Cell transplantation method and cell production method exhibiting high choroidal reconstruction rate

Abstract

The purpose of the present invention is to provide a method for reconstructing a degenerated or thinned choroid.　The present invention provides a choroid reconstruction agent containing, as an active ingredient, stem cells that have the ability to differentiate into both blood cells and vascular endothelial cells and that are to be transplanted into a degenerated or thinned choroid.

Description

Cell transplantation method and cell preparation method with a high rate of choroid reconstruction

[0001] The present invention relates to transplantation techniques for disease treatment, and more particularly to a method for reconstructing the choroid in eye diseases. More specifically, it relates to a technique for transplanting hemangioblasts alone or mixed with other cells into the choroid.

[0002] The choroid is a vascular-rich tissue that covers the posterior two-thirds of the eyeball. The choroid includes the Hallar layer and the Satller layer through which branches of arteries and veins run, and the choroid capillary plate composed of capillaries. The choroid capillary plate is formed by a single layer of capillaries running extremely densely, and this sheet has a lobular structure centered on arteries. Also, the endothelial cells of the choroid capillary plate are fenestrated endothelial cells that have a molecular sieve only on the retinal side. Such blood vessels with these characteristics are not found in other sites and are a very special vascular network.

[0003] Since the choroid nourishes photoreceptor cells and retinal pigment epithelial cells, it is important for maintaining normal visual function. Because it can change its thickness by changing blood flow volume, it also contributes to focusing adjustment. Also, the rich blood flow is considered to function in suppressing the temperature rise of the retina. The ciliary nerves and the long posterior ciliary arteries run within the choroid. These nerves and blood vessels are essential for the control of the ciliary body and the iris.

[0004] The development of the choroid is different in the Hallar layer, the Satller layer, and the choroid capillary plate. Hemangioblasts appear on the outer periphery of the developing eye cup, and the choroid capillary plate is formed by constructing a local vascular network. The arteries and veins of the Hallar layer and the Satller layer branch and extend from existing blood vessels and invade the choroid.

[0005] Cells other than vascular endothelial cells, such as pericytes, melanocytes, and stromal cells, are also important for the normal development and function of the choroid. Pericytes are involved in controlling blood flow and vascular elongation. Melanocytes not only block excessive light but have also been reported to be necessary for the elongation of arteries and veins in the Hallar and Satller layers. Stromal cells control the formation of vortex veins that drain blood from the choroid. Pericytes, melanocytes, and stromal cells are known to differentiate from mesenchymal stem cells and neural crest cells.

[0006] Li M, Wang P, Huo ST, et al. Human Pluripotent Stem Cells Derived Endothelial Cells Repair Choroidal Ischemia. Adv Sci (Weinh). 2024;11(9):e2302940.

[0007] In many diseases involving retinal degeneration, the progression of choroidal degeneration is known to worsen the condition. Examples of such diseases include pathological myopia, myopic macular degeneration, myopic optic neuropathy, age-related macular degeneration, retinitis pigmentosa, high myopia, rhegmatogenous retinal detachment, and macular dystrophy. Traditionally, these diseases have been treated with anti-VEGF antibodies, photodynamic therapy, direct photocoagulation, transpupillary thermotherapy, and surgery. However, these treatments have difficulty restoring the structure and function of the choroid.

[0008] Furthermore, choroidal degeneration affects not only blood vessels but also perivascular cells and melanocytes that support choroidal function. In diseases causing choroidal degeneration, the stem cells that give rise to these cells and tissues are likely to be exhausted and depleted. Therefore, stem cell transplantation to the choroid is considered an effective treatment for the diseases described above.

[0009] Therefore, the present invention aims to provide a method for reconstructing a degenerated or thinned choroid.

[0010] Therefore, the present invention provides a method for reconstructing the choroid by stem cell transplantation. The stem cells to be transplanted are hemangioblasts, a mixture of hemangioblasts and mesenchymal stem cells, a mixture of hemangioblasts and neural crest cells, or a mixture of hemangioblasts, mesenchymal stem cells, and neural crest cells. By transplanting these cells into the degenerated choroid, the aim is to reconstruct not only the blood vessels but the entire choroid and obtain a high therapeutic effect.

