JP2005278641A - Method for inducing differentiation of nerve stem cell and differentiation-inducing culture medium and differentiation-inducing agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for inducing the differentiation of a nerve stem cell, by which the differentiation of the nerve stem cell into neurons, especially acetylcholinergic neurons, can be induced at a high rate, and to provide a culture medium for inducing the differentiation. <P>SOLUTION: This method for inducing the differentiation of the nerve stem cell comprises adding chondroitinase to a nerve stem cell culture medium during culturing, or adding neurocan and chondroitinase to the nerve stem cell. The chondroitinase is preferably chondroitinase ABC or chondroitinase AC. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for inducing differentiation of neural stem cells in vitro, a medium capable of inducing differentiation, and a differentiation inducing agent, and particularly relates to a differentiation inducing method, a differentiation inducing medium and a differentiation inducing agent having a high differentiation ratio into neurons.

  It has been reported that transplantation of fetal nerve tissue is effective in the treatment of neurological diseases such as Parkinson's disease and spinal cord injury. However, due to lack of donors and ethical issues, nerves instead of fetal nerve tissue are reported. Transplantation of stem cells is also being considered.

  As a method of treating nerve regeneration by transplantation, 1) a method in which neural stem cells amplified in vitro are transplanted and differentiated into a target damaged nerve cell / tissue in a patient body, and 2) a target neural cell in advance in vitro. There is a method of differentiating into tissues and transplanting the obtained nerve cells / tissues.

  When neural stem cells themselves are transplanted, it is difficult to accurately control the final differentiation of the transplanted cells, and the target nerve cells may not be sufficiently induced to differentiate in vivo. For example, it has been reported that when neural stem cells are transplanted into the hippocampus, they can differentiate into neurons, but when transplanted into the striatum or cerebral cortex, they remain almost undifferentiated and only a few can differentiate into neurons ( Non-patent document 1). Therefore, in order to enhance the transplantation effect, a method of inducing differentiation of neural stem cells into certain nerve cells in vitro and transplanting them in advance is desired.

  As a method for inducing differentiation of neural stem cells in culture, for example, neural stem cell growth factors (LIF, EGF, FGF2, etc.) are excluded from the typical neurosphere method as a neural stem cell proliferation culture method, and 1% bovine serum is used instead. And 1 μM retinoic acid in a culture medium, it has been reported that neurons are about 20% and astrocytes are about 80% (Non-patent Document 2).

  On the other hand, as nerve cells / tissues for treating neurological diseases, the types of damaged nerve cells differ depending on the type of disease. For example, vascular diseases such as cerebral infarction and cerebral contusion and traumatic diseases require neurons and glial cells, and degenerative diseases such as Parkinson's disease and Alzheimer's disease require specific neurons. Therefore, in order to supply neurons for the treatment of degenerative diseases such as Parkinson's disease and Alzheimer's disease, a differentiation induction method with a high ratio of inducing differentiation into neurons is desired.

  Furthermore, these degenerative diseases differ in the target neurons. For example, Parkinson's disease requires dopaminergic neurons, Huntington's disease requires GABAergic neurons, and Alzheimer's disease requires cholinergic neurons. Therefore, in order to increase the productivity of nerve tissue for nerve cell / tissue treatment, it is desired to establish a method for inducing differentiation into specific neurons. Here, when differentiation is induced using a medium supplemented with the above-mentioned serum, it is known that most of the induced neurons are GABAergic neurons, and Alzheimer's disease has a high proportion of diseased persons. For the treatment of Parkinson's disease, a differentiation induction method capable of preferentially inducing dopaminergic neurons and cholinergic neurons is strongly desired.

    As a differentiation induction method for obtaining a large number of specific neurons, for example, Patent Document 1 discloses that embryonic stem cells are cultured in suspension in the presence or absence of noggin protein to form embryoid bodies, which are used as fibroblasts. A method of selectively producing motor neurons and GABAergic neurons by floating culture in the presence of a growth factor and sonic hedgehog protein, culturing them into neural stem cells, and then differentiating them is disclosed.

  Regarding the induction of differentiation into dopaminergic neurons, it has been reported that dopaminergic neurons were differentiated when mouse ES cells were co-cultured with stromal cells (PA6 cells) in serum-free medium in the absence of retinoic acid. (Non-patent Document 3).

  It has also been reported that cholinergic neurons can be obtained when cultured in a medium containing bFGF, heparin, and laminin (Non-patent Document 4).

JP 2002-291469 A Fricker, R.M. A. Et al. Neurosci. 1999, 19: 5990-6005. Kanemura, Y et al. Neurosci. Res. , 2002, 69: 869-879. Kawasaki, H et al .: Neuron. 2000, 28: 31-40 Wu, P .; Et al .: Nature Neuroscience, 2002, 5: 1271-1278.

  The present invention has been made in view of the circumstances as described above, and an object thereof is to provide a new differentiation induction method from neural stem cells, particularly a method capable of inducing differentiation of neurons at a high rate, a medium, and a differentiation inducer. It is to provide.

  The first method for inducing differentiation of neural stem cells of the present invention is a method of adding chondroitinase to the neural stem cell culture medium in culture.

