JP2003125759A - Human neural stem cell derived from human amniotic mesenchymal cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a human neural stem cell stably suppliable without causing problems in compatibility during transplantation. SOLUTION: This cell is separated from a human amniotic mesenchymal cell layer and capable of expressing nestin and vimentin which are markers of the neural stem cell and incorporating 5-bromo-2'-deoxy-uridine into the cell by culturing the cell in the presence of the 5-bromo-2'-deoxy-uridine. Thereby, the cell can be collected in a large amount together with placenta and the collection of the cell is free from ethical problems. The cell can stably be supplied. Since the cell has immunological tolerance, there is no problem about the compatibility when the cell is transplanted to a patient. Therefore, the cell exhibits excellent effects as a drug delivery system, etc., for the substance produced with a neurocyte by transplanting the cell into the brain of the patient suffering from an intractable nervous disease such as Parkinson's disease and metabolic nervous disease.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel neural stem cell isolated from human amniotic membrane. The cells of the present invention are
It is useful as a source of substances produced by nerve cells, and also as a drug delivery system for substances produced by nerve cells by transplanting into the brain of patients with intractable neurological diseases such as Parkinson's disease and metabolic nerve disease. It is useful.

[0002]

2. Description of the Related Art Pluripotent stem cells are undifferentiated cells that can differentiate to form various tissues, and are important in the fields of regenerative medicine and tissue engineering. As stem cells, bone marrow stem cells obtained from bone marrow and cord blood stem cells have been conventionally known, but these are difficult to stably supply. Also, this year, it was announced that a large amount of pluripotent stem cells could be collected from human placenta. However, since the placenta is maternally derived, when transplanting cells differentiated from placenta-derived stem cells, compatibility must be investigated to prevent rejection, and for patients who are not compatible, There is a problem that it cannot be transplanted.

[0003]

An object of the present invention is to provide neural stem cells which can be stably supplied and whose compatibility during transplantation does not matter.

[0004]

As a result of earnest research, the present inventors have found that neural stem cells are present in the mesenchymal cell layer of human amnion, and have completed the present invention.

That is, the present invention shows that when isolated from human amnion mesenchymal cell layer, expressing the neural stem cell markers nestin and vimentin, and cultured in the presence of 5-bromo-2'-deoxy-uridine, 5-bromo- It provides cells that take up 2'-deoxy-uridine into the cells. Further, the present invention provides a neuron-forming cell containing the cell of the present invention.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION As described above, the cells of the present invention are those separated from the human amnion mesenchymal cell layer. The mesenchymal cell layer is located between the chorion layer and the amniotic epithelial cell layer. Although the amniotic membrane is a tissue derived from a fetus, it can be collected in a state of being attached to a placenta derived from a mother, and since it is a large tissue that covers the entire inner wall of the uterus, a large amount can be collected. Furthermore, since the placenta and the amniotic membrane attached to it are treated as medical waste, there is no ethical problem in collection.

The cells of the present invention are separated by detaching the amniotic epithelial cell layer + the mesenchymal cell layer of human amniotic membrane from the chorion layer, treating this with trypsin to remove amniotic epithelial cells, and treating with a protease. can do. Here, preferred examples of the protease treatment include treatment with papain, collagenase, neutral protease) and DNase mixed solution (see Examples below), but it is not limited to this. Not something.