[0011] In other words, the present invention provides the following: [Aspect A-1] A choroidal reconstruction agent comprising, as an active ingredient, stem cells having the ability to differentiate into both blood cells and vascular endothelial cells for transplantation into a degenerated or thinned choroid. [Aspect A-2] The choroidal reconstruction agent according to Aspect A-1, wherein the stem cells having the ability to differentiate into both blood cells and vascular endothelial cells are hemangioblasts. [Aspect A-3] The choroidal reconstruction agent according to Aspect A-1, comprising, as an active ingredient, a mixture of hemangioblasts and mesenchymal stem cells for transplantation into a degenerated or thinned choroid. [Aspect A-4] The choroidal reconstruction agent according to Aspect A-1, comprising, as an active ingredient, a mixture of hemangioblasts and neural crest cells for transplantation into a degenerated or thinned choroid. [Aspect A-5] A choroidal reconstruction agent according to Aspect A-1, comprising a mixture of hemangioblast, mesenchymal stem cells, and neural crest cells as an active ingredient for transplantation into a degenerated or thinned choroid. [Aspect A-6] A non-human animal as a choroidal degeneration model, which has been administered an oxidizing agent. [Aspect A-7] A non-human animal according to Aspect A-6, in which an oxidizing agent has been administered to the choroid. [Aspect A-8] A non-human animal according to Aspect A-6, in which the oxidizing agent is an iodate salt. [Aspect A-9] A non-human animal according to Aspect A-8, in which the iodate salt is an alkali metal iodate salt or an alkaline earth metal iodate salt. [Aspect A-10] A non-human animal according to Aspect A-9, in which the alkali metal is sodium or potassium, and the alkaline earth metal is calcium or barium. [Aspect A-11] A non-human animal according to Aspect A-6, in which the non-human animal is a mammalian non-human animal. [Aspect A-12] A non-human mammal belonging to the order Rodentia, as described in Aspect A-11. [Aspect A-13] A non-human mammal belonging to the family Muridae, as described in Aspect A-12. [Aspect A-14] A non-human mammal belonging to the subfamily Murinae, as described in Aspect A-13. [Aspect A-15] A non-human mammal belonging to the family Mouse or rat, as described in Aspect A-11. [Aspect B-1] A method for reconstructing a choroid, comprising the step of transplanting stem cells having the ability to differentiate into both blood cells and vascular endothelial cells into a degenerated or atrophied choroid.[Aspect B-2] The method according to aspect B-1, wherein the stem cells having the ability to differentiate into both blood cells and vascular endothelial cells are hemangioblasts. [Aspect B-3] The method according to aspect B-1, wherein in the step of transplanting stem cells having the ability to differentiate into both blood cells and vascular endothelial cells into a degenerated or thinned choroid, a mixture of hemangioblasts and mesenchymal stem cells is transplanted into the degenerated or thinned choroid. [Aspect B-4] The method according to aspect B-1, wherein in the step of transplanting stem cells having the ability to differentiate into both blood cells and vascular endothelial cells into a degenerated or thinned choroid, a mixture of hemangioblasts and neural crest cells is transplanted into the degenerated or thinned choroid. [Aspect B-5] The method according to aspect B-1, wherein, in the step of transplanting stem cells having the ability to differentiate into both blood cells and vascular endothelial cells into a degenerated or thinned choroid, a mixture of hemangioblasts, mesenchymal stem cells and neural crest cells is transplanted into the degenerated or thinned choroid. [Aspect C-1] Use of stem cells having the ability to differentiate into both blood cells and vascular endothelial cells for the production of a pharmaceutical composition for transplanting into a degenerated or thinned choroid to reconstruct the choroid. [Aspect C-2] The use according to aspect C-1, wherein the stem cells having the ability to differentiate into both blood cells and vascular endothelial cells are hemangioblasts. [Aspect C-3] Use of a mixture of hemangioblasts and mesenchymal stem cells for the production of a pharmaceutical composition for transplanting into a degenerated or thinned choroid to reconstruct the choroid. [Aspect C-4] Use of a mixture of hemangioblasts and neural crest cells for producing a pharmaceutical composition for reconstructing a choroid by transplantation into a degenerated or thinned choroid. [Aspect C-5] Use of a mixture of hemangioblasts, mesenchymal stem cells and neural crest cells for producing a pharmaceutical composition for reconstructing a choroid by transplantation into a degenerated or thinned choroid.

[0012] The choroidal reconstruction agent of the present invention can reconstruct the choroid by transplanting it into a degenerated or thinned choroid. The non-human animal of the present invention can be used as a choroidal degeneration model, and for example, by identifying candidate substances that increase choroidal thickness upon administration to this model as substances that regenerate the choroid, it is possible to screen for substances that regenerate the choroid.

[0013] Figure 1 shows the results of extracting eyeballs from choroidal degeneration model mice after injecting them with hemangioblast suspension, preparing frozen sections, and staining them with HE. Figure 2 shows the results of measuring the choroidal thickness of the stained images shown in Figure 1. PBS represents the results of injecting PBS into control mice, Non-Transplant represents the results of injecting PBS into choroidal degeneration model mice, and Transplant represents the results of injecting hemangioblast suspension into choroidal degeneration model mice. In the hemangioblast transplantation group, the choroidal thickness increased in both the nasal and temporal directions compared to the non-transplantation group, indicating choroidal regeneration. Figure 3 is a graph showing the effect of mixed cell transplantation. H+N: hemangioblast + neural crest cells, H+M: hemangioblast + mesenchymal stem cells, H+N+M: hemangioblast + neural crest cells + mesenchymal stem cells. Figure 4 is a graph showing the effect of mixed cell transplantation. H: hemangioblast, H+N: hemangioblast + neural crest cells, H+M: hemangioblast + mesenchymal stem cells, H+N+M: hemangioblast + neural crest cells + mesenchymal stem cells.