  In the first and second methods, the neural stem cell medium preferably contains no serum, and the basic medium contains basic fibroblast growth factor, epidermal growth factor or leukocyte migration inhibitory factor It is preferable that

  The second method of inducing differentiation of neural stem cells of the present invention is a method of adding neurocan and chondroitinase to neural stem cells, and the third method of inducing differentiation is obtained by degrading neurocan by chondroitinase. It is the method of adding the protein obtained.

  In the first to third differentiation induction methods, the chondroitinase is preferably chondroitinase ABC or chondroitinase AC.

  The method for inducing differentiation of neural stem cells of the present invention is a method for inducing differentiation of cholinergic neurons, GABAergic neurons, and glutamatergic neurons.

  The neural stem cell differentiation-inducing medium of the present invention contains a conditioned medium and chondroitinase in which neural stem cells and / or neural precursor cells are cultured. The chondroitinase is preferably chondroitinase ABC or chondroitinase AC.

  The neural stem cell differentiation inducer of the present invention contains, as a main component, a protein obtained by treating chondroitinase with chondroitin sulfate proteoglycan secreted by neural stem cells and / or neural precursor cells. The chondroitinase is preferably chondroitinase ABC or chondroitinase AC.

  In another aspect of the present invention, a neural stem cell differentiation inducer contains a supernatant of a neural stem cell growth medium in which neural stem cells and / or neural precursor cells have been cultured overnight or longer and chondroitinase.

  Furthermore, the neural stem cell differentiation inducer according to another aspect of the present invention contains chondroitinase and neurocan, and may contain a protein obtained by degrading neurocan chondroitinase as an active ingredient. .

  The “neural stem cell” referred to in the present invention refers to a cell having self-proliferating ability and the ability to differentiate by differentiation induction, and the “neural progenitor cell” is a cell divided from a neural stem cell. A cell that has not differentiated but differentiates after one or two divisions.

  The method for inducing differentiation of neural stem cells according to the present invention is a simple method such as adding chondroitinase or a combination of chondroitinase and neurocan, or a chondroitinase degradation product of neurocan, and also neurons, particularly cholinergic agonists. High differentiation rate into sex neurons. In addition, when the neural stem cell differentiation-inducing medium of the present invention is used, neurons can be obtained in a higher proportion as differentiated cells than in a medium supplemented with conventional serum. Furthermore, if the neural stem cell differentiation inducer of the present invention is used, it is possible to induce differentiation with a high neuron ratio.

The neural stem cells and neural progenitor cells used in the present invention are both human-derived.
The first differentiation inducing method of the present invention is a method for inducing differentiation of neural stem cells by adding chondroitinase to the neural stem cell medium in culture.

  As the neural stem cell medium that can be used in the method of the present invention, a known medium used as a neural stem cell growth medium can be used. 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) as basic growth factors (bFGF), epidermal growth factor (EGF) or leukocyte migration inhibitory factor ( A medium supplemented with at least one of LIF) is used, and preferably contains all of these growth factors. In addition, in order to increase the growth rate, an additive of insulin, progesterone, plarescine, transferrin, selenite, or a B27 additive or an N2 additive containing an additive for nerve cell culture, and if necessary, heparin Or heparan sulfate or these desulfated glycosaminoglycans may be contained. Moreover, the antibiotic may be contained as needed.

  Examples of commercially available products include NS-A medium manufactured by Euroclone, Neutral Progenitor Basal Medium (NPBMTM) manufactured by Cambridge, and the like.

  On the other hand, it is preferable that the medium does not contain serum. This is because the serum contains an unknown differentiation-inducing agent, and the neuron ratio of the present invention can inhibit differentiation. Furthermore, since the induction ratio of GABAergic neurons can be increased by the presence of serum, it is preferable that no serum is present when it is desired to induce neurons other than GABAergic neurons preferentially.

  The culture time in the medium having the above composition is not particularly limited, but it may be cultured for at least −night, preferably −day and night, more preferably about 2 days after replanting with a new medium. preferable. As will be described later, in the differentiation induction method of the present invention, the added chondroitinase is collectively referred to as “hNSPC” when it does not distinguish between neural stem cells and / or neural progenitor cells in culture (hereinafter, neural stem cells and neural progenitor cells). The chondroitin sulfate proteoglycan secreted by the enzyme, and the resulting degradation product acts as a differentiation inducer of hNSPC. Therefore, the chondroitin sulfate proteoglycan needs to be cultured only for the time required for secretion of chondroitin sulfate proteoglycan. is there.

  Chondroitinase is added to such culture medium. Chondroitinase is a kind of enzyme that decomposes and removes chondroitin sulfate groups from chondroitin sulfate proteoglycans. The reaction mechanism is chondroitin sulfate A, chondroitin sulfate C, dermatan sulfate, chondroitin and hyaluronic acid N-acetylhexosamini. The bond is broken to give disaccharides and oligosaccharides containing unsaturated hexuronic acid at the non-reducing end. As chondroitinase, various chondroitinases such as chondroitinase B, chondroitinase C, chondroitinase AC-I and chondroitinase AC-II are used in addition to chondroitinase ABC derived from Proteus vulgaris cells. Of these, chondroitinase ABC and chondroitinase AC are preferably used because of their high differentiation-inducing activity.

