JP2002325571A - Method for inducing differentiation of retina - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for inducing differentiation of a retinal nerve cell, capable of making a cell derived from a nerve stem cell express its function by making the retina take the nerve stem cell and making the taken nerve stem cell differentiate, so that a marker of optic cell or a marker of bipolar cell can be expressed concretely, and to provide the retinal nerve cell capable of expressing the marker of optic cell or the marker of bipolar cell, so that the retinal nerve cell is suitably used to be transplanted to the retina or the like. SOLUTION: This method for inducing the differentiation of the retinal nerve cell comprises obtaining the nerve stem cell or a nerve stem precursor cell from a cell derived from the eyeball tissue or an embryonic stem cell, introducing a homeobox gene specific to the retina into the nerve stem cell or the nerve stem precursor cell, and culturing the obtained cell under a differentiation- inducing condition. The retinal nerve cell is obtained by the method for inducing the differentiation of the retinal nerve cell.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for inducing differentiation of retinal nerve cells, which can provide cells suitable for transplantation into the retina, particularly autologous transplantation, and to retinal nerve cells obtained by the method.

[0002]

2. Description of the Related Art In recent years, the presence of neural stem cells in the brain and spinal cord has been recognized, and there are expectations for transplantation therapy for Parkinson's disease and retinal pigment degeneration, nerve regeneration medicine, and the like using these stem cells.

[0003] Regarding neural stem cells, it has been reported in a transplantation experiment using rat or mouse neural stem cells that the neural stem cells transplanted into the brain move to an appropriate position and differentiate into neurons and glia.

[0004] In regenerative medicine in the field of ophthalmology, when adult rat hippocampus-derived neural stem cells are injected into the vitreous cavity of a 2-7 day old rat whose nerve differentiation is in progress, the neural stem cells migrate to the retina. It is known that they engraft, differentiate into various retinal cell-like cells, and regenerate nerve fibers.

[0005] However, at present, the retinal nerve cells do not differentiate and function to recognize light until they function as retinal nerve cells.

[0006] Therefore, there is a need for means capable of engrafting and differentiating neural stem cells in the retina and allowing cells derived from the neural stem cells to exhibit functions as retinal neural cells.

[0007]

DISCLOSURE OF THE INVENTION The present invention relates to engrafting and differentiating neural stem cells in the retina and expressing functions in cells derived from the neural stem cells. An object of the present invention is to provide a method for inducing differentiation of retinal nerve cells, which is capable of expressing a bipolar cell marker. Another object of the present invention is to provide a retinal nerve cell exhibiting a photoreceptor cell marker or a bipolar cell marker, which is suitable for transplantation into the retina or the like.

[0008]

That is, the present invention provides:
[1] Neural stem cells or neural progenitor cells are obtained from eye tissue-derived cells or embryonic stem cells, a retinal-specific homeobox gene is introduced into the neural stem cells or neural progenitor cells, and the obtained cells are subjected to differentiation-inducing conditions. A method for inducing differentiation of retinal nerve cells, wherein the cells derived from eye tissue are selected from the group consisting of fetal neural retina, ciliary pigment epithelial cells, and retinal pigment epithelial cells The method for inducing differentiation according to the above [1], wherein the retina-specific homeobox gene is at least one selected from the group consisting of Crx, Chx10, Pax and Rax.
The method for inducing differentiation according to the above [1] or [2], [4] the method according to any one of the above [1] to [3], wherein the conditions for inducing differentiation are culturing in the presence of retinoic acid and serum. Differentiation Inducing Method, [5] Differentiation Inducing Condition: DM Including N ₂ Supplements in the Presence of Retinoic Acid and Serum
The method for inducing differentiation according to the above [4], wherein the method is a culture with EM / F12, [6] any one of the above [1] to [5].
Retinal nerve cells obtained by the method for inducing differentiation of retinal nerve cells as described in [7], and [7] opsin-positive or PK
The present invention relates to the retinal nerve cell according to the above [6], which exhibits C positivity.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION According to the method for inducing differentiation of retinal nerve cells of the present invention, neural stem cells or neural progenitor cells are obtained from eye tissue-derived cells, and a retinal-specific homeobox gene is added to the neural stem cells or neural precursor cells. There is one major feature in introducing and culturing the obtained cells under differentiation-inducing conditions.

