Retinal progenitor cells having ability to differentiate into retinal pigment epithelial cells, photoreceptor cells, and crystalline lens epithelial cells, and mass production method therefor

Abstract

The present invention relates to retinal progenitor cells simultaneously exhibiting characteristics of retinal pigment epithelial cells, photoreceptor cells, and lens epithelial cells, and a mass production method therefor. When retinal progenitor cells are isolated and cultured using the method according to one aspect, the retinal progenitor cells can be mass-produced in a short time, and thus the method is economical. In addition, the isolated retinal progenitor cells exhibit all of the differentiation characteristics of retinal pigment epithelial cells, photoreceptor cells and lens epithelial cells. Therefore, the present invention can be effectively used in the fields of research on retinal diseases such as macular degeneration, and research and development of therapeutic agents.

Description

Retinal progenitor cells having the ability to differentiate into retinal pigment epithelial cells, photoreceptor cells, and lens epithelial cells, and a method for mass production thereof

[0001] The present invention relates to retinal progenitor cells having the ability to differentiate into retinal pigment epithelial cells, photoreceptor cells, and lens epithelial cells, and a method for mass production thereof.

[0002]

[0003] Degenerative eye diseases, particularly dry macular degeneration, are one of the major causes of vision loss, yet effective treatments involving the regeneration of the damaged retina are limited. In these diseases, damage to retinal pigment epithelium (RPE) and photoreceptor cells is known to be a key pathological mechanism of disease progression. Therefore, regenerative medicine approaches that repair or replace damaged RPE and photoreceptor cells are considered the most promising therapeutic strategies.

[0004] Based on current research and early clinical trial results, regenerative therapy using RPE and photoreceptor cell transplantation has demonstrated efficacy and safety, presenting new possibilities for the treatment of degenerative eye diseases. However, existing cell sources and technologies still have limitations that need to be overcome.

[0005] In this regard, in the case of RPE transplantation derived from post-mortem donated eyes, mass production is difficult during the process of securing RPE, and there are technical limitations in securing high-purity photoreceptor cells as well. Furthermore, in the case of RPE transplantation derived from embryonic stem cells and induced pluripotent stem cells, the differentiation process takes at least three months, which limits rapid therapeutic application, and there is a lack of efficient processes for mass production. Moreover, while organoid-based photoreceptor differentiation technology can enhance physiological similarity and functionality by reproducing complex 3D structures, mass production of high-purity and high-functionality photoreceptor cells is difficult, and additional research is required to improve the reproducibility and productivity of organoid differentiation technology.

[0006] To overcome these technical limitations, it is necessary to develop more efficient and stable methods for supplying RPE and photoreceptor cells, while simultaneously requiring innovative technologies capable of ensuring mass production and clinical safety. A method based on one aspect proposes a novel cell-based therapeutic strategy to address these issues and aims to contribute to the treatment of degenerative eye diseases.

[0007]

[0008] One aspect is to provide isolated retinal progenitor / precursor cells (RPCs) expressing one or more selected from the group consisting of retinal pigment epithelium (RPE) development factor, photoreceptor cell development factor, and lens epithelial cell (LEC) development factor.

[0009] Another aspect is to provide retinal pigment epithelium (RPE) differentiated from the aforementioned retinal progenitor cells.

[0010] Another aspect is to provide photoreceptor cells differentiated from the aforementioned retinal progenitor cells.

[0011] Another aspect is to provide lens epithelial cells (LECs) differentiated from the aforementioned retinal progenitor cells.

[0012] Another aspect is to provide a pharmaceutical composition for the prevention or treatment of retinal diseases comprising the above-mentioned retinal progenitor cells.

[0013] Another aspect is to provide the use of the retinal progenitor cells for the manufacture of drugs for the prevention or treatment of retinal diseases.

[0014] Another aspect is to provide a pharmaceutical composition for the prevention or treatment of retinal disease comprising the step of administering the above-mentioned retinal progenitor cells to an individual in need thereof.

[0015] Another aspect provides a method for proliferating retinal progenitor cells (RPCs), comprising the step of culturing isolated RPCs in a medium containing an EMT (Epithelial-Mesenchymal Transition) inhibitor.