  Such chondroitinase is preferably added to the culture medium in a range of final concentration of about 1 mU / ml.

  Thus, the medium in which chondroitinase is added during the cultivation of hNSPC corresponds to the neural stem cell differentiation induction medium of the present invention. That is, the differentiation-inducing medium of the present invention contains a conditioned medium for culturing hNSPC and chondroitinase.

  The conditioned medium contains chondroitin sulfate proteoglycan, which is a secretion resulting from the cultivation of hNSPC. Therefore, the differentiation-inducing medium of the present invention to which chondroitinase is added contains a protein obtained as a result of degrading and removing chondroitin sulfate of chondroitin sulfate proteoglycan secreted by neural stem cells. This protein corresponds to the neural stem cell differentiation inducer of the present invention.

  As described above, according to the differentiation induction method of the present invention, that is, when the differentiation induction medium of the present invention is used or the differentiation inducer of the present invention is used, differentiation of neural stem cells is started. As a result of differentiation, neurons: astrocytes differentiate at a ratio of approximately 4: 5. This means that the differentiation rate into neurons is higher than in the differentiation induction using conventional serum. Furthermore, neurons obtained by differentiation induction are GABAergic neurons, cholinergic neurons, and glutamatergic neurons, and it is possible to obtain cholinergic neurons at a higher rate compared to conventional neural stem cell differentiation induction methods. it can.

  The second method for inducing differentiation of neural stem cells of the present invention is a method of adding neurocan and chondroitinase to neural stem cells. In other words, the second differentiation induction method uses a combination of neurocan and chondroitinase as a differentiation inducer. Neurocan and chondroitinase may be added separately to neural stem cells, or both may be mixed and added immediately before the addition.

  Neurocan is the substance of chondroitin sulfate proteoglycan secreted from hNSPC, and its composition and structure are disclosed in Christa K et al .: GENE, 1998, 221: 199-205, and the amino acid sequence of the protein portion. No. in the sequence list. 1.

  In the second differentiation induction method of the present invention, a neurocan that can be separated and collected from the conditioned medium of hNSPC can be used separately, so there is no need to wait for chondroitin sulfate proteoglycan to be secreted by culture. Therefore, neural stem cells to be induced to differentiate can also be applied to a state in which a cell that has just been separated and collected from a medium is seeded in a new medium. However, since neurocan needs to be degraded by chondroitinase, the target neural stem cells must exist under conditions that do not denature chondroitinase.

  The third differentiation induction method of the present invention is a method of adding a protein obtained by degrading neurocan with chondroitinase to neural stem cells. Focusing on the fact that differentiation induction is due to the action of a protein obtained by removing the sugar chain of neurocan, which is a chondroitin sulfate proteoglycan, in the third method of the present invention, the differentiation induction entity is used. A protein that is a degradation product of a chondroitinase of a certain neurocan is added as a differentiation inducer.

  While the degradation of neurocan by chondroitinase takes about one night, the third differentiation inducing method works directly on the protein part of neurocan that can induce differentiation, so it is effective for inducing differentiation early. is there.

  The protein portion obtained by the neurocan decomposition treatment with chondroitinase is shown in SEQ ID NO: 1. A protein having an amino acid sequence as shown in 1. Further, chondroitinases that can be used in the third differentiation induction method are those listed in the first differentiation induction method.

  In the second and third differentiation inducing methods, cholinergic neurons can be obtained at a high rate as in the first differentiation inducing method.

[Neural stem cells]
Neural stem cells were obtained by substituting hNSPCs taken from the forebrain of the 9th week of pregnancy with the approval of the National Hospital Organization Osaka Medical Center Ethics Committee and the National Institute of Advanced Industrial Science and Technology. Neurospheres obtained by culturing for about 4 days in a stem cell growth medium were converted into single cells by trypsin treatment and used for measurement.

[Neural stem cell culture medium]
The medium composition used in the following examples is as follows.
(A) Medium used in neurosphere method DMEM / F12 (1: 1 mixture, Sigma)
Human recombination (hereinafter abbreviated as “hr-”) EGF (Pepro Tech) 20 ng / ml
hr-FGF2 (Pepro Tech) 20 ng / ml
hr-LIF (Chemicon International) 10 ng / ml
Heparin (Sigma) 5mg / ml
B27 (Invitrogen)
HEPES15mM
Antibiotic-antimycotic (Invitrogen)

(B) hNSPC differentiation induction medium DMEM / F12 (1: 1 mixture, Sigma)
Heparin (Sigma) 5mg / ml
B27 (Invitrogen)
HEPES 15 mM (Invitrogen)
Antibiotic-antimycotic (Invitrogen)
1% fetal bovine serum (JPH Biscience)
Retinoic acid 1μM (Sigma)

(C) CMCH culture solution DMEM / F12 (1: 1 mixture, Sigma)
Heparin (Sigma) 5mg / ml
B27 (Invitrogen)
15 mM HEPES (Invitrogen)
Antibiotic-antimycotic (Invitrogen)

[Confirmation that neural stem cells synthesize chondroitin sulfate proteoglycans]
After culturing in the neural stem cell culture medium (a) for 4 days, the culture solution was collected. This culture solution was centrifuged at 20000 × g for 15 minutes to remove cells and cell debris, thereby obtaining a culture supernatant.

