JP2005218308A - Proliferation auxiliary for neural stem cell and culture medium for neural stem cell containing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a proliferation auxiliary increasing the proliferation rate of a neural stem cell, having a low anticoagulant activity and safe even when administered into the body of a patient or accumulated in the body. <P>SOLUTION: The proliferation auxiliary is a glycosaminoglycan in which the 6-position of D-glucosamine constituting a heparin is substantially desulfated and has ≤50 s APTT (activated partial thromboplastin time) when measurement is made by adding the glycosaminoglycan to the standard plasma at 30 μg/ml final concentration and ≤100 s, more preferably ≤80 s APTT when the measurement is made by adding the glycosaminoglycan at 100 μg/ml final concentration. The proliferation auxiliary is suitably used for culturing the neural stem cell for transplantation into the living body in vitro. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention is capable of enhancing the proliferation ability of neural stem cells that can be used for the treatment of neurological diseases, in particular, neural stem cells for transplantation, and proliferation that can efficiently provide safe growth assistants and safe neural stem cells to transplant patients. Relates to the medium.

  In recent years, transplantation of neural stem cells to patients has been proposed as a treatment method for central nervous system diseases caused by acute brain injury such as stroke, trauma, and hypoxia, such as Alzheimer's disease, Huntington's disease, and Parkinson's disease. Yes.

  Neural stem cells are transplanted into a living body such as a patient, and then engraft in the patient, where they differentiate and supply damaged or lost neural tissue, such as supplying dopaminergic neurons that are lost in Parkinson's disease. It becomes possible to renew in the patient's body. Therefore, such a treatment method using neural stem cell transplantation is expected as a treatment method for neurological diseases.

  The neural stem cells used for transplantation are preferably human-derived stem cells from the viewpoint of few side effects and rejection. In some cases, human-derived neural stem cells can be collected from the patient's own nerve tissue, for example, peripheral nerve tissue. There are many cases to use.

  At present, fetal-derived neural stem cells cannot obtain a sufficient amount of neural stem cells that meet the requirements due to a lack of donors. Even if it can be collected from the patient itself, the amount is very small.

  For this reason, in treatment, there is no choice but to proliferate a sufficient amount of neural stem cells in vitro and transplant a part thereof.

  For culturing neural stem cells, a medium in which basic factors including serum, sugar, amino acids, vitamins, and salts necessary for cell growth are usually added to growth factors such as basic fibroblast growth factor (bFGF). Incubate.

  Furthermore, in recent years, it has been proposed to culture in a medium supplemented with other growth factors or glycosaminoglycans as components for increasing the growth rate. For example, Patent Document 1 discloses the addition of epidermal growth factor (EGF) and heparan sulfate as a method for regulating proliferation of neural stem cells in vitro. Patent Document 2 proposes to add leukocyte migration inhibitory factor (LIF) in addition to EGF and bFGF. Further, Patent Document 2 proposes adding heparin in addition to EGF, bFGF, and LIF.

  It is possible to increase the proliferation rate of neural stem cells by adding such various growth factors and glycosaminoglycans. However, the rate of increase in proliferation rate differs depending on the type of glycosaminoglycan, and the growth rate used in combination. The degree of increase in growth rate varies depending on the type of factor.

  On the other hand, it may be preferable to remove components that are preferably added as components that promote cell growth during the culture. For example, if glycosaminoglycan having high anticoagulability such as heparin is administered to a patient's body while attached to neural stem cells, it is very dangerous in the case of intracerebral hemorrhage.

  Therefore, when cultured cells are used for transplantation, washing is essential after cell separation from the medium, but growth factor receptors exist on the cell surface, and growth factors bound to the receptors are completely removed by washing. Often cannot be adsorbed on the cell surface. Furthermore, since many glycosaminoglycans have high affinity with growth factors, they cannot be removed by washing with physiological saline. Although the number of washings can be increased and the washing solution can be devised, new problems such as damage to neural stem cells and a decrease in yield due to washing are caused.