According to the method for inducing differentiation of retinal nerve cells of the present invention, the function of photoreceptor cells or bipolar cells, which was difficult to express when hippocampus-derived neural stem cells were transplanted and differentiated into the retina, was expressed. It has an excellent effect of being able to do so.

Further, according to the method for inducing differentiation of retinal nerve cells of the present invention, the method expresses the function of photoreceptor cells or bipolar cells.
Specifically, since an opsin-positive or PKC-positive cell can be obtained, an excellent effect that a cell useful for retinal regenerative medicine can be developed is exhibited.

In the differentiation-inducing method of the present invention, a tissue that can be used to obtain neural stem cells or neural progenitor cells can be obtained by introducing a retinal-specific homeobox gene into the obtained cells, thereby obtaining a marker for photoreceptor cells, for example, , Opsin,
Alternatively, any tissue capable of expressing a bipolar cell marker and PKC may be used, and specific examples include eyeball tissues, more specifically, an inner layer of an eye embryo. Further, such a tissue may be derived from an adult or a fetal individual.

Further, in the method for inducing differentiation of the present invention,
Using embryonic stem cells, neural stem cells or neural progenitor cells may be obtained from the embryonic stem cells.

Methods for inducing neural stem cells or neural progenitor cells from the embryonic stem cells are described in, for example, Kawasaki, H., Sasai Y., Neuron., 2000 Oc.
t; 28 (1): 31-40].
The conditions for culturing and maintaining embryonic stem cells include, for example,
"Molecular Biology Protocol", published by Nankodo, etc.

The tissue-derived cells include fetal neural retina, ciliary pigment epithelial cells, retinal pigment epithelial cells, and the like.

The above-mentioned tissue-derived cells are obtained, for example, by treating a tissue excised by an appropriate means with Dispase, EDTA, etc., and then treating it with trypsin to separate single cells, and further confluent with an appropriate medium. The cells can be harvested by culturing to the extent possible and treating the resulting cells with trypsin treatment and collagenase treatment. Here, when culturing is performed at the time of cell collection, a medium such as a medium containing basic fibroblast growth factor, a medium containing epidermal cell growth factor, and a medium containing LIF (leukocyte migration inhibitory factor) described below. Can be used.

Neural stem cells or neural progenitor cells express markers such as nestin.

Such ciliary pigment epithelial cell-derived neural stem cells or neural progenitor cells are also included in the scope of the present invention.

The neural stem cells or neural progenitor cells may be obtained as a neural sphere containing the neural stem cells. In the present invention, such neural spheres may be subjected to the following gene transfer and induction of differentiation into nerves. Good.

The basic fibroblast growth factor (bFG)
F) -containing serum-free medium (hereinafter referred to as bFGF-containing serum-free medium) includes D2 containing an N ₂ supplement.
MEM / F12 and the like. The content of the basic fibroblast growth factor in such a medium was 10 ng /
ml or more, preferably 20 ng / ml or more, more preferably 40 ng / ml or more.

N ₂ supplements include insulin
5 μg / ml, transferrin 100 μg / ml, progestron 20 nM, putrescine 100 μ
M, 30 nM sodium selenate.

Conditions such as temperature, oxygen concentration and carbon dioxide concentration in the culture in the serum-free medium containing bFGF are as follows:
It can be set appropriately according to the cells.