(1) Culture supernatant The obtained culture supernatant was blotted on a nitrocellulose membrane (Advantec) in 10 μl increments using Immunodot (ATTO).

  In order to suppress non-specific adsorption of the antibody to the nitrocellulose membrane, a nitrocellulose membrane in a solution containing 0.1% Tween 20 and PBS (hereinafter referred to as “PBS-T”) and 1% BSA is used. Was immersed for 1 hour at room temperature to perform a blocking treatment.

  A primary anti-chondroitin sulfate monoclonal antibody monoclonal anti-chondroitin sulfate clone CS-56 (Sigma) diluted 1000-fold with PBS-T was used as the primary antibody solution. A blocking-treated nitrocellulose membrane was immersed in this primary antibody solution at 4 ° C. overnight to react with the primary antibody. After the reaction, the nitrocellulose membrane was washed with PBS-T, and then a secondary antibody reaction was performed.

As a secondary antibody, goat anti-mouse IgM (μ) F (ab) ₂ antibody was used, and this secondary antibody and HRP (a cross-linked product of immunoglobulin G and horseradish peroxidase, American Qualex) were added with PBS-T. The secondary antibody solution diluted 10,000 times was reacted at room temperature for 1 hour.

  After the secondary antibody reaction, it was washed with PBS-T, chemiluminescent using an ECL • Plus kit (Amersham Biosciences), exposed to a cassette together with an X-ray film, and developed. The results are shown in FIG.

(2) Control As a control, a neural stem cell culture solution (a) in which cells were not cultured was similarly blotted on a nitrocellulose membrane, similarly blocked, and then reacted with an antibody. FIG. 1 shows the result of light emission after the antibody reaction in the same manner as (1).

  From FIG. 1, it can be confirmed that the control has no change and does not contain chondroitin sulfate proteoglycan. On the other hand, in the neural stem cell culture supernatant, dots were observed, and it was confirmed that chondroitin sulfate proteoglycan was present. Therefore, it can be confirmed that the neural stem cells secrete chondroitin sulfate proteoglycan by culture.

[Relationship between chondroitinase and differentiation induction]
Example 1:
After culturing hNSPC in neural stem cell culture medium (a) for 4 days, the culture solution was collected, and this culture solution was centrifuged at 10,000 × g for 15 minutes to obtain a culture supernatant. This culture supernatant was divided into two, and one was fractionated using a separation membrane capable of separating a compound having a molecular weight of 100,000 or more and less than 100,000. Protease-free chondroitinase ABC (Seikagaku Corporation) was added to the fraction (i) having a molecular weight of less than 100,000 and the fraction (ii) not having been heat-treated with a molecular weight of 100,000 or more so as to be 1 mU / ml. And reacted for 1.5 hours.

  To the other culture supernatant, protease-free chondroitinase ABC (Seikagaku Corporation) was added to 1 mU / ml, reacted at 37 ° C. for 1.5 hours, and then heat-treated at 100 ° C. for 10 minutes. (This heat-treated culture supernatant is referred to as (iii)).

  (I) When hNSPC was cultured in each chondroitinase-treated culture solution of (ii) and (iii), hNSPC started to differentiate only in the fraction of (ii). In the fraction (ii), 2 hours after the start of differentiation, almost all of the neurospheres in culture stuck to the bottom of the cell culture dish, and after 24 hours of culture, cells that extended protrusions were confirmed from these neurospheres. (FIG. 2). In general, since chondroitin sulfate has a molecular weight of 100,000 or less, the fraction (i) contains chondroitin sulfate, and the fraction (ii) and (iii) contains proteins from which chondroitin sulfate has been decomposed and removed by chondroitinase. include. In the fraction (iii), it is considered that the degradation product of chondroitin sulfate proteoglycan, which is considered to be a differentiation inducer produced by the added chondroitinase, has lost its differentiation induction ability by heat treatment.

  After 2 days of culture, an equal amount of the above-mentioned CMCH culture solution was added and cultured for 4 days, and then half of the CMCH solution was converted and cultured for 4 more days. Ten days after the start of the culture, as shown in FIG. 3, it was confirmed that many cells migrated and spread from the neurosphere and differentiated into cells having monopolar and bipolar processes.

Comparative Example 1:
Protease-free chondroitinase ABC (Seikagaku Corporation) was added to a new neural stem cell culture medium (a) at 1 mU / ml, and hNSPC was cultured in this culture solution.
hNSPC started to grow after the start of culture and formed neurospheres, but after 3 days, cells having protrusions were confirmed, and it was confirmed that differentiation was induced.
It can be seen that differentiation is not induced by simply adding chondroitinase to the hNSPC culture medium (a), and that secretion by hNSPC is necessary for differentiation induction.

Comparative Example 2:
The hNSPC in culture was transferred to a new neural stem cell culture medium (a), 1 mU / ml chondroitinase ABC was added, and reacted at 37 ° C. for 1 hour 30 minutes.
After the reaction, the culture solution containing chondroitinase ABC was removed, and the cells were separated. The cells were washed 3 times with DMEM / F12 (1: 1 mixture) to remove chondroitinase ABC. The washed cells were cultured in a new neural stem cell culture medium (a). After culture, growth started and neurospheres were formed. Even after 3 days from the start of culture, no differentiation was observed.
Therefore, it can be seen that even if cells are pretreated with chondroitinase ABC, differentiation cannot be induced unless chondroitinase ABC is present in the conditioned medium.