[0016]

[0017] One aspect provides isolated retinal progenitor / precursor cells (RPCs) expressing one or more selected from the group consisting of retinal pigment epithelium (RPE) development factor, photoreceptor cell development factor, and lens epithelial cell (LEC) development factor.

[0018] In one embodiment, the retinal progenitor cell may express all of the retinal pigment epithelial cell development factor, the photoreceptor cell development factor, and the lens epithelial cell development factor.

[0019] The aforementioned “Retinal Progenitor / Precursor Cells (RPCs)” refer to origin cells possessing multipotent potential capable of determining differentiation into cells present in the retina and retinal pigment epithelial cells. Retinal progenitor cells may be cells induced by differentiation from various stem cells, such as retinal stem cells, bone marrow stem cells, adipose stem cells, neural stem cells, embryonic stem cells, induced pluripotent stem cells, and somatic cell nuclear transfer stem cells, or they may be retinal origin cells present within the retina or extracted. Retinal origin cells are generally capable of symmetric or asymmetric division, and as a result, they may differentiate into various retinal cells or retinal pigment epithelial cells, or form other retinal progenitor cells through a proliferative process.

[0020] In one embodiment, the retinal progenitor cell may be a retinal progenitor cell isolated from an individual, and specifically, may be a retinal progenitor cell derived from an ectopic embryo. The ectopic embryo may be an ectopic embryo in the first or second trimester of pregnancy at 6 to 8 weeks of gestation, and specifically, the ectopic embryo may be an ectopic embryo at 6.5 weeks of gestation.

[0021] In one embodiment, the cell may be a single cell, a cell cluster formed by two or more cells, or a cell population formed by aggregating multiple cells. Additionally, the cell may be in the form of a cell aggregate, a cell mass, or a tissue-like structure composed of cells. Furthermore, the cell may include a homogeneous cell population or a heterogeneous cell population, and the cell may be a naturally derived cell, a transformed cell, or a genetically modified cell.

[0022] In one embodiment, stillborn fetal tissue was isolated from the excised fetus of the first trimester of ectopic pregnancy and skin tissue was removed from the developing ocular tissue, and then a single cell was isolated by treating it with a proteolytic enzyme such as collagenase, and karyotype analysis and chromosomal microarray were performed on the isolated single cell to confirm that the copy number and karyotype were normal (see Example 1).

[0023] In another embodiment, as a result of analyzing the characteristics of the retinal progenitor cells, it was confirmed that they express not only retinal pigment epithelial cell development factor but also photoreceptor cell development factor and lens epithelial cell development factor (see Example 3.1).

[0024] The aforementioned “Retinal Pigment Epithelium (RPE)” refers to cells forming a single layer between the retina and the choroid capillaries, specifically referring to a single layer of pigment epithelial cells. These cells are characterized by performing important roles in maintaining retinal function and are known to be capable of absorbing scattered light energy, interacting with photoreceptors to maintain the visual cycle, maintaining the binding structure between photoreceptors and the choroid capillary layer, secreting various growth factors, transporting ions, water, and metabolites, and removing degenerated photoreceptor outer segments through phagocytosis.

[0025] In one embodiment, the retinal pigment epithelium (RPE) development factor may be one or more selected from the group consisting of SYNPR, FLRT3, PAX2, ELN, PMEL, FRZB, GRIA4, LRP2, IGF2, NEFM, LYPD1, ROM1, DPP10, SOX6, PAX8, HMX1, SFRP2, NCAM1, PMEL, DCT, PAX8, SOX6, ROM1, CD24, CD248, THY1, SOX2, and PAX6, and specifically, the retinal pigment epithelium development factor may be SYNPR, PAX2, ELN, PMEL, FRZB, GRIA4, LRP2, IGF2, NEFM, LYPD1, PROM1, SOX6, HMX1, SFRP2, NCAM1, DCT, PAX8, CD248, BMP7, It may be one or more selected from the group consisting of OCA2, TTR, SOX, and PAX6, and more specifically, the retinal pigment epithelial cell development factor may be one or more selected from the group consisting of SOX2 and PAX6.