[0014] Choroidal Reconstruction Agent: In the present invention, reconstruction includes restoring at least a part of the structure and / or function of tissue damaged or lost due to disease, disability, or other factors, and such restoration is not limited to complete morphological regeneration. Examples of stem cells in the present invention that have the ability to differentiate into both blood cells and vascular endothelial cells include hemangioblasts. Hemangioblasts are known to express markers such as VEGFR2, PODXL, VE-CAD (VE-cadherin), GATA1, and CD34, and can be identified by these markers, but the markers used for identification are not limited to these and may be known hemangioblast identification markers. Also, although not limited, in the present invention, hemangioblasts may be cells that are positive for at least one, more preferably two or more, selected from the group consisting of VEGFR2, PODXL, VE-CAD, GATA1, and CD34. Positivity for each marker can be determined by methods well known to those skilled in the art, such as flow cytometry, immunostaining, or RNA expression analysis.

[0015] In this invention, hemangioblasts used for transplantation can be collected from the patient's own bone marrow aspiration sample or peripheral blood after G-CSF administration. They can also be isolated from umbilical cord blood. They can also be differentiated from pluripotent stem cells and isolated and purified. As for isolation methods, magnetic bead precipitation and cell sorting methods, which use proteins specifically expressed in hemangioblasts as indicators, can be used, but other methods may also be used.

[0016] Isolated hemangioblasts can be used for transplantation directly or after being cultured and grown. An example of culture conditions is culture at 35°C and 5% CO2 using a culture medium such as DMEM containing 15% serum, to which SCF is added at a concentration of 100 ng / ml, bFGF at 1 ng / ml, LIF at 10 ng / ml, and Oncostatin M at 10 ng / ml. However, the conditions are not limited to these.

[0017] In other words, in an example of culture conditions, for example, the basal medium such as DMEM should be 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 16% or more, 17% or more, 18% or more, 19% or more, 20% or more, 21% or more, 22% or more, 23% or more, 24% or more, 25% or more, 26% or more, 27% or more, 28% or more, 29% or more , or containing 30% or more serum, and containing 30% or less, 29% or less, 28% or less, 27% or less, 26% or less, 25% or less, 24% or less, 23% or less, 22% or less, 21% or less, 20% or less, 19% or less, 18% or less, 17% or less, 16% or less, 15% or less, 14% or less, 13% or less, 12% or less, 11% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less serum.

[0018] In an example of culture conditions, for instance, the culture medium contains SCF at concentrations of 10 ng / ml or higher, 20 ng / ml or higher, 30 ng / ml or higher, 40 ng / ml or higher, 50 ng / ml or higher, 60 ng / ml or higher, 70 ng / ml or higher, 80 ng / ml or higher, 90 ng / ml or higher, 100 ng / ml or higher, 110 ng / ml or higher, 120 ng / ml or higher, 130 ng / ml or higher, 140 ng / ml or higher, and 150 ng / ml. g / ml or higher, 160 ng / ml or higher, 170 ng / ml or higher, 180 ng / ml or higher, 190 ng / ml or higher, 200 ng / ml or higher, 210 ng / ml or higher, 220 ng / ml or higher, 230 ng / ml or higher, 240 ng / ml or higher, 250 ng / ml or higher, 260 ng / ml or higher, 270 ng / ml or higher, 280 ng / ml or higher, 290 ng / ml or higher, or 300 ng / ml or higher SCF is added to achieve the following levels: 300 ng / ml or less, 290 ng / ml or less, 280 ng / ml or less, 270 ng / ml or less, 260 ng / ml or less, 250 ng / ml or less, 240 ng / ml or less, 230 ng / ml or less, 220 ng / ml or less, 210 ng / ml or less, 200 ng / ml or less, 190 ng / ml or less, 180 ng / ml or less, 170 ng / ml or less, 160 ng / ml or less, 150 ng / ml or less, 140 ng / ml or less, 130 ng / ml or less, 120 ng / ml or less, 110 ng / ml or less, 100 ng / ml or less, 90 ng / ml or less, 80 ng / ml or less, 70 ng / ml or less, 60 ng / ml or less, 50 ng / ml or less, 40 ng / ml or less, 30 ng / ml or less, 20 ng / ml or less, or 10 ng / ml or less.