[Analysis of differentiation induction]
Example 2:
For hNSPC cultured for 10 days in Example 1 above, the culture solution was removed, and the cell population induced to differentiate was collected. This cell population was added with 4% paraformaldehyde and allowed to react at 4 ° C. for 20 minutes. After fixation with paraformaldehyde, the cells were washed 3 times with PBS for 10 minutes.

  The washed cells were mixed with PBS containing goat serum (10%) and Triton X-100 (0.01%), and a blocking reaction was performed at room temperature for 1 hour. After blocking, a primary antibody solution was added and reacted at 4 ° C. overnight. Here, as a primary antibody solution, PBS containing 10% goat serum and 0.01% Triton X-100 was used as a diluent, βIII (monoclonal antibody against βIII tubulin: BABCO) 500 times, GFAP (anti-antibody). A solution obtained by diluting glial cell fibrillary acidic protein polyclonal antibody (Sigma) by 80 times was used.

After the reaction with the primary antibody solution, the reaction solution was removed and washed with PBS. Subsequently, it was made to react with a secondary antibody solution at room temperature for 1 hour. Here, as the secondary antibody solution, Alexa anti-mouse Ig antibody 488 and Alexa anti-mouse Ig antibody 568 (both molecular probes), goat serum (10%) and Triton X-100 (0.01%) A 500-fold diluted PBS was used. Moreover, TOPRO-3 (Molecular Probes) was added to the secondary antibody solution, and nuclear staining was performed during the reaction with the secondary antibody solution.
After the reaction, the culture solution was removed, washed with PBS, further washed with ultrapure water, and sealed.

  The cells were observed using a confocal laser scanning microscope (LSM510, manufactured by Carl Zeiss). Each staining result is shown in FIGS. Moreover, as a result of analyzing the ratio of βIII positive cells that are neuronal marker molecules and GFAP positive cells that are astrocyte marker molecules based on microscopic observation, the results are shown in Table 1.

Comparative Example 3:
Neural stem cells are cultured for 10 days in the same manner as in Example 2 using a conventional differentiation-inducing medium (see (b) above) excluding growth factors from the medium used for the neurosphere method, which is a neural stem cell growth culture method. did. After the culture, primary antibody reaction and secondary antibody reaction were performed in the same manner as in Example 1, and the cells were observed using a confocal laser scanning microscope (LSM510, manufactured by Carl Zeiss). Each staining result is shown in FIGS. Moreover, as a result of analyzing the ratio of βIII positive cells that are neuron marker molecules and GFAP positive cells that are astrocyte marker molecules based on microscopic observation, the results are shown in Table 1.

Reference example 1:
As a control, the primary antibody reaction and the secondary antibody reaction were performed on the cells before being subjected to the differentiation induction medium, and the existence ratio of the initial nerve cells was examined. The results are shown in Table 1.

  From Table 1, in the conventional differentiation-inducing medium, the ratio of differentiation into glial cells was high (neuron: astrocyte = 2: 7), whereas in the differentiation-inducing medium of the present invention, the ratio of differentiation into neurons was high ( Neuron: astrocyte = 4: 5). Therefore, it can be seen that the differentiation inducing method of the present invention is a method for inducing differentiation of neurons from neural stem cells with high efficiency.

[Analysis of differentiation-induced neurons]
In order to confirm the types of β-III positive cells (neurons) obtained in Example 2, the following experiment was performed.

  For hNSPC cultured for 10 days in Example 1 above, the culture solution was removed, and the cell population induced to differentiate was collected. This cell population was added with 4% paraformaldehyde and allowed to react for 20 minutes at room temperature. After fixation with paraformaldehyde, the cells were washed 3 times with PBS for 10 minutes.

  The washed cells were subjected to a blocking reaction for 1 hour at room temperature using PBS containing 0.25% gelatin and 0.01% Triton X-100. After blocking, a primary antibody solution was added and reacted at 4 ° C. overnight. Here, the reaction of the anti-acetylcholine transferase antibody was caused to react based on the operation flow shown in FIG. 10, and the reaction with the anti-GABA antibody and the anti-glutamic acid antibody was caused to react based on the operation flow shown in FIG.

