.0001). In addition, in the case of B27 supplementation (M2), the frequency of expression of GFAP and Tuj1 together was lower than in the absence of B27 supplement (M1) (under hypoxic conditions: P <0.003, Oxygen condition: P < 0.001).

In Fig. 4E, the expression of Dapi water was significantly increased (P < 0.0001) in the fetal neural progenitor cells when Diel-alpha-tocopherol acetate, alpha-tocopherol, or both were added compared to the normal culture conditions. In addition, the addition of B27 supplement (M2) showed higher Dapi (normal oxygen condition: Ps <0.008) compared to M1 without addition of B27 supplement. In the fetal neural progenitor cells, Dapi-expressing cells were significantly higher (M4: P <0.006) when compared with the normal oxygen condition under hypoxic condition when α-tocopherol alone was added.

Therefore, when DIA-alpha-tocopherol acetate, alpha-tocopherol, or both were added, expression of GFAP-positive cells, a neuronal cell marker, and Tuj1-positive cells, which are neuronal markers, was lower than that of a normal culture condition and Dapi was highly expressed Respectively.

Claims (12)

Cultured human fetal midbrain-derived neural progenitor cells in a serum-free medium containing a mixture of tocopherol at a concentration of 0.01 μg / ml to 10 μg / ml and tocopherol acetate at a concentration of 0.01 μg / ml to 10 μg / ml , Wherein the medium does not contain lecithin and phosphatidylcholine. &Lt; Desc / Clms Page number 19 &gt; The method according to claim 1, wherein the human fetal midbrain-derived neural progenitor cells are cultured under a hypoxia condition. delete The method according to claim 1, wherein the human fetal midbrain-derived neural progenitor cells are proliferated in an undifferentiated state. delete The method according to claim 1, wherein the mixing ratio of tocopherol and tocopherol acetate in the medium is 1: 5 to 5: 1. The method of claim 1, wherein the medium further comprises a basic fibroblast growth factor (bFGF) and an epidermal growth factor (EGF). 8. The method of claim 7, wherein the medium further comprises FGF4 (fibroblast growth factor 4). 3. The method of claim 2, wherein the hypoxic condition is 0.1% to 10% oxygen in air. delete delete Wherein the composition comprises a mixture of tocopherol in a concentration from 0.01 μg / ml to 10 μg / ml and tocopherol acetate in a concentration from 0.01 μg / ml to 10 μg / ml and does not comprise lecithin and phosphatidylcholine. A serum-free medium composition for undifferentiated proliferation of human fetal midbrain-derived neural progenitor cells.

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