[0019] In an example of culture conditions, for instance, the culture medium contains bFGF at concentrations of 0.1 ng / ml or higher, 0.2 ng / ml or higher, 0.3 ng / ml or higher, 0.4 ng / ml or higher, 0.5 ng / ml or higher, 0.6 ng / ml or higher, 0.7 ng / ml or higher, 0.8 ng / ml or higher, 0.9 ng / ml or higher, 1 ng / ml or higher, 1.1 ng / ml or higher, 1.2 ng / ml or higher, 1.3 ng / ml or higher, and 1.4 ng / ml or higher. Above 1.5 ng / ml, 1.6 ng / ml, 1.7 ng / ml, 1.8 ng / ml, 1.9 ng / ml, 2 ng / ml, 2.1 ng / ml, 2.2 ng / ml, 2.3 ng / ml, 2.4 ng / ml, 2.5 ng / ml, 2.6 ng / ml, 2.7 ng / ml, 2.8 ng / ml, 2.9 ng / ml, or 3 ng / ml or higher. It is added to achieve the following levels of bFGF: 3 ng / ml or less, 2.9 ng / ml or less, 2.8 ng / ml or less, 2.7 ng / ml or less, 2.6 ng / ml or less, 2.5 ng / ml or less, 2.4 ng / ml or less, 2.3 ng / ml or less, 2.2 ng / ml or less, 2.1 ng / ml or less, 2 ng / ml or less, 1.9 ng / ml or less, 1.8 ng / ml or less, 1.7 ng / ml or less, 1.6 ng / ml or less. It is added to achieve concentrations of 1.5 ng / ml or less, 1.4 ng / ml or less, 1.3 ng / ml or less, 1.2 ng / ml or less, 1.1 ng / ml or less, 1 ng / ml or less, 0.9 ng / ml or less, 0.8 ng / ml or less, 0.7 ng / ml or less, 0.6 ng / ml or less, 0.5 ng / ml or less, 0.4 ng / ml or less, 0.3 ng / ml or less, 0.2 ng / ml or less, or 0.1 ng / ml or less.

[0020] In an example of culture conditions, for instance, the culture medium should contain LIF of 1 ng / ml or more, 2 ng / ml or more, 3 ng / ml or more, 4 ng / ml or more, 5 ng / ml or more, 6 ng / ml or more, 7 ng / ml or more, 8 ng / ml or more, 9 ng / ml or more, 10 ng / ml or more, 11 ng / ml or more, 12 ng / ml or more, 13 ng / ml or more, 14 ng / ml or more, 15 ng / ml or more, 16 ng / ml or more, 17 ng / ml or more, 18 ng / ml or more, 19 ng / ml or more, 20 ng / ml or more, 21 ng / ml or more, 22 ng / ml or more, 23 ng / ml or more, 24 ng / ml or more, 25 ng / ml or more, 26 ng / ml or more, 27 ng / ml or more, 28 ng / ml or more, 29 ng / ml or more, or 30 ng / ml or more. The LIF is added to achieve the following levels: 30 ng / ml or less, 29 ng / ml or less, 28 ng / ml or less, 27 ng / ml or less, 26 ng / ml or less, 25 ng / ml or less, 24 ng / ml or less, 23 ng / ml or less, 22 ng / ml or less, 21 ng / ml or less, 20 ng / ml or less, 19 ng / ml or less, 18 ng / ml or less, 17 ng / ml or less, 16 ng / ml or less, 15 ng / ml or less, 14 ng / ml or less, 13 ng / ml or less, 12 ng / ml or less, 11 ng / ml or less, 10 ng / ml or less, 9 ng / ml or less, 8 ng / ml or less, 7 ng / ml or less, 6 ng / ml or less, 5 ng / ml or less, 4 ng / ml or less, 3 ng / ml or less, 2 ng / ml or less, or 1 ng / ml or less.

[0021] In an example of culture conditions, for instance, the culture medium is prepared by adding Oncostatin M to a concentration of 1 ng / ml or more, 2 ng / ml or more, 3 ng / ml or more, 4 ng / ml or more, 5 ng / ml or more, 6 ng / ml or more, 7 ng / ml or more, 8 ng / ml or more, 9 ng / ml or more, 10 ng / ml or more, 11 ng / ml or more, 12 ng / ml or more, 13 ng / ml or more, 14 ng / ml or more, 15 ng / ml or more, 16 ng / ml or more, 17 ng / ml or more, 18 ng / ml or more, 19 ng / ml or more, 20 ng / ml or more, 21 ng / ml or more, 22 ng / ml or more, 23 ng / ml or more, 24 ng / ml or more, 25 ng / ml or more, 26 ng / ml or more, 27 ng / ml or more, 28 ng / ml or more, 29 ng / ml or more, or 30 ng / ml or more. M is added to achieve concentrations of 30 ng / ml or less, 29 ng / ml or less, 28 ng / ml or less, 27 ng / ml or less, 26 ng / ml or less, 25 ng / ml or less, 24 ng / ml or less, 23 ng / ml or less, 22 ng / ml or less, 21 ng / ml or less, 20 ng / ml or less, 19 ng / ml or less, 18 ng / ml or less, 17 ng / ml or less, 16 ng / ml or less, 15 ng / ml or less, 14 ng / ml or less, 13 ng / ml or less, 12 ng / ml or less, 11 ng / ml or less, 10 ng / ml or less, 9 ng / ml or less, 8 ng / ml or less, 7 ng / ml or less, 6 ng / ml or less, 5 ng / ml or less, 4 ng / ml or less, 3 ng / ml or less, 2 ng / ml or less, or 1 ng / ml or less.