(1) Reaction with anti-acetylcholine transferase antibody As a primary antibody solution, goat anti-choline acetyltransferase (chat) polyclonal antibody (Chemicon) was added with PBS containing 0.25% gelatin and 0.01% Triton X-100 in 200%. A diluted solution was used. After making it react with this primary antibody solution at 4 degreeC overnight, the reaction liquid was removed and it wash | cleaned 3 times for 10 minutes by PBS. Subsequently, it was made to react with a secondary antibody solution at room temperature for 1 hour. Here, as the secondary antibody solution, a diluted solution obtained by diluting Alexa anti-goat Ig antibody 568 with PBS containing 0.25% gelatin and 0.01% Triton X-100 was used. After the reaction, the reaction solution was removed and washed 3 times with PBS for 10 minutes. Subsequently, after blocking reaction (room temperature, 60 minutes) with PBS containing goat serum (10%) and Triton X-100 (0.01%), β-III (monoclonal antibody against β-tubulin: Babco Was reacted with a primary antibody reaction solution diluted with PBS containing goat serum (10%) and Triton X-100 (0.01%) (room temperature, 2 hours). After the reaction, the reaction solution was removed and washed with PBS three times for 10 minutes, and then Alexa anti-goat IgG antibody 568 was washed with PBS containing goat serum (10%), Triton X-100 (0.01%), and TOPRO3. The diluted secondary antibody solution was reacted at room temperature for 60 minutes. After removing the reaction solution, the cells were washed with PBS three times for 10 minutes, and then observed using a confocal laser scanning microscope (LSM510, manufactured by Carl Zeiss).

(2) Reaction with anti-GABA antibody and anti-glutamic acid antibody First, a mixed solution of rabbit anti-GABA polyclonal antibody (Sigma) and βIII (monoclonal antibody against β-tubulin: Babco), rabbit anti-glutamic acid polyclonal antibody (Sigma) ) And β-III (Babco), a diluted solution obtained by diluting these mixtures with PBS containing goat serum (10%) and Triton X-100 (0.01%) is a primary antibody. Used as a reaction solution.

  After making this primary reaction liquid react at 4 degreeC overnight, the reaction liquid was removed and it wash | cleaned 3 times for 10 minutes by PBS. The secondary antibody reaction was then performed at room temperature for 60 minutes. As secondary antibody reaction solutions, Alexa anti-goat IgG antibody 568, Alexa anti-rabbit antibody 488 (both molecular probes), goat serum (10%), Triton X-100 (0.01%) and TOPRO- A diluted solution of 3 (Molecular Probes) diluted with PBS was used. After the reaction, the reaction solution was removed, washed with PBS three times for 10 minutes, and then the cells were observed using a confocal laser scanning microscope (LSM510, manufactured by Carl Zeiss).

(3) Observation result The existence ratio of each antibody in β-III positive cells was examined based on each labeled secondary antibody by microscopic observation. Cholinergic neurons were 33.3 ± 6.76%, GABAergic neurons were 56.2 ± 9.43%, and glutamatergic neurons were 11 ± 3.61%.

[Relationship between chondroitinase types and differentiation induction]
After culturing hNSPC in the neural stem cell culture medium (a) for 14 days, the culture solution was collected, and this culture solution was centrifuged at 1000 × g for 15 minutes to obtain a culture supernatant.

  The culture supernatant was divided into three groups, and protease free chondroitinase ABC, chondroitinase AC-I, or chondroitinase B (all from Seikagaku Corporation) was added to each group, and hNSPC Was added and cultured. The state after culturing for 24 hours was observed with a microscope. FIG. 12 shows a photomicrograph when chondroitinase ABC is added, FIG. 13 shows a photomicrograph when chondroitinase AC-I is added, and FIG. 14 shows a photomicrograph when chondroitinase B is added. .

  As can be seen by comparing FIGS. 12 to 14, sufficient differentiation induction occurred in the culture supernatant containing chondroitinase ABC and chondroitinase AC-I, but in the culture supernatant containing chondroitinase B, The differentiation induction rate was small.

[Isolation of differentiation-inducing factor]
(1) Determination of fraction having differentiation-inducing ability hNSPC was seeded in neural stem cell medium (a), cultured for 14 days, centrifuged at 10,000 xg for 15 minutes, and the supernatant was collected. This supernatant was transferred to an ultrafiltration membrane Amicon Ultra-15 (Millipore) that fractionated at a molecular weight of 1000000, and separated into a molecular weight of 1000000 or more and less than 1000000. Here, for fractions having a molecular weight of 1,000,000 or more, protein concentration was performed during fractionation, and the solvent was replaced with DEAE column equilibration buffer (50 mM Tris, 300 mM NaCl).

  The obtained fraction with a molecular weight of 1,000,000 or more was filtered through Mikeless 0.45 μm (Millipore) to remove insolubles, and then charged into an anion exchange column Hitrap DEAE FF (Amersham Biosciences). This ion exchange column was washed with DEAE column equilibration buffer, then eluted with DEAE column elution buffer 1 (50 mM Tris, 600 mM NaCl), and then eluted with DEAE column elution buffer 2 (50 mM Tris, 1 M NaCl). Surfactant polyoxyethylene laurin ether (Brij35) was added to the fraction eluted with DEAE column elution buffer 1 to a final concentration of 0.01%. To this fraction, 5 times the amount of one fraction of heparin column equilibration buffer (50 mM Tris, 0.01% Brij35) was added, and the resultant was loaded into an affinity column Hitrap heparin HP (Amarsham Biosciences). After washing with a heparin column equilibration buffer, it was connected to a chromatography system AKTAexplorer10XT (Amersham Bioscience). Using this system, a gradient elution of a heparin column elution buffer (50 mM Tris, 1 M NaCl, 0.01% Brij35) was performed from the heparin column equilibration buffer, and an elution fraction containing hNSPC differentiation-inducing molecules was collected. The elution fraction containing a differentiation-inducing molecule is contained in a fraction eluted from an elution buffer of 50 mM Tris, 350 mM NaCl, 0.01% Brij35 with an elution buffer of 50 mM Tris, 420 mM NaCl, 0.01% Brij35.