Special table flat 10-509592 Special table 2002-518990

  The present invention has been made in view of the circumstances as described above, and an object thereof is to increase the proliferation rate of neural stem cells, and even when administered into a patient or to accumulate in the body. It is to provide a safe growth aid even if it is done.

  The inventors of the present invention have completed the present invention as a result of intensive studies on the content of sulfate groups, the position of sulfate groups, the ability to promote proliferation of neural stem cells, anticoagulant activity, etc. for various glycosaminoglycans.

  That is, the neural stem cell proliferation aid of the present invention contains a desulfated glycosaminoglycan in which the 6-position sulfate group of the D-glucosamine residue constituting the sulfated glycosaminoglycan is substantially desulfated. .

Specifically, the desulfated glycosaminoglycan preferably has the following characteristics (a), (b) and (c).
(A) 2-acetamido-2-deoxy-4-O- (4-) in an unsaturated disaccharide composition obtained by disaccharide analysis in which degradation by glycosaminoglycan degrading enzyme and analysis by high performance liquid chromatography are combined. Deoxy-α-L-threo-hex-4-enopyranosyluronic acid) -6-O-sulfo-D-glucose, 2-deoxy-2-sulfamino-4-O- (4-deoxy-α-L -Threo-hex-4-enopyranosyluronic acid) -6-O-sulfo-D-glucose, 2-acetamido-2-deoxy-4-O- (4-deoxy-2-O-sulfo-α- L-threo-hex-4-enopyranosyluronic acid) -6-O-sulfo-D-glucose and 2-deoxy-2-sulfamino-4-O- (4-deoxy-2-O-sulfo- -L-threo-hex-4-enopyranosyluronic acid) -6-O-sulfo-D-glucose is 10 mol% or less, and 2-deoxy-2-sulfamino-4-O- ( 4-deoxy-2-O-sulfo-α-L-threo-hex-4-enopyranosyluronic acid) -6-O-sulfo-D-glucose is 1.5 mol% or less, The sugar yield is 60% or more. (B) The weight average molecular weight is 3000 to 30000 Dalton.

(C) The activated partial thromboplastin time (APTT) when measured by adding to standard plasma at a final concentration of 30 μg / ml is 50 seconds or less, or measured by adding to standard plasma at a final concentration of 100 μg / ml The activated partial thromboplastin time (APTT) is 100 seconds or less, more preferably 80 seconds or less.

    The neural stem cells are preferably derived from humans, and are preferably used for culturing neural stem cells for living transplantation in vitro.

  Moreover, it is preferable that the neural stem cell proliferation assistant of the present invention is used when neural stem cells are cultured in vitro in the presence of bFGF, EGF or LIF.

  The neural stem cell culture medium of the present invention contains the neural stem cell growth aid of any of the above inventions, and is preferably a known medium containing inorganic salts, carbohydrates, hormones, essential amino acids, and vitamins necessary for the proliferation of neural stem cells. BFGF is added to the basic growth medium, and the above-mentioned neural stem cell growth assistant of the present invention is added at a final concentration of 0.01 to 100 μg / ml.

  The neural stem cell proliferation aid of the present invention has a growth rate equal to or higher than that of heparin, which is conventionally known as a neural stem cell proliferation aid, and has low anticoagulant activity. Alternatively, even if transplanted into a living body while adhering to neural stem cells and remaining without being metabolized in the body thereafter, blood coagulation does not occur, and there is no adverse effect such as showing a bleeding tendency. Therefore, the neural stem cells cultured using the nerve growth aid of the present invention can be used for transplantation for the treatment of human neurological diseases with a washing that does not cause cell damage or a decrease in yield.

  In the neural stem cell proliferation aid of the present invention, the O-6 position sulfate group of the D-glucosamine residue having a sulfate group constituting a sulfated glycosaminoglycan such as heparin, heparan sulfate, sulfated hyaluronic acid is desulfated. Desulfated glycosaminoglycan is an active ingredient. This is because the desulfated glycosaminoglycan in which the O-6 sulfate group of the D-glucosamine residue is desulfurized has low anticoagulant activity and has little effect on intracerebral hemorrhage and the like.