The cells of the present invention are confirmed to express nestin and vimentin by immunohistostaining. Nestin and vimentin are neural stem cell markers, and it is recognized in this field that cells expressing them can be determined to be pluripotent neural stem cells (Ana Villaet
al., Experimental Neurology 161, 67-84 (2000)). Therefore, the cells of the present invention are pluripotent neural stem cells. In addition, the cells of the present invention are characterized by 5-bromo-2'-deoxy-
When cultured in the presence of uridine (5BrdU), uptake of 5BrdU was detected, indicating that it is in the mitotic phase. It is common to examine the growth state of cells using 5BrdU, and it can be easily performed using a commercially available 5BrdU measurement kit. In addition, cells of the present invention, B-27 (Brewer, GJ et al., (1993) J. Neuroscien which is an additive for hippocampal cell culture.
When cultured in a culture medium containing ce Res. 35, 567), nestin became negative, expression of vimentin was almost disappeared, and differentiation into nerve cells was observed. B-27 is biotin, L-carnitine, corticosterone, ethanolamine, D (+)-galactose, glutathione (reduced form), linolenic acid, progesterone, putrescine, retinyl acetate, selenium, T3 (triode- 1-Tyroneine), DL-
Additive for hippocampal cell culture consisting of α-tocopherol, DL-α-tocopherol acetate, bovine albumin, catalase, insulin, superoxide dismutase, and transferrin. US Invitrog
It is marketed by en. Further, when the cells of the present invention are suspension-cultured in a culture medium containing a mitogenic agent such as fibroblast growth factor (FGF) and epidermal growth factor (EGF), cell spheres are formed, and a part of this sphere is formed. When collected and similarly subjected to suspension culture, spheres are formed again (secondary spheres). Therefore, the cells of the present invention can be cultured in an undifferentiated state and have self-renewal properties.

It is to be noted that the scope of the present invention also includes cultured cells obtained by subjecting the above-mentioned cells of the present invention to primary culture or subsequent subculture, which express the above-mentioned nestin and vimentin and take in 5BrdU. Be done.

The cells of the present invention are derived from human amniotic membrane, and amniotic membrane is derived from a fetus, which shows immunological tolerance. That is, immunohistochemical staining showed a positive reaction for HLA Class I,
There is no stainability of II. In addition, Fas ligand positive cells are present. Recently, the reason why amniotic tissue is difficult to induce rejection is considered to be that HLA Class Ib (HLA-G) expression and the presence of Fas ligand-positive cells contribute to the suppression of rejection (Ophthalmology 42: 257- 269, 2000). Therefore, it is possible to transplant without making the compatibility of HLA a problem.

As specifically described in the following examples, the cells of the present invention form spheres by suspension culture in the presence of βFGF (fibroblast growth factor) and / or EGF (epithelial growth factor). Then, when a part of it is collected and subjected to suspension culture, spheres (secondary spheres) are formed again.
For example, among the cells separated by the above protease treatment, cells of the present invention can be easily isolated by a method such as suspension culture of cells that are positive for nestin and Musashi-1 to form secondary spheres. It is possible to

The cells of the present invention are differentiated into nerve cells by culturing in the presence of the above hippocampal cell culture additive such as B-27. In addition, the cells of the present invention can be cultured on uncoated dishes in the presence of cytokines such as NGF (nerve growth factor) or NT-3 (neutrophin-3) to give oligodendrocytes or astrocytes. Differentiate into cells. Therefore,
The cells of the present invention can be used for such neuronal cell formation. The differentiated cultured nerve cells can be used as a source of various substances such as dopamine and choline acetyltransferase produced by the nerve cells. In addition, dopamine is a substance known to be significantly reduced in Parkinson's disease patients, and choline acetyltransferase is a substance known to be significantly reduced in Alzheimer's disease patients. Further, since the cells of the present invention exhibit immunological tolerance, transplantation to a part damaged in dementia or the like (for example, hippocampus, basal ganglia, brain striatum in Alzheimer's disease, etc.) It can be used as a drug delivery system (DDS) such as produced choline acetyltransferase, and can be used for treatment of dementia, Parkinson's disease, metabolic nerve disease and the like. In addition, by introducing a desired foreign gene into a cell of the present invention by a known method (for example, Examples 1 to 3 of JP-A-9-94092), and transplanting the substance, a substance encoded by the foreign gene is introduced. DDS
Can also be used as

[0013]

EXAMPLES The present invention will be described more specifically below based on examples. However, the present invention is not limited to the following examples.

Example 1, Comparative Example 1 1. Cell Separation and Culture From the placenta after delivery of a pregnant woman who obtained informed consent, the amnion epithelial cell layer + mesenchymal cell layer was separated from the chorion layer and separated. 37 with 0.125% trypsin solution + 1.3 mM EDTA
Treated at 15 ° C for 15 minutes. This is repeated 4 times, the trypsin solution is centrifuged to collect the cells, and the phosphate buffer (PBS) is added.
It was washed 3 times with (trypsin-treated fraction (Comparative Example 1)). After washing the undigested tissue mass with phosphate buffer, mixed enzyme (0.01% papain, 1 mg / ml collagenase, 0.01% DNa
se, 0.1% neutral protease) at 37 ° C. for 1 hour. The sediment was suspended in PBS by centrifugation at 1000 rpm for 10 minutes. After passing through a 20 μm filter, PBS
It was washed 3 times with (mixed enzyme treatment fraction (Example 1)).