Examples of the retina-specific homeobox gene include a gene that has a region-specific expression pattern in the eye during development and controls region-specific morphogenesis, and a gene that is involved in the expression of differentiation traits. Can be Specific examples include Crx, Chx10, Pax6, and Rax. In the differentiation induction method of the present invention, from the viewpoint that the photoreceptor cell marker can be expressed by introduction into neural stem cells or neural progenitor cells, preferably, Crx or Chx10 is used, and the bipolar cell marker can be expressed. From the viewpoint, preferably, Chx10 is used. The base sequence of the Crx was GenBank.
k accession number: shown in U77615.

The method for introducing the retinal-specific homeobox gene into neural stem cells or neural progenitor cells includes, for example, a gene transfer method using an adenovirus vector,
Examples include electroporation, a gene transfer method using a retroviral vector, a gene transfer method using an adeno-associated virus, lipofection, electroporation, and the like. From the viewpoint of transfer efficiency, a gene transfer method using an adenovirus vector and a gene transfer method using a retrovirus vector are preferred.

Next, the transfected neural stem cells or neural progenitor cells are cultured under differentiation-inducing conditions suitable for differentiation into retinal nerve cells.

The conditions for inducing differentiation include culturing in the presence of retinoic acid and serum. Here, examples of the medium that can be used for culture include a DMEM / F12 medium containing an N ₂ supplement.
Further, conditions such as temperature, oxygen concentration, carbon dioxide concentration and the like during the culturing can be appropriately set according to the cells.

The amount of retinoic acid used is 0.1 μM
It is preferably 0.5 μM or more, more preferably 10 μM or less, and preferably 5 μM or less.

The amount of the serum used is preferably about 1% at the time of induction of differentiation.

Cells differentiated by culture of neural stem cells or neural progenitor cells into which a gene has been introduced under differentiation-inducing conditions, when the introduced gene is a Crx gene, exhibit opsin positive as a photoreceptor cell marker, and Β-III tubulin positive cell marker, GFAP
When the introduced gene is a Chx10 gene, it exhibits PKC positivity which is a marker of bipolar cells, and β-III tubulin positive which is a marker of nerve cells, and GFAP positivity. It has the characteristic of presenting. Therefore, such cells can be used as retinal nerve cells. The present invention also includes retinal nerve cells obtained by such a method for inducing differentiation.

The retinal nerve cells obtained by the method of inducing differentiation according to the present invention can be used as transplant cells for patients with retinal degenerative diseases, such as retinal pigment degeneration, age-related macular degeneration, retinal detachment, glaucoma, and retinal vascular occlusion. The application as is expected. Furthermore, the neural stem cells obtained by the present invention have the advantage that a uniform and sufficient amount of cells can be obtained and can be cryopreserved, thereby enabling planned treatment.

Further, according to the method for inducing differentiation of the present invention,
The addition of cytokines and the introduction of foreign genes are also expected to change the direction of differentiation.

[0032]

EXAMPLES Example 1 (1) Culture of Retina Fetal 18-day-old rat retinas or fetal 11-week-old human retinas were isolated. Serum-free medium containing 20 ng / ml basic fibroblast growth factor (bFGF) [Composition: DMEM
/ F12 + N ₂ supplement] using 5% CO ₂ ,
The cells were cultured at 37 ° C. under 20% O ₂ . When the cells had grown sufficiently, the culture was harvested with 0.125% trypsin [Gi
bco) at 37 ° C. for 5 minutes,
On a collagen-coated dish [medium composition: DMEM /
F12 + N ₂ supplement + bFGF] with 5% CO ₂ ,
The cells were subcultured at 37 ° C. under 20% O ₂ for 30 days. The study on human fetal retina was approved by the Kyoto University Medical Ethics Committee. Hereinafter, the N ₂ supplement is composed of insulin 5 μg / ml, transferrin 100 μg / ml, progestron 20 nM, putrescine 100 μM, sodium selenate 30 n
M.

Upon induction of differentiation, b
5% CO ₂ , 20% O ₂ in a medium (composition: DMEM / F12 + N ₂ supplement + 0.5 μM RA) supplemented with fetal bovine serum (FBS) and retinoic acid after removing FGF
And cultured at 37 ° C for 2 weeks.