  Here, the sulfated glycosaminoglycan is a glycosaminoglycan in which an amino sugar residue is an N-acetyl, N-sulfuric acid and / or O-sulfuric acid substitute of D-glucosamine and has a sulfate group. The hexuronic acid residue means D-glucuronic acid or L-iduronic acid, or those in which these hydroxyl groups are sulfated, and specifically include heparin, heparan sulfate, sulfated hyaluronic acid. The In particular, heparin is a glycosaminoglycan that is mainly present in mammalian mast cells, and consists of repeating units of disaccharides of hexuronic acid (D-glucuronic acid or L-iduronic acid) and glucosamine, and N-2 of glucosamine. In addition to the position, most of the O-6 position of glucosamine and the O-2 position of hexuronic acid are sulfated and have 2.5 to 3 sulfate groups per carboxyl group 1.

  In the present invention, “desulfated” does not mean only artificially removing the sulfate group of sulfated glycosaminoglycan, but usually at a specific position that is often sulfated. It means that there is no sulfate group. Therefore, as the compound in which the 6-position sulfate group of the D-glucosamine residue having a sulfate group constituting sulfated glycosaminoglycan is desulfated, naturally occurring heparan sulfate; artificially desulfated sulfate group Heparin: Desulfated glycosaminoglycan (hereinafter referred to as “6DSH”), which is artificially desulfurized only at the O-6 sulfate group of the D-glucosamine residue, is mentioned below. From the viewpoint of contribution to increase in the growth rate, 6DSH is preferably used.

6DSH can be produced by the method disclosed in WO00 / 06608. For example, a pyridine soluble salt of heparin is bound to the 6-position hydroxyl group of a glucosamine residue in a chemical disaccharide analysis method at 100 ° C. or higher in pyridine in the presence of N-methyl-N- (trimethylsilyl) trifluoroacetamide (MTSTFA). The mixture is heated for a sufficient time so that substantially no sulfate groups are detected (90 to 150 minutes at about 100 to 115 ° C.), pyridine is distilled off from the resulting reaction mixture, water is added, and (Usually 10 ⁻² to 10 ⁻⁴ Torr).

  The chemical disaccharide analysis method referred to in this specification is described in WO96 / 01278, WO00 / 06608 and J.P. Biochem. (1998) 123, 240-246.

  The 6DSH produced in this manner is digested with heparin digestion enzymes (heparinase, heparitinase I, II), and the resulting unsaturated disaccharide is analyzed by high performance liquid chromatography (HPLC). Total amount of unsaturated disaccharide [2-acetamido-2-deoxy-4-O- (4-deoxy-α-L-threo-hex-4-enopyracyluronic acid) -D-glucose (hereinafter “ΔDiHS-0S”) 2-acetamido-2-deoxy-4-O- (4-deoxy-α-L-threo-hex-4-enopyrazyluronic acid) -6-O-sulfo-D-glucose (hereinafter “Δ”). DiHS-6S ”), 2-deoxy-2-sulfamino-4-O- (4-deoxy-α-L-threo-hex-4-enopyracyluronic acid) -D Glucose (hereinafter referred to as “ΔDiHS-NS”), 2-acetamido-2-deoxy-4-O- (4-deoxy-2-O-sulfo-α-L-threo-hex-4-enopyracyluronic acid) -D-glucose (hereinafter referred to as “ΔDiHS-US”), 2-deoxy-2-sulfamino-4-O- (4-deoxy-α-L-threo-hex-4-enopyracyluronic acid) -6 -O-sulfo-D-glucose (hereinafter referred to as "ΔDiHS-di (6, N) S"), 2-deoxy-2-sulfamino-4-O- (4-deoxy-2-O-sulfo- α-L-threo-hex-4-enopyracyluronic acid) -D-glucose (hereinafter referred to as “ΔDiHS-di (U, N) S”), 2-acetamido-2-deoxy-4-O— (4-deoxy 2-O-sulfo-α-L-threo-hex-4-enopyracyluronic acid) -6-O-sulfo-D-glucose (hereinafter referred to as “ΔDiHS-di (U, 6) S”), 2 -Deoxy-2-sulfamino-4-O- (4-deoxy-2-O-sulfo-α-L-threo-hex-4-enopyrazyluronic acid) -6-O-sulfo-D-glucose Mole% total of “ΔDiHS-tri (U, 6, N) S”) is defined as 100%, and an unsaturated disaccharide (ΔDiHS-6S, ΔDiHS) containing a glucosamine residue having a sulfate group at the 6-position -Di (6, N) S, ΔDiHS-di (U, 6) S and ΔDiHS-tri (U, 6, N) S) is 10 mol% or less, preferably 5 mol% or less, ΔDiHS-tri (U, 6, N) S is 1.5 mol% or less , Preferably 1 mol% or less, and most preferably undetectable. Usually, the 6-position desulfation rate is 90% or more when calculated on the basis of standard heparin analyzed by enzymatic disaccharide analysis.