Both fractions contained DMEM: F-1 containing 10% fetal bovine serum (FBS)
2 (1: 1) culture medium (human leukemia inhibitory factor (hLIF, alomone labo, Israel) 1000 U / ml, and 2-mercaptoethanol (2-ME, Sigma), collagen-coated culture dish Primary culture was performed at 37 ° C. in a 5% CO ₂ incubator above, using DMEM: F-12 (1: 1) used here.
The culture medium is a 1: 1 mixture of Dulbecco's modified Eagle medium (DMEM) and Ham's F-12 nutrient mixture (F-12), which is commercially available as a serum-free medium for general mammalian cell culture (Sigma, USA). )
It has been done. After 3 days, after becoming confluent, treated with 0.125% trypsin + 1.3 mM EDTA, 24
Secondary culture was performed in the same culture solution in a collagen-coated culture dish with holes, and B-27 (B-27 Suppl manufactured by Invitrogen) was used.
ement (50x) diluted 50 times at final concentration) containing DMEM: F-12 (1:
1) The medium was converted into a culture solution and cultured. After 3 to 5 days, immunohistological staining was performed by the method described below.

Next, the primary cultured cells were treated with 0.125% trypsin +
N2 additive (progesterone 0.63 μg / ml, putrescine 1611 μg / ml, selenite 0.52 μg / ml, insulin 500 μg at a final concentration in a culture dish treated with 1.3 mM EDTA and treated with poly (2-hydroxyethyl methacrylate) (HEMA). / ml, human transferrin 10000 μg / ml, commercially available from Invitrogen),
DME containing 20 μg / ml bovine basic FGF and 20 μg / ml EGF
Suspension culture was performed in M: F-12 (1: 1) culture medium. The culture dish is
Coated with poly 2-hydroxyethyl methacrylate.
After 2 to 5 days, spheres with a diameter of 50 to 200 μm were formed. A specimen was prepared from this sphere on a cover glass with a cell collecting centrifuge, and immunostaining was performed by the following method. Add this sphere to 0.125% trypsin + 1.3 mM EDTA
When the suspension culture was carried out again with the above culture medium after the treatment with, the secondary spheres were formed. To study differentiation, suspension culture cells were treated with several cytokines such as NT-3 and NGF. That is, specifically, it carried out as follows. Transfer the amniotic spheres to the attachment culture system and
The cells were cultured in a serum-free medium containing 0 ng / ml) and NT-3 (50 ng / ml).

2. Immunostaining Cryostat fragments of amniotic membrane including amniotic epithelial cell layer and amniotic mesenchymal cell layer and suspension cultured cells were immunostained. For immunohistological staining, anti-human nestin polyclonal antibody or anti-human Musashi-1 monoclonal antibody was used as the primary antibody, and anti-rabbit IgG rhodamine was used as the secondary antibody.
(1: 100 Chemicon) and anti-rabbit IgG FITC (Z
YMED) was used in a conventional manner. Specifically, it carried out as follows. Cultured cells or amniotic tissue were fixed with 4% paraformaldehyde for 1 minute and incubated with the above primary antibody at room temperature for 2 hours. Then, the cells were incubated with a secondary antibody diluted with 0.3% triton X100 (trade name) for 2 hours. The immunoblotted cells were observed with an Olympus IX 10 fluorescence microscope and analyzed by confocal images using an Olympus Fluoview laser scanning microscope. further,
Other cell markers CK19 (Santa Cruz), vimentin (PROGEN), Gal C (Sigma) and β-tub
-III (manufactured by Sigma) was also subjected to immunohistological staining in the same manner as above using a commercially available monoclonal antibody against each cell marker. Anti-Fas ligand (manufactured by SANTA CRUZ), anti-HLA Class I (HLA-A, B, C; manufactured by Ansel) and anti-HLA Class II (HLA-DP, DQ, DR; manufactured by Ansel) as primary antibodies. Using. Further, the above culture is carried out in the presence of 5-bromo-2'-deoxy-uridine (5BrdU) (manufactured by Roche Diagnostics KK) (concentration in medium 10 μM),
Intracellular 5BrdU was detected using a commercial kit (Roche Diagnostics Co., Ltd.)