Confirmation of whether or not the elution fraction contains a differentiation-inducing molecule (confirmation of differentiation-inducing ability) is carried out by adding 2.5% to 5% of the confirmation medium with the elution fraction in a culture dish containing CMCH medium ( 50 mM Tris + 1 M NaCl + 0.01% Brij35 as a solvent) and 1 mU protease-free chondroitinase ABC (Seikagaku Corporation) were added and allowed to stand overnight in a CO ₂ incubator. This was done by observing the morphology and adhesion to the culture dish. When suspended hNSPC cell mass adheres to the bottom of the culture dish, extends the protrusion, and migration from the cell mass is observed, it is determined that hNSPC has differentiated, that is, the eluted fraction has differentiation-inducing ability. did.

(2) Isolation and purification of molecule having differentiation-inducing ability The elution fraction with differentiation-inducing ability collected in (1) was diluted 10-fold with a heparin column equilibration buffer. This diluted solution was packed in an anion column MONOQ column (Amersham Bioscience). After washing the column with the heparin column equilibration buffer, gradient elution of the heparin column elution buffer from the heparin column equilibration buffer was performed. According to the method for confirming differentiation inducing ability performed in (1), the ability to induce differentiation into hNSPC was evaluated. As a result, from the elution buffer fraction of 50 mM Tris, 950 mM NaCl, 0.01% Brij35, 50 mM Tris, 1 M NaCl, 0 In the fraction eluted with an elution buffer of .01% Brij35, differentiation induction was observed as shown in FIG. In addition, when the medium was added with 50 mM Tris + 1 M NaCl + 0.01% Brij35 + 1 mU protease-free chondroitinase ABC (Seikagaku Corporation) as a control and left in a CO ₂ incubator overnight, FIG. As shown in FIG. 4, no protrusion extending from the cell mass was observed. The length of the scale bar in FIGS. 15 and 16 is 50 μm.

  Among the fractions packed in the heparin column of (1), the fraction packed in the MONOQ column, and the fraction eluted with MONOQ, SDS polyacrylamide gel electrophoresis (SDS-- The results of PAGE) are shown in FIG. In the MONOQ fraction having differentiation-inducing ability, one band with a molecular weight of 578,000 daltons was observed. It was found that the molecule having the differentiation-inducing activation ability contained in the conditioned medium of hNSPC was a 578,000 dalton molecule.

[Identification of differentiation-inducing factor]
The purified 578,000 dalton molecule was identified using a mass spectrometer (Ultraflex from BRUKERDALTONICS) as follows.

  First, 1 mU protease-free chondroitinase ABC was added to a 578,000 dalton molecule and reacted at 37 ° C. for 60 minutes to cleave the chondroitin sulfate chain.

  The protein molecule obtained by the chondroitinase treatment was subjected to SDS-PAGE, and after staining with Coomassie, the stained band was cut out. The excised band was trypsinized and the resulting fragment was identified by MS / MS analysis. The results of MS / MS analysis (mass spectrum) are shown in FIGS. 18 (a), (b) and (c). From these, it was found that the isolated molecule was neurocan (sequence list No. 1). 18 (a) shows “GIEDEQDLVPLEVTGVVFHYR” corresponding to amino acid sequence 145 to 165th, FIG. 18 (b) shows “ELGGEVFYVGPAR” corresponding to 257-269th, and FIG. This is “DFQWTDNTGLQFENWR” corresponding to the 1170th.

[Confirmation of neuronal differentiation-inducing ability of isolated substance]
The cell population induced to differentiate was collected using the fraction containing the molecule having differentiation-inducing ability (578,000 daltons) isolated and purified as described above. This cell population was added with 4% paraformaldehyde and allowed to react at 4 ° C. for 20 minutes. After fixation with paraformaldehyde, the cells were washed 3 times with PBS for 10 minutes.

  The washed cells were mixed with PBS containing goat serum (10%) and Triton X-100 (0.01%), and a blocking reaction was performed at room temperature for 1 hour. After blocking, a primary antibody solution was added and reacted at 4 ° C. overnight. Here, as a primary antibody solution, PBS containing 10% goat serum and 0.01% Triton X-100 was used as a diluent, βIII (monoclonal antibody against βIII tubulin: BABCO) 500 times, GFAP (anti-antibody). A solution obtained by diluting glial cell fibrillary acidic protein polyclonal antibody (Sigma) by 80 times was used.

After the reaction with the primary antibody solution, the reaction solution was removed and washed with PBS. Subsequently, it was made to react with a secondary antibody solution at room temperature for 1 hour. Here, as the secondary antibody solution, Alexa anti-mouse Ig antibody 488 and Alexa anti-mouse Ig antibody 568 (both molecular probes), goat serum (10%) and Triton X-100 (0.01%) A 500-fold diluted PBS was used. Moreover, TOPRO-3 (Molecular Probes) was added to the secondary antibody solution, and nuclear staining was performed during the reaction with the secondary antibody solution.
After the reaction, the culture solution was removed, washed with PBS, further washed with ultrapure water, and sealed.