  The average molecular weight measured using gel filtration is preferably 3000 to 30000 Dalton (Da), more preferably 5000 to 20000 Da, and most preferably 7000 to 16000 Da.

  Further, the activated partial thromboplastin time (APTT) when measured by adding to standard plasma at a final concentration of 30 μg / ml is 50 seconds or less. Since the APTT time of a normal specimen is about 25 to 40 seconds, it is necessary not to exceed 100 seconds even if a large amount is accumulated in the body. Therefore, the activated partial thromboplastin time (APTT) when measured by adding to standard plasma at a final concentration of 100 μg / ml is more preferably 100 seconds or less, and still more preferably 80 seconds or less. It is 6DSH that forcibly desulfurizes nearly 100% of the O-6 position of D-glucosamine that satisfies such safety conditions.

  The neural stem cell growth aid having the above-described configuration is suitably used as a neural stem cell growth aid in vitro. The nerve cell growth aid may contain water, a buffer solution, etc. in addition to the desulfated glycosaminoglycan.

  The applied neural stem cell culture medium contains bFGF as a neural stem cell growth factor in the basic growth medium. Specifically, a basic medium (for example, Iscove modified Dulbecco medium (IMDM), RPMI, DMEM, Fischer medium, α medium) containing components (inorganic salts, carbohydrates, hormones, essential amino acids, vitamins) necessary for viable growth of cells. , Leibovitz medium, L-15 medium, NCTC medium, F-12 medium, MEM, McCoy medium) and bFGF at a final concentration of 0.001 to 0.1 μg / ml. EGF and LIF may be further added as growth factors.

  The neural stem cell growth assistant of the present invention is added to the above medium at a final concentration of 0.01 to 100 μg / ml, preferably 0.1 to 20 μg / ml. The neural stem cell proliferation aid of the present invention, particularly 6DSH, can contribute to doubling of the proliferation rate by coexistence with bFGF, but it hardly contributes to the increase of proliferation rate at less than 0.01 μg / ml, and 100 μg / ml. This is because even if the amount exceeds ml, the degree of increase in the growth rate tends to decrease, and the remaining adverse effect becomes larger.

  The culture is preferably performed for 10 days or longer. The reason is not clear, but the effect of increasing the proliferation rate by adding the neural stem cell proliferation aid of the present invention is only slightly observed in less than 10 days.

  When cultured for 2 weeks in a medium containing the neural stem cell growth aid of the present invention, the growth rate was about 1.5 times that of the case without the neural stem cell growth aid of the present invention. For example, 10 times or more cells can be obtained.