As a result, surprisingly, as shown in FIG. 1, neural spheres, which are thought to contain neural stem cells, are isolated and cultured from rat and human fetal retinas in the same manner as hippocampal-derived neural stem cells. We can see that we can do it.

Then, the neural sphere was changed to 4
After fixing to a slide glass with% paraformaldehyde, frozen sections were prepared.

With respect to the obtained section, an antibody against nestin which is a marker for neural stem cells [Pharmi
ngen].

As a result, almost all cells are nestin-positive. Furthermore, after induction of differentiation with retinoic acid, β-III tubulin and GFAP-positive cells were observed, indicating that the cells differentiated into young nerves and glial cells.

(2) Induction of Neuronal Differentiation from Ciliary Pigment Epithelial Cells The eyes of 3-4 week-old DA rats were enucleated, and the anterior segment was separated by incising the sclera slightly anterior to the equator in a circular shape. . After complete removal of the retina, the lens and ciliary pigment-free epithelial cells were removed. After isolating the ciliary tissue, each tissue was subjected to Dispase (1000 U / ml) for 3 hours.
The mixture was treated at 7 ° C. for 15 minutes, and then treated with 0.05% EDTA at room temperature for 20 minutes.

Some ciliary tissues were adhered on a laminin-coated dish so that the pigment epithelial cell side was on the lower side, and the serum-free medium containing bFGF was used for 5% CO ₂ , 20% O ₂ . Adherent culture was performed at 37 ° C. for 20 days under ₂ .

Subsequently, for induction of differentiation into nerves, the medium was changed to a medium containing fetal calf serum [composition: DMEM / F
12 + N ₂ supplement + retinoic acid]
Culture was continued similarly.

As a result, neu was obtained from ciliary pigment epithelial cells.
ral sphere-like cells were obtained. Nestin-positive cells were confirmed before induction of nerve differentiation, and after induction of differentiation, β-III tubulin and GFAP-positive cells were still present in a small number.

(3) Preparation of Recombinant Adenovirus Vector and Gene Transfer Hereinafter, preparation of a recombinant adenovirus vector and gene transfer using the vector are performed according to the method of Kanegae and Saito et al. [Literature name: Gene transfer & expression analysis experiment method] Pp. 27-42,
(1997), published by Yodosha].

The Crx gene, which is a specific homeobox gene for photoreceptors, and the GFP gene, which is a reporter gene, were amplified, and the respective genes cut out with restriction enzymes were incorporated into an expression cosmid cassette. In addition,
For the Crx gene, Chx10 gene and GFP gene, respectively, a plasmid holding Crx, Chx
Amplification was performed using transformants obtained by transforming host Escherichia coli with each of the plasmid holding GFP and the plasmid holding GFP.

Next, adenoviruses expressing the respective genes were prepared by the homologous recombination method. Subsequently, the obtained recombinant adenovirus was infected to 293 cells and proliferated, and the virus was purified by a step gradient method using cesium chloride.

Next, each of the ciliary pigment epithelial cell-derived neural stem cells (or neural progenitor cells) was infected with the purified adenovirus expressing Crx gene, Chx10 gene or adenovirus expressing GFP gene. .

The resulting infected cells were subjected to neural differentiation conditions [DM
EM / F12 + N ₂ supplement + 1% FBS + 20
ng / ml bFGF conditions].

The effect on neural differentiation of each of the adenovirus-infected cells expressing the Crx gene, the adenovirus infected with the Chx10 gene and the adenovirus infected with the GFP gene was determined by immunocytochemical analysis as described below.

For the cultured cells, after removing the medium by suction, 4
% Paraformaldehyde, fixed on a slide glass, and blocked with skim milk.