[0022] In an example of culture conditions, for instance, culture is carried out at 25°C or higher, 26°C or higher, 27°C or higher, 28°C or higher, 29°C or higher, 30°C or higher, 31°C or higher, 32°C or higher, 33°C or higher, 34°C or higher, 35°C or higher, 36°C or higher, 37°C or higher, 38°C or higher, 39°C or higher, 41°C or higher, 42°C or higher, 43°C or higher, 44°C or higher, or 45°C or higher. Culture is carried out at 45°C or lower, 44°C or lower, 43°C or lower, 42°C or lower, 41°C or lower, 40°C or lower, 39°C or lower, 38°C or lower, 37°C or lower, 36°C or lower, 35°C or lower, 34°C or lower, 33°C or lower, 32°C or lower, 31°C or lower, 30°C or lower, 29°C or lower, 28°C or lower, 27°C or lower, 26°C or lower, or 25°C or lower.

[0023] In an example of culture conditions, for instance, the culture is carried out under CO2 concentrations of 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 16% or more, 17% or more, 18% or more, 19% or more, or 20% or more, and under CO2 concentrations of 20% or less, 19% or less, 18% or less, 17% or less, 16% or less, 15% or less, 14% or less, 13% or less, 12% or less, 11% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less.

[0024] In this invention, mesenchymal stem cells used for transplantation can be collected from bone marrow aspiration samples, adipose tissue, dental pulp tissue, etc. Since mesenchymal stem cells have low immunogenicity, they do not need to be derived from the patient's own tissue. These tissues can be cultured under appropriate conditions, adhere to the bottom of the culture vessel, and isolated as proliferating cells. They can also be differentiated from pluripotent stem cells, isolated, and purified. As for isolation methods, magnetic bead precipitation and cell sorting methods, which use proteins specifically expressed in mesenchymal stem cells as indicators, can be used, but other methods may also be used. Products commercially available for medical use can also be used.

[0025] Isolated mesenchymal stem cells can be used for transplantation directly or after being cultured and proliferated. An example of culture conditions is culturing at 35°C and 5% CO2 using a basal medium such as DMEM containing 20% ​​serum, but the conditions are not limited to this.

[0026] In other words, in an example of culture conditions, for example, the basal medium such as DMEM should be 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 16% or more, 17% or more, 18% or more, 19% or more, 20% or more, 21% or more, 22% or more, 23% or more, 24% or more, 25% or more, 26% or more, 27% or more, 28% or more, 29% or more , or containing 30% or more serum, and containing 30% or less, 29% or less, 28% or less, 27% or less, 26% or less, 25% or less, 24% or less, 23% or less, 22% or less, 21% or less, 20% or less, 19% or less, 18% or less, 17% or less, 16% or less, 15% or less, 14% or less, 13% or less, 12% or less, 11% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less serum.

[0027] In an example of culture conditions, for instance, culture is carried out at 25°C or higher, 26°C or higher, 27°C or higher, 28°C or higher, 29°C or higher, 30°C or higher, 31°C or higher, 32°C or higher, 33°C or higher, 34°C or higher, 35°C or higher, 36°C or higher, 37°C or higher, 38°C or higher, 39°C or higher, 41°C or higher, 42°C or higher, 43°C or higher, 44°C or higher, or 45°C or higher. Culture is carried out at 45°C or lower, 44°C or lower, 43°C or lower, 42°C or lower, 41°C or lower, 40°C or lower, 39°C or lower, 38°C or lower, 37°C or lower, 36°C or lower, 35°C or lower, 34°C or lower, 33°C or lower, 32°C or lower, 31°C or lower, 30°C or lower, 29°C or lower, 28°C or lower, 27°C or lower, 26°C or lower, or 25°C or lower.

[0028] In an example of culture conditions, for instance, the culture is carried out under CO2 concentrations of 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 16% or more, 17% or more, 18% or more, 19% or more, or 20% or more, and under CO2 concentrations of 20% or less, 19% or less, 18% or less, 17% or less, 16% or less, 15% or less, 14% or less, 13% or less, 12% or less, 11% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less.

[0029] In this invention, neural crest cells used for transplantation can be collected from the patient's oral mucosa, skin tissue, etc. They can also be differentiated from pluripotent stem cells, isolated, and purified. As for isolation methods, magnetic bead precipitation and cell sorting methods, which use proteins specifically expressed in neural crest cells as indicators, can be used, but other methods may also be used.