  When neural stem cells cultured in a medium containing the stem cell growth aid of the present invention are used for transplantation into a living body, cells separated from the medium and washed about 1 to 3 times with physiological saline or the like are used. In this degree of washing, most of the bFGF bound to the membrane surface remains attached, and further, the growth assistant bound to bFGF also remains attached. However, the rate of increase in the amount of liberated growth aid relative to the increase in the number of washings is small, and the adverse effects of neural stem cell damage and yield reduction due to the increase in the number of washings are greater. It is preferable. On the other hand, the neural stem cell proliferation aid of the present invention has low anticoagulant activity and has almost no effect on the blood coagulation time at about 50 μg / ml. With 6DSH, the blood coagulation time is doubled compared to normal samples. 100 μg / ml or more is required for this. Therefore, not only when the growth assistant of the present invention is transplanted together with neural stem cells, but also when it accumulates in the brain as a result of continuous administration of neural stem cells cultured in a medium containing the growth assistant of the present invention over a long period of time. Even so, it is possible to avoid the adverse effect of excessively increasing the risk of intracerebral hemorrhage or the like as compared with a normal sample.

〔Measuring method〕
First, the measurement method used in the examples of the present invention will be described.
(A) Disaccharide analysis by enzymatic digestion 1.0 mg of a sample was dissolved in 220 μl of 20 mM sodium acetate (pH 7.0) containing 2 mM calcium acetate, and 20 mU heparinase, 20 mU heparitinase I and II were added at 37 ° C. The reaction was performed for 2 hours. The obtained enzyme digestion solution, that is, the produced unsaturated disaccharide was analyzed by high performance liquid chromatography. The peak area of each unsaturated disaccharide was calculated, and the peak area relative to the total area was expressed as%.

  For analysis by HPLC, 15 μl of enzyme digestion solution was analyzed using HPLC (Rika Rika, model 852). Absorbance at 232 nm was measured using an ion exchange column (Dionex, CarboPac PA-1 column 4.0 mm × 250 mm). Based on an unsaturated disaccharide standard (manufactured by Seikagaku Corporation) (Yamada et al., J. Biol. Chem., 270, 8696-8706, (1995)), lithium chloride was used at a flow rate of 1 ml / min. The method was based on a method using a gradient system (50 mM → 2.5 M) (Kariya et al., Comp. Biochem. Physiol., 103B, 473, (1992)).

(B) Weight average molecular weight 5 μl of a 1% solution was analyzed by gel filtration by HPLC. The column was TSKgel- (G4000 + G3000 + G2500) PWX (Tosoh, 7.8 mm × 30 cm) and eluted with 0.2 M sodium chloride at 40 ° C. and a flow rate of 0.6 ml / min. For the detection, a differential refractometer (Shimadzu Corporation, AID-2A) was used.

(C) APTT time 100 μl of plasma obtained by centrifuging blood collected at 1/10 volume of 3.2% citrate from rat lower aorta at 1000 × g for 10 minutes and 100 μl of various concentrations of each sample were measured. The bottle was placed in a cup and kept warm at 37 ° C for 1 minute. Thereafter, 100 μl of actin (trade name: Mitsubishi Pharma Co., Ltd.) that had been kept warm at 37 ° C. in advance was added, and the temperature was further kept for 2 minutes. Next, 100 μl of a 0.02 M calcium chloride solution kept at 37 ° C. was added, and the time until coagulation occurred at this time was measured with a blood coagulation automatic measuring device (KC-10A: manufactured by Amelung).

(D) Endotoxin Measured by LAL kinetic method. That is, Seikagaku Corporation LS-50M set was used. 50 μl of each of the specimen, standard solution (dilution series), and blank was dispensed into the wells of a microplate (endotoxin, β-glucan free) with a chip (endotoxin, β-glucan free) or the like. 50 μl of the prepared LAL reagent was added to each well into which the specimen, standard solution, and blank were dispensed with a syringe (endotoxin, β-glucan free) or the like. The microplate was covered and set on the well reader SK601. After 1 minute of stirring, warming and measurement was started. That is, the change in absorbance with time [mAbs / min] at 37 ° C. for 30 minutes was measured at a measurement wavelength of 405 nm and a control wavelength of 492 nm.

(E) Proliferation measurement It measured by ATP method. Specifically, a 96-well plate seeded with 100 μl / well of neural stem cells at a cell density of 1 × 10 ⁵ cells / ml was used for a cell proliferation measurement kit (Celltiter-Glo ^™ Luminescent Cell Viability Assay: Promega). 75 μl / well or 100 μl / well was added, and after 30 minutes, measurement was performed with a luminescence measurement plate reader (1420 ARVO-D: manufactured by Perkin Elmer).