Various primary antibodies known as various nerve markers [anti-nestin antibody, anti-neurofilament 2
00 antibody, anti-MAP5 antibody, anti-β-III tubulin antibody, anti-GFAP antibody, anti-opsin antibody] at the optimal dilution ratio, and react with the immobilized cells. I let it. Subsequently, visualization was performed using a fluorescent-labeled secondary antibody (manufactured by Amersham).

The visualized marker was observed with a fluorescence microscope, and part of the marker was photographed and recorded using a confocal microscope. The results are shown in FIG.

As shown in FIG. 2, when differentiation was induced after introducing Crx or Chx10 using an adenovirus vector, when Crx was introduced, opsin-positive cells, which are photoreceptor markers, were observed. It can be seen that when introduced, PCK activity, which is a marker for bipolar cells, is observed. On the other hand, when the gene was not introduced or when GFP (green
When the differentiation was induced after the introduction of the fluorescein protein) gene, no opsin-positive cells were observed.

The above results suggest that undifferentiated neural stem cells (or neural progenitor cells) can be obtained from rat fetal retina, human fetal retina, and rat ciliary epithelium. In addition, by inducing differentiation, neural stem cells (or neural progenitor cells)
Are suggested to differentiate into nerves and glia.
Furthermore, by introducing a homeobox gene, for example, a Crx gene, opsin-positive cells, which are photoreceptor markers, can be obtained. For example, by introducing a Chx10 gene, PKC-positive cells, which are bipolar cell markers, can be obtained. Is obtained.

[0053]

According to the method for inducing differentiation of the present invention, neural stem cells can be engrafted and differentiated in the retina and functions can be expressed in cells derived from the neural stem cells. An excellent effect that a marker or a bipolar cell can be expressed is exhibited. In addition, the retinal nerve cells of the present invention are expected to be applied as transplant cells to patients with retinal degenerative diseases, such as retinitis pigmentosa, age-related macular degeneration, retinal detachment, glaucoma, and retinal vascular occlusion.

[Brief description of the drawings]

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram of neuro obtained from fetal rat retina.
It is a figure which shows al sphere.

FIG. 2 shows the expression of Cr using an adenovirus vector.
It is a figure which shows the cell which induced differentiation after introduction of x gene. Cells expressing opsin, a photoreceptor cell marker, are observed.

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (reference) A61P 27/06 C12N 15/00 A C12N 5/10 5/00 B F term (reference) 4B024 AA01 BA80 CA01 CA04 DA02 EA02 EA04 FA02 FA10 GA11 HA17 4B065 AA90X AA93Y AB01 BA01 BB25 BB32 BB34 CA44 4C084 AA13 NA14 ZA332 4C087 AA01 AA02 AA03 BB56 BB57 CA04 NA14 ZA33

Claims (7)

[Claims] Claims 1. An ocular tissue-derived cell or an embryonic stem cell,
Obtaining a neural stem cell or neural progenitor cell, introducing a retinal-specific homeobox gene into the neural stem cell or neural progenitor cell, and culturing the obtained cell under differentiation-inducing conditions. Differentiation induction method. 2. The method for inducing differentiation according to claim 1, wherein the ocular tissue-derived cells are one selected from the group consisting of fetal neural retina, ciliary pigment epithelial cells, and retinal pigment epithelial cells. 3. The retinal-specific homeobox gene is C
The differentiation induction method according to claim 1 or 2, wherein the method is at least one selected from the group consisting of rx, Chx10, Pax6, and Rax. 4. The method according to claim 1, wherein the differentiation-inducing condition is culturing in the presence of retinoic acid and serum.
4. The method for inducing differentiation as described in the above item. 5. The condition for inducing differentiation is that DMEM / F containing N ₂ supplement in the presence of retinoic acid and serum.
The method for inducing differentiation according to claim 4, which is a condition for culturing in step (12). 6. A retinal nerve cell obtained by the method for inducing differentiation of a retinal nerve cell according to any one of claims 1 to 5. 7. The retinal nerve cell according to claim 6, which exhibits opsin positive or PKC positive.