[0030] Isolated neural crest cells can be used for transplantation directly or after being cultured and grown. An example of culture conditions is culture at 35°C and 5% CO2 using a culture medium prepared by adding 20 ng / ml of EGF and 20 ng / ml of bFGF to a serum-free basal medium such as DMEM / F12, and a low-adhesion culture vessel, but the conditions are not limited to this. Neural crest cells are known to express markers such as p75 and SOX10, and can be identified by such markers, but the markers used for identification are not limited to these and may be any known neural crest cell identification markers. Also, although not limited, in the present invention, neural crest cells may be cells that are positive for at least one, more preferably two or more, markers selected from the group consisting of p75 and SOX10. Here, positivity for each marker can be determined by methods well known to those skilled in the art, such as flow cytometry, immunostaining, or RNA expression analysis.

[0031] In other words, in an example of culture conditions, for example, the culture medium contains EGF at concentrations of 1 ng / ml or higher, 2 ng / ml or higher, 3 ng / ml or higher, 4 ng / ml or higher, 5 ng / ml or higher, 6 ng / ml or higher, 7 ng / ml or higher, 8 ng / ml or higher, 9 ng / ml or higher, 10 ng / ml or higher, 11 ng / ml or higher, 12 ng / ml or higher, 13 ng / ml or higher, 14 ng / ml or higher, 15 ng / ml or higher, 16 ng / ml or higher, 17 ng / ml or higher, 18 ng / ml or higher, 19 ng / ml or higher, 20ng / ml or more, 21ng / ml or more, 22ng / ml or more, 23ng / ml or more, 24ng / ml or more, 25ng / ml or more, 26ng / ml or more, 27ng / ml or more, 28ng / ml or more, 29ng / ml or more, 30ng / ml ml or more, 31ng / ml or more, 32ng / ml or more, 33ng / ml or more, 34ng / ml or more, 35ng / ml or more, 36ng / ml or more, 37ng / ml or more, 38ng / ml or more, 39ng / ml or more, or 40ng / ml or more EGF is added so that it is below 40ng / ml, below 39ng / ml, below 38ng / ml, below 37ng / ml, below 36ng / ml, below 35ng / ml, below 34ng / ml, below 33ng / ml, above 32ng / ml. Bottom, 31ng / ml or less, 30ng / ml or less, 29ng / ml or less, 28ng / ml or less, 27ng / ml or less, 26ng / ml or less, 25ng / ml or less, 24ng / ml or less, 23ng / ml or less, 22ng / ml or less, 21ng It is added to achieve a concentration of 1 ng / ml or less, 20 ng / ml or less, 19 ng / ml or less, 18 ng / ml or less, 17 ng / ml or less, 16 ng / ml or less, 15 ng / ml or less, 14 ng / ml or less, 13 ng / ml or less, 12 ng / ml or less, 11 ng / ml or less, 10 ng / ml or less, 9 ng / ml or less, 8 ng / ml or less, 7 ng / ml or less, 6 ng / ml or less, 5 ng / ml or less, 4 ng / ml or less, 3 ng / ml or less, 2 ng / ml or less, or 1 ng / ml or less.

[0032] In other words, as an example of culture conditions, for example, the culture medium contains bFGF at concentrations of 1 ng / ml or higher, 2 ng / ml or higher, 3 ng / ml or higher, 4 ng / ml or higher, 5 ng / ml or higher, 6 ng / ml or higher, 7 ng / ml or higher, 8 ng / ml or higher, 9 ng / ml or higher, 10 ng / ml or higher, 11 ng / ml or higher, 12 ng / ml or higher, 13 ng / ml or higher, 14 ng / ml or higher, 15 ng / ml or higher, 16 ng / ml or higher, 17 ng / ml or higher, 18 ng / ml or higher, and 19 ng / ml or higher. , 20ng / ml or more, 21ng / ml or more, 22ng / ml or more, 23ng / ml or more, 24ng / ml or more, 25ng / ml or more, 26ng / ml or more, 27ng / ml or more, 28ng / ml or more, 29ng / ml or more, 30ng / ml ml or more, 31ng / ml or more, 32ng / ml or more, 33ng / ml or more, 34ng / ml or more, 35ng / ml or more, 36ng / ml or more, 37ng / ml or more, 38ng / ml or more, 39ng / ml or more, or 40ng / ml or more bFGF is added so as to be above 40ng / ml, 39ng / ml or less, 38ng / ml or less, 37ng / ml or less, 36ng / ml or less, 35ng / ml or less, 34ng / ml or less, 33ng / ml or less, 32ng / ml Below, 31ng / ml or less, 30ng / ml or less, 29ng / ml or less, 28ng / ml or less, 27ng / ml or less, 26ng / ml or less, 25ng / ml or less, 24ng / ml or less, 23ng / ml or less, 22ng / ml or less, 21n It is added to achieve a concentration of g / ml or less, 20 ng / ml or less, 19 ng / ml or less, 18 ng / ml or less, 17 ng / ml or less, 16 ng / ml or less, 15 ng / ml or less, 14 ng / ml or less, 13 ng / ml or less, 12 ng / ml or less, 11 ng / ml or less, 10 ng / ml or less, 9 ng / ml or less, 8 ng / ml or less, 7 ng / ml or less, 6 ng / ml or less, 5 ng / ml or less, 4 ng / ml or less, 3 ng / ml or less, 2 ng / ml or less, or 1 ng / ml or less.