[Glycosaminoglycan]
The following (1) to (6) sulfated glycosaminoglycans or desulfurized oxides thereof (2DSH, 6DSH) were used. The composition of heparin glycosaminoglycan (heparin, 2DSH, 6DSH, heparan sulfate) is as shown in Table 2, and the weight average molecular weight and endotoxin of each glycosaminoglycan are as shown in Table 3.

(1) Heparin An injection grade product derived from pig small intestine manufactured by SPL of the United States was used.

(2) 2-O-desulphated-heparin (2DSH)
It was prepared by selectively desulfating the sulfate group at the O-2 position of hexuronic acid using the above heparin as a raw material. Selective desulfation is achieved by dissolving heparin sodium salt in an alkali metal hydroxide aqueous solution or alkaline earth metal hydroxide aqueous solution such as sodium hydroxide, and then freezing and lyophilizing the solution left for 20 to 30 minutes. It was prepared by the method of performing. The sample was subjected to activated carbon treatment to remove endotoxin.

(3) 6-O-desulphated-heparin (6DSH)
Using the above heparin as a raw material, the sulfate group at the O-6 position of glucosamine was prepared by selectively desulfating according to the method described in WO00 / 06608. Selective desulfation involves the pyridine soluble salt of heparin in the presence of N-methyl-N- (trimethylsilyl) trifluoroacetamide (MTSTFA) in pyridine at 100 ° C. or higher. Heat for a sufficient period of time so that the sulfate group bonded to the hydroxyl group is substantially not detected (90 to 150 minutes at about 100 to 115 ° C.), and after removing pyridine from the resulting reaction mixture, add water. Then, the pressure was reduced at normal temperature (usually 10 ⁻² to 10 ⁻⁴ Torr). The sample was subjected to activated carbon treatment to remove endotoxin.

(4) Chondroitin sulfate C (CS)
An injection grade product derived from shark cartilage manufactured at Kurihama Plant of Seikagaku Corporation was used.

(5) Dermatan sulfate (DS)
It is a chicken crown-derived material prepared at Seikagaku Corporation Central Research Laboratory.
The sample was subjected to activated carbon treatment to remove endotoxin.

(6) Heparan sulfate (HS)
After alkaline extraction from acetone defatted bovine kidney manufactured by Seikagaku Corporation, protease-digested cells were obtained, nucleic acid was removed, alcohol fraction was treated with chondroitinase ABC, and Schiller (J. Biol. Chem., 236, 983, (1961) and purified by chromatography.

[Neural stem cells]
Neural stem cells are derived from human fetuses at the age of 9 and 10 weeks of pregnancy with the approval of the Ethics Committee of the National Hospital Osaka Medical Center and Tissue Engineering Research Center. A neurosphere made into a single cell by trypsin treatment was used for measurement.

[Relationship between type of glycosaminoglycan and proliferation rate of neural stem cells]
1 × 10 ⁵ cells / ml neural stem cells were seeded in a 96-well plate and cultured.
As a culture medium, DEMEM / F12 is used as a basic medium, anti-bioticmicotic (invitrogen antibiotics, containing penicillin, streptomycin, amphotericin B), plasmocin (Invivogen antimycoplasma agent), B27 (Invitrogen serum). Trade name of substitute), FGF-2 (final concentration 0.02 μg / ml), EGF (final concentration 0.02 μg / ml), LIF (final concentration 0.01 μg / ml) and glycosamino shown in Table 3 above A medium containing no glycosaminoglycan was used as a medium to which any one of glycans (final concentration 10 μg / ml) was added, and as a control medium.