  The cells were observed using a confocal laser scanning microscope (LSM510, manufactured by Carl Zeiss). The staining results of βIII and GFAP are shown in FIG. FIG. 19 shows that there are βIII positive cells that are neuronal marker molecules and GFAP positive cells that are astrocyte marker molecules. The length of the scale bar in FIG. 19 is 50 μm.

  In order to confirm the type of βIII-positive cells (neurons), the reaction was carried out according to the above “(1) Reaction with anti-acetylcholine transferase antibody (ChAT)”. After the reaction, the result of a confocal laser scanning microscope (LSM510, manufactured by Carl Zeiss) photograph is shown in FIG. As can be seen from FIG. 20, acetylcholine transferase positive cells were observed, confirming the presence of cholinergic neurons. The length of the scale bar in FIG. 20 is 50 μm.

According to the method for inducing differentiation of neural stem cells of the present invention, a chondroitinase is simply added to hNSPC in culture, or a combination of neurocan and chondroitinase or a chondroitinase of neurocan is added to hNSPC just subcultured. By adding a protein molecule generated by degrading itinase, differentiation into neurons can be induced with high efficiency, and not only GABAergic neurons but also cholinergic neurons can be obtained at a high rate.
Therefore, the differentiation-inducing method, differentiation-inducing agent, and differentiation-inducing medium of the present invention can be used as a method for easily producing neurons for transplantation of neurological diseases, particularly neurons for treating Alzheimer's in vitro.

It is a figure which shows the result of the luminescent reaction with an anti-chondroitin sulfate monoclonal antibody. 2 is an optical micrograph (magnification 100 times) showing a state 24 hours after the start of differentiation in Example 1. 2 is an optical micrograph (magnification 100 times) showing a state 10 days after the start of differentiation in Example 1. 3 is an optical micrograph (magnification 200 times) showing the βIII staining result of Example 2. 2 is an optical micrograph (magnification 200 times) showing the result of staining GFAP of Example 2. 2 is an optical micrograph (magnification 200 times) showing the result of nuclear staining of Example 2. 4 is an optical micrograph (magnification 200 times) showing the βIII staining result of Comparative Example 3. It is an optical microscope photograph (magnification 200 times) which shows the dyeing | staining result of GFAP of the comparative example 3. 4 is an optical micrograph (magnification 200 times) showing the result of nuclear staining in Comparative Example 3. It is a flowchart which shows reaction operation with an anti- acetylcholine transferase antibody. It is a flowchart which shows reaction operation with an anti- GABA antibody and an anti- glutamic acid antibody. It is a microscope picture (200-times multiplication factor) at the time of adding chondroitinase ABC. It is a microscope picture (200-times multiplication factor) at the time of adding chondroitinase AC-I. It is a microscope picture (magnification 200 times) at the time of adding chondroitinase B. It is a microscope picture (100 times) which shows the result of hNSPC which induced differentiation with the MONOQ elution fraction which has differentiation-inducing ability. It is a microscope picture (100 time) which shows the differentiation-inducing ability evaluation test result of control. It is a result of SDS-PAGE of heparin filling liquid, MONOQ filling liquid, and differentiation-inducing activity fraction of MONOQ. It is a result (mass spectrum) of MS analysis of an isolated substance. It is an optical microscope photograph (magnification 200 times) showing the βIII and GFAP staining results of the MONOQ differentiation-inducing activity fraction. It is an optical micrograph (magnification 200 times) showing the result of staining βIII-positive cells with ChAT.

Claims (13)

A method of inducing differentiation of neural stem cells by adding chondroitinase to a neural stem cell medium in culture. The induction method according to claim 1, wherein the medium does not contain serum. The induction method according to claim 1 or 2, wherein the medium is a basic medium containing a basic fibroblast growth factor, epidermal growth factor or leukocyte migration inhibitory factor. A method of inducing neural stem cell differentiation by adding neurocan and chondroitinase to neural stem cells. A method of inducing differentiation of neural stem cells by adding a protein obtained by degrading neurocan chondroitinase to neural stem cells. The differentiation induction method according to claim 1, wherein the chondroitinase is chondroitinase ABC or chondroitinase AC. The method according to any one of claims 1 to 6, which induces differentiation of cholinergic neurons, GABAergic neurons, and glutamatergic neurons. A neural stem cell differentiation-inducing medium containing a conditioned medium in which neural stem cells and / or neural precursor cells are cultured and chondroitinase. The neural stem cell differentiation induction medium according to claim 7, wherein the chondroitinase is chondroitinase ABC or chondroitinase AC. An agent for inducing differentiation of neural stem cells, the main component of which is a protein obtained by treating chondroitin sulfate proteoglycans secreted by neural stem cells and / or neural precursor cells with chondroitinase. Neural stem cell differentiation-inducing agent containing neural stem cell and / or neural progenitor cells cultured overnight or above for culture of neural stem cell growth medium and chondroitinase. A neural stem cell differentiation inducer comprising neurocan and chondroitinase. A neural stem cell differentiation inducer comprising as an active ingredient a protein obtained by degrading neurocan chondroitinase.