[0033] In an example of culture conditions, for instance, culture is carried out at 25°C or higher, 26°C or higher, 27°C or higher, 28°C or higher, 29°C or higher, 30°C or higher, 31°C or higher, 32°C or higher, 33°C or higher, 34°C or higher, 35°C or higher, 36°C or higher, 37°C or higher, 38°C or higher, 39°C or higher, 41°C or higher, 42°C or higher, 43°C or higher, 44°C or higher, or 45°C or higher. Culture is carried out at 45°C or lower, 44°C or lower, 43°C or lower, 42°C or lower, 41°C or lower, 40°C or lower, 39°C or lower, 38°C or lower, 37°C or lower, 36°C or lower, 35°C or lower, 34°C or lower, 33°C or lower, 32°C or lower, 31°C or lower, 30°C or lower, 29°C or lower, 28°C or lower, 27°C or lower, 26°C or lower, or 25°C or lower.

[0034] In an example of culture conditions, for instance, the culture is carried out under CO2 concentrations of 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 16% or more, 17% or more, 18% or more, 19% or more, or 20% or more, and under CO2 concentrations of 20% or less, 19% or less, 18% or less, 17% or less, 16% or less, 15% or less, 14% or less, 13% or less, 12% or less, 11% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less.

[0035] When performing transplantation, the above cells are preferably used suspended in a suitable substrate. Examples of substrates include, but are not limited to, physiological saline and phosphate-buffered physiological saline. Physiologically active factors such as hormones, growth factors, small molecule compounds, and extracellular matrix may be added to the substrate. Possible and not limited to added factors include, but are not limited to, VEGFA, bFGF, collagen IV, and laminin.

[0036] The transplantation is preferably performed by injecting the cell suspension into the body. It is preferable to inject it into the patient's superior choroidal space using a syringe, but injection may be performed by other methods.

[0037] Non-human animals as choroidal degeneration models: The present invention provides non-human animals as choroidal degeneration models, which are administered with an oxidizing agent. The non-human animals of the present invention are preferably those to which the oxidizing agent has been administered to the choroid. The oxidizing agent is preferably an iodate. The iodate is preferably an alkali metal iodate or an alkaline earth metal iodate. The alkali metal is preferably sodium or potassium, and the alkaline earth metal is preferably calcium or barium. The administered oxidizing agent may be a solution. The concentration of the oxidizing agent in the solution is, for example, 0.01 ug / ul or more, 0.1 ug / ul or more, or 1 ug / ul or more, for example, 1000 ug / ul or less, 100 ug / ul or less, 50 ug / ul or less, or 10 ug / ul or less. The non-human animals are preferably mammalian non-human animals. Mammalian non-human animals are preferably rodents. Mammalian non-human animals are preferably murids. Non-human mammals preferably belong to the Murinae subfamily. Non-human mammals preferably are mice or rats.

[0038] In this specification, the presence of an upper and lower limit for a numerical value is considered self-evident, as it is understood that the numerical range defined by that upper and lower limit is also described. If an upper limit and a lower limit are selectively described, it is considered self-evident that the numerical range defined by the arbitrarily selected upper and lower limits is also described.

[0039] The present invention will be described more specifically below with reference to examples, but the present invention is not limited to these examples.

[0040] Test Example 1: Isolation and Culture of Hemangioblasts. Heads were collected from C57BL / 6J mouse embryos at gestation day 10.5 and treated with collagenase 1 to disperse them at the single-cell level. A biotinylated antibody against the hemangioblast-specific protein PODXL was added to this dispersion and reacted. Then, streptavidin-coated magnetic beads were added to precipitate the hemangioblasts, which were then collected.

[0041] The harvested hemangioblasts were cultured in DMEM containing fetal bovine serum, penicillin, streptomycin, LIF, bFGF, SCF, and Oncostatin M. The hemangioblasts obtained in this way were positive for VEGFR2, PODXL, VE-CAD, and GATA1.

[0042] Test Example 2: Isolation and Culture of Neural Crest Cells The hair follicles of C57BL / 6J mice were collected, and the hair bulbs were cultured in DMEM / F12 containing B-27 supplement, bFGF, and EGF using dishes coated with collagen I. After 7 days, the cells that had migrated to the culture bottom were detached by trypsin treatment, transferred to new collagen I-coated dishes, and cultured in the same culture medium as above. The neural crest cells obtained in this way were positive for p75 and SOX10.

[0043] Test Example 3: Isolation and Culture of Mesenchymal Stem Cells The femurs of C57BL / 6J mice were collected, washed to remove bone marrow, and then crushed. The crushed bone marrow was cultured in DMEM containing fetal bovine serum using dishes coated with fibronectin. After 7 days, the cells that had migrated to the culture bottom were detached by trypsin treatment, cultured and passaged under the same conditions as above, and maintained until only fibroblast-like cells remained morphologically.