According to the ATP method, the relative cell numbers on the 1st, 6th, and 14th days of culture were measured. The results are shown in FIG.
As can be seen from FIG. 1, in chondroitin sulfate (CS) and dermatan sulfate (DS), cell growth tends to be inferior to control, and on day 14, the number of cells increased more than 6 times in control. In contrast, chondroitin sulfate and dermatan sulfate were less than 6 times. Therefore, it can be seen that the addition of heparin-based glycosaminoglycan is effective in increasing the proliferation rate of neural stem cells. Furthermore, those exceeding 8 times on the 14th day are 2DSH, 6DSH, and heparan sulfate. It can be seen that a compound having a lower sulfuric acid content than heparin can contribute to an increase in growth rate.

[Comparison of anticoagulant activity of heparin glycosaminoglycans]
Regarding the heparin glycosaminoglycan shown in Table 2, the relationship between the addition concentration and the APTT time was examined. The results are shown in Table 4 and FIG.

  Heparin is a minute amount of 1 μg / ml, and the APTT time exceeds 50 seconds of a normal sample, which proves dangerous. All the desulfated glycosaminoglycans obtained by desulfating heparin had a shorter APTT time than heparin, but 2DSH showed anticoagulant activity when it exceeded 3 μg / ml. You can see that the position is important.

  On the other hand, 6DSH and heparan sulfate were both 30 μg / ml and the APTT time was 50 seconds or less. However, looking at the concentration at which the APTT time of the normal specimen is doubled, 6DSH is not doubled even at 100 μg / ml, but heparan sulfate is doubled.

[Relationship between 6DSH concentration and growth rate]
Using DEMEM / F12 as a basic medium, anti-bioticicotic (Invitrogen antibiotics, containing penicillin, streptomycin, amphotericin B), plasmocin (Invitrogen anti-mycoplasma agent), B27 (Invitrogen's serum substitute for Invitrogen) ), LIF (final concentration 0.01 μg / ml), bFGF (final concentration 0.02 μg / ml), and 6DSH to a final concentration of 20, 2, 0.2, or 0.02 μg / ml 1 × 10 ⁵ cells / ml neural stem cells were seeded in each medium and cultured for 17 days.

For each concentration of 6DSH, the relative number of cells on the 2nd, 7th, and 17th days of culture was measured by the ATP method. The results are shown in FIG.
Further, the final concentration of 6DSH is 20, 2.0, 0.2, or 0.02 μg / ml in the same manner as described above except that EGF is added instead of bFGF as a growth factor to be added to the medium. For each medium thus added, the relative cell numbers on the second, seventh, and 17th days of culture were measured by the ATP method. The results are shown in FIG. 3 and 4, “□ (white square)” represents the second day of culture, “▲ (black triangle)” represents the seventh day of culture, and “◯ (white circle)” represents the 17th day of culture.

In FIG. 3, on the 17th day, the number of cells was different depending on the concentration of 6DSH. However, in FIG. This shows that 6DSH can contribute to an increase in proliferation rate in the coexistence with bFGF.
Moreover, it was recognized from FIG. 3 that the effect of increasing the proliferation rate was particularly great when the final concentration of 6DSH was 0.1 to 20 μg / ml.

  The neural stem cell proliferation aid of the present invention is useful for doubling the proliferation rate of neural stem cells in vitro, and is administered in a large amount or continuously in a minute amount and accumulates in the brain. Even if it is a case, the risk for intracerebral hemorrhage etc. is low, so it can be administered directly with neural stem cells as a neural stem cell growth aid, or when cultured human neural stem cells for in vitro treatment of neural diseases It can be used effectively as a growth aid.

It is a graph which shows the proliferation result of the cell by various glycosaminoglycans. It is a graph which shows the relationship between the density | concentration of various glycosaminoglycans, and APTT time. It is a graph which shows the density | concentration of 6DSH in bFGF presence, and the relationship of cell proliferation. It is a graph which shows the relationship between the density | concentration of 6DSH in the presence of EGF, and cell proliferation.