3. Results The cells of the present invention (Example 1) obtained from the mixed enzyme-treated fraction were subjected to collagen for 3 days in a DMEM: F-12 (1: 1) culture medium containing 10% FBS (containing hLIF and 2-ME). When the cells were adherently cultured in a coated culture dish, the immunostaining characteristics of the cells were CK19- / vimentin ++ / nestin + / musashi-1 + (about 15% in total cells).
%). As mentioned above, cells expressing nestin and vimentin are recognized in the art as neural stem cells. Therefore, it was revealed that the cells of the present invention are neural stem cells. It was also found that 5BrdU was positive and was in the mitotic phase in the culture in the presence of 5BrdU. In addition, when differentiated with B-27-containing culture medium, vimentin ± / nestin- / musashi-1- / Gal C ± / β-tub-III ++ was obtained, and the neural stem cell marker disappeared, suggesting differentiation into neural cells. . In addition, as a result of treating suspension-cultured cells with several cytokines such as NT-3 and NGF, when cultured in NGF-containing medium, GFAP-positive cells increased, suggesting differentiation into astroglia (astrocytic cells). Was done. When cultured in a medium containing NT-3, GFAP and Gal C positive cells were increased, suggesting differentiation into astroglia and oligoglia (oligodendrocytes).

Further, the cells of the present invention are treated with N2 and bovine basic FG.
When suspension culture was carried out in a culture medium containing F, EGF and 1% human serum albumin (HAS), the diameter was 50 to 200μ after 2 to 3 days from the start of culture
m amniotic spheres were formed, and when a part thereof was taken and cultured in the same manner, similar secondary spheres were formed. Thereby, the cell of the present invention has self-renewal property,
It was revealed that the cells can be cultured in an undifferentiated state in the presence of mitogens such as FGF and EGF.

On the other hand, cells obtained from the trypsin-treated fraction (Comparative Example 1) were adherent-cultured in a collagen-coated culture dish for 3 days in a DMEM: F-12 (1: 1) culture medium containing 10% FBS.
Immunostaining characteristics of the cells were CK19 + / vimentin- / nestin- / musashi-1-. Further, by the liquid culture,
No spheres were formed. From this, it was revealed that there are no neural stem cells in the amniotic epithelial cells.

[0021]

According to the present invention, stable supply is possible,
Neural stem cells were offered for the first time, whose compatibility during transplantation did not matter. The cells of the present invention can be collected in large amounts together with the placenta, and can be stably supplied without ethical problems. Moreover, since the cells of the present invention have immunotolerance,
Compatibility when transplanted to a patient does not matter. Therefore,
By transplanting the cells of the present invention into the brains of patients with intractable neurological diseases such as Parkinson's disease and metabolic nerve diseases, it is considered that they exert an excellent effect as a drug delivery system for substances produced by nerve cells.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 41/00 A61P 43/00 105 43/00 105 111 111 C12N 5/00 E (72) Inventor Nobuo Sakuragawa 2-1254-25 Ogawamachi, Kodaira-shi, Tokyo (72) Inventor Ayako Uchida 2-1306-44 Ogawamachi, Kodaira-shi, Tokyo Coat Flora C101 F term (reference) 4B065 AA93X AC14 BB13 CA24 CA44 4C087 BB58 CA04 NA14 ZA01 ZA02 ZA15 ZA16 ZB21 ZC41

Claims (3)

[Claims] 1. When isolated from human amnion mesenchymal cell layer, expressing the neural stem cell markers nestin and vimentin, and cultured in the presence of 5-bromo-2′-deoxy-uridine, 5-bromo-2′-deoxy is obtained. -A cell that takes up uridine into the cell. 2. The cell according to claim 1, which expresses Musashi-1. 3. A nerve cell-forming cell containing the cell according to claim 1 or 2.