[0044] Example 1: Preparation of Choroidal Degeneration Model Mice and Effect of Hemangioblast Transplantation 1 μl of a 8 μg / μl sodium iodate solution was injected into the choroid of 3-week-old mice to prepare choroidal degeneration model mice. Mice injected with PBS instead of the sodium iodate solution were used as controls. Three days after the preparation of the model mice, 1 μl of the hemangioblast suspension (5x10 4 cells / μl) obtained in Test Example 1 or 1 μl of PBS was injected into the suprachoroidal space of the model mice and control mice. Fourteen days after the preparation of the model mice, the eyeballs were enucleated, frozen sections were prepared, stained with HE, and the thickness of the choroid was measured on the nasal and temporal sides respectively.

[0045] The results are shown in Figures 1 and 2. As shown in Figure 1, HE staining revealed choroidal recovery in the hemangioblast transplant group. Furthermore, as shown in Figure 2, the hemangioblast transplant group showed increased choroidal thickness in both the nasal and temporal regions compared to the non-transplant group, indicating regeneration of choroidal tissue.

[0046] Example 2: Effects of Mixed Cell Transplantation The left eye of 3-week-old mice was treated with sodium iodate, and on day 3, a mixed suspension of PBS and two types of cells (hemangioblasts + neural crest cells, or hemangioblasts + mesenchymal stem cells) or three types of cells (hemangioblasts + neural crest cells + mesenchymal stem cells) was injected into the choroid. The right eye was used as an undamaged control. Neural crest cells were obtained from Experiment 2, and mesenchymal stem cells were obtained from Experiment 3. Two weeks later, the eyeballs were extracted from the mice, and the thickness of the choroid on the temporal side was compared with that of the undamaged eye. Damage caused by sodium iodate was detected in the PBS-treated eye, but recovery of choroid thickness was observed in the group injected with the mixed cell suspension (Figure 3). In Figure 3, H+N represents hemangioblasts + neural crest cells, H+M represents hemangioblasts + mesenchymal stem cells, and H+N+M represents hemangioblasts + neural crest cells + mesenchymal stem cells.

[0047] Example 3: Effects of Mixed Cell Transplantation The effects of transplantation of hemangioblasts alone were compared with the effects of mixed transplantation of a mixed suspension of two types of cells (hemangioblasts + neural crest cells, or hemangioblasts + mesenchymal stem cells) or three types of cells (hemangioblasts + neural crest cells + mesenchymal stem cells). The experiment was conducted in the same manner as in Example 2. The results are shown in Figure 4. In all combinations, mixed transplantation showed a higher effect than transplantation of hemangioblasts alone. In Figure 4, H represents hemangioblasts, H+N represents hemangioblasts + neural crest cells, H+M represents hemangioblasts + mesenchymal stem cells, and H+N+M represents hemangioblasts + neural crest cells + mesenchymal stem cells.

Claims

1. A choroidal reconstruction agent containing, as an active ingredient, stem cells capable of differentiating into both blood cells and vascular endothelial cells, for transplantation into degenerated or thinned choroids.

2. The choroidal reconstructor according to claim 1, wherein the hemangioblast is a stem cell having the ability to differentiate into both blood cells and vascular endothelial cells.

3. The choroidal reconstruction agent according to claim 1, comprising a mixture of hemangioblast and mesenchymal stem cells as an active ingredient for transplantation into a degenerated or thinned choroid.

4. The choroidal reconstruction agent according to claim 1, comprising as an active ingredient a mixture of hemangioblasts and neural crest cells for implantation into a degenerated or thinned choroid.

5. The choroidal reconstruction agent according to claim 1, comprising a mixture of hemangioblasts, mesenchymal stem cells, and neural crest cells as an active ingredient for transplantation into a degenerated or thinned choroid.

6. Non-human animals used as a model for choroidal degeneration, which have been administered an oxidizing agent.

7. A non-human animal according to claim 6, wherein an oxidizing agent is administered to the choroid.

8. The non-human animal according to claim 6, wherein the oxidizing agent is an iodate salt.

9. The non-human animal according to claim 8, wherein the iodate is an alkali metal iodate or an alkaline earth metal iodate.

10. The non-human animal according to claim 9, wherein the alkali metal is sodium or potassium, and the alkaline earth metal is calcium or barium.

11. The non-human animal according to claim 6, wherein the non-human animal is a non-human mammal.

12. The non-human mammal according to claim 11, wherein the non-human mammal belongs to the order Rodentia.

13. A non-human animal belonging to the family Muridae, as described in claim 12.

14. A non-human animal belonging to the Murinae subfamily, as described in claim 13.

15. The non-human animal according to claim 11, wherein the mammalian non-human animal is a mouse or a rat.

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