Claims (9)

A prosthetic agent for proliferation of neural stem cells, comprising a desulfated glycosaminoglycan in which the 6-position sulfate group of the D-glucosamine residue having a sulfate group constituting the sulfated glycosaminoglycan is substantially desulfated Neural stem cell proliferation aid. The neural stem cell proliferation aid according to claim 1, wherein the desulfated glycosaminoglycan has the following characteristics (a), (b) and (c).
(A) 2-acetamido-2-deoxy-4-O- (4-) in an unsaturated disaccharide composition obtained by disaccharide analysis in which degradation by glycosaminoglycan degrading enzyme and analysis by high performance liquid chromatography are combined. Deoxy-α-L-threo-hex-4-enopyranosyluronic acid) -6-O-sulfo-D-glucose, 2-deoxy-2-sulfamino-4-O- (4-deoxy-α-L -Threo-hex-4-enopyranosyluronic acid) -6-O-sulfo-D-glucose, 2-acetamido-2-deoxy-4-O- (4-deoxy-2-O-sulfo-α- L-threo-hex-4-enopyranosyluronic acid) -6-O-sulfo-D-glucose and 2-deoxy-2-sulfamino-4-O- (4-deoxy-2-O-sulfo- Total -L-threo-hex-4- perilla pyranosyl uronic acid) -6-O-sulfo -D- glucose is 10 mol% or less,
And 2-deoxy-2-sulfamino-4-O- (4-deoxy-2-O-sulfo-α-L-threo-hex-4-enopyranosyluronic acid) -6-O-sulfo-D- Glucose is 1.5 mol% or less, and the effective disaccharide yield is 60% or more,
(B) the weight average molecular weight is 3000 to 30000 Dalton;
(C) The activated partial thromboplastin time (APTT) when measured by adding to standard plasma at a final concentration of 30 μg / ml is 50 seconds or less. The neural stem cell proliferation aid according to claim 1, wherein the desulfated glycosaminoglycan has the following characteristics (a), (b) and (c).
(A) 2-acetamido-2-deoxy-4-O- (4-) in an unsaturated disaccharide composition obtained by disaccharide analysis in which degradation by glycosaminoglycan degrading enzyme and analysis by high performance liquid chromatography are combined. Deoxy-α-L-threo-hex-4-enopyranosyluronic acid) -6-O-sulfo-D-glucose, 2-deoxy-2-sulfamino-4-O- (4-deoxy-α-L -Threo-hex-4-enopyranosyluronic acid) -6-O-sulfo-D-glucose, 2-acetamido-2-deoxy-4-O- (4-deoxy-2-O-sulfo-α- L-threo-hex-4-enopyranosyluronic acid) -6-O-sulfo-D-glucose and 2-deoxy-2-sulfamino-4-O- (4-deoxy-2-O-sulfo- Total -L-threo-hex-4- perilla pyranosyl uronic acid) -6-O-sulfo -D- glucose is 10 mol% or less,
And 2-deoxy-2-sulfamino-4-O- (4-deoxy-2-O-sulfo-α-L-threo-hex-4-enopyranosyluronic acid) -6-O-sulfo-D- Glucose is 1.5 mol% or less, and the effective disaccharide yield is 60% or more,
(B) the weight average molecular weight is 3000 to 30000 Dalton;
(C) The activated partial thromboplastin time (APTT) when measured by adding to standard plasma at a final concentration of 100 μg / ml is 100 seconds or less. The neural stem cell proliferation aid according to any one of claims 1 to 3, wherein the neural stem cell is derived from a human. The neural stem cell proliferation aid according to any one of claims 1 to 4, which is used for in vitro culture. The neural stem cell proliferation aid according to any one of claims 1 to 5, wherein the neural stem cell is used for transplantation into a living body. The neural stem cell proliferation aid according to any one of claims 1 to 6, which is used when culturing neural stem cells in vitro in the presence of a basic fibroblast growth factor, epidermal growth factor or leukocyte migration inhibitory factor. A culture medium for neural stem cells comprising the neural stem cell growth aid according to any one of claims 1 to 7. A nerve in which basic fibroblast growth factor is added to the basic growth medium, and the neural stem cell growth assistant according to any one of claims 1 to 7 is added at a final concentration of 0.01 to 100 µg / ml. Medium for stem cells.

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