[0026] In one embodiment, the SOX2 expression amount of the retinal progenitor cells may be 1 to 20%, 1 to 15%, 1 to 9%, 5 to 20%, 5 to 15%, 5 to 9%, 8 to 20%, 8 to 15%, or 8 to 9% relative to the SOX2 expression amount of the embryonic stem cells.

[0027] In one embodiment, the number of retinal progenitor cells expressing SOX2 may be 20 to 90%, 20 to 70%, 20 to 60%, 20 to 50%, 30 to 90%, 30 to 70%, 30 to 60%, 30 to 50%, 40 to 90%, 40 to 70%, 40 to 60%, or 40 to 50% relative to the total number of retinal progenitor cells.

[0028] In one embodiment, the number of retinal progenitor cells expressing PAX6 may be 20 to 90%, 20 to 70%, 20 to 60%, 20 to 50%, 30 to 90%, 30 to 70%, 30 to 60%, 30 to 50%, 40 to 90%, 40 to 70%, 40 to 60%, or 40 to 50% relative to the total number of retinal progenitor cells.

[0029] In one embodiment, the number of retinal progenitor cells expressing SOX2 or PAX6 may be 20 to 90%, 20 to 70%, 20 to 60%, 20 to 50%, 30 to 90%, 30 to 70%, 30 to 60%, 30 to 50%, 40 to 90%, 40 to 70%, 40 to 60%, or 40 to 50% relative to the total number of retinal progenitor cells.

[0030] In one embodiment, the number of retinal progenitor cells expressing SOX2 and PAX6 may be 20 to 90%, 20 to 70%, 20 to 60%, 20 to 50%, 30 to 90%, 30 to 70%, 30 to 60%, 30 to 50%, 40 to 90%, 40 to 70%, 40 to 60%, or 40 to 50% relative to the total number of retinal progenitor cells.

[0031] In one embodiment, the expression ratio of SOX2 / PAX6 may act as a regulatory factor for the differentiation and functional characteristics of the retinal progenitor cells. For example, if SOX2 is overexpressed, the tendency toward neural progenitor cells may persist, and if PAX6 is overexpressed, differentiation into specific retinal cells may be promoted. Therefore, the expression ratio of SOX2 / PAX6 can be considered and regulated as an important factor in artificial retinal tissue culture or retinal regeneration therapy.

[0032] In one embodiment, when the retinal progenitor cells are a group of two or more clustered cells, a cell population, a cell aggregate, or a cell mass, i.e., a group of retinal progenitor cells, the proportion of the group of cells expressing SOX2 within the group of retinal progenitor cells may be 20 to 60%, specifically, the proportion of the group of cells expressing SOX2 within the group of retinal progenitor cells may be 20 to 60%, 20 to 50%, 30 to 60%, 30 to 50%, 40 to 60%, 40 to 50%, 45 to 50%, and more specifically, the proportion of the group of cells expressing SOX2 within the group of retinal progenitor cells may be 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50%.

[0033] Additionally, the proportion of the cell group expressing PAX6 within the retinal progenitor cell group may be 20 to 60%, and specifically, the proportion of the cell group expressing SOX2 within the retinal progenitor cell group may be 20 to 60%, 20 to 50%, 30 to 60%, 30 to 50%, 40 to 60%, 40 to 50%, 45 to 50%, and more specifically, the proportion of the cell group expressing SOX2 within the retinal progenitor cell group may be 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50%.

[0034] Furthermore, the proportion of the cell group expressing both SOX2 and PAX6 within the retinal progenitor cell group may be 5 to 50%, and specifically, the proportion of the cell group expressing both SOX2 and PAX6 within the retinal progenitor cell group may be 5 to 50%, 5 to 40%, 5 to 30%, 10 to 50%, 10 to 40%, 10 to 30%, 20 to 50%, 20 to 40%, or 20 to 30%, and more specifically, the proportion of the cell group expressing both SOX2 and PAX6 within the retinal progenitor cell group may be 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%.

[0035] In another embodiment, it was confirmed that the surface markers expressed by the retinal progenitor cells, as well as SOX2 and PAX6, were expressed at high levels. In another embodiment, it was found that the retinal progenitor cells expressed CD44, CD73, and CD166, did not express HLA-DR molecules at all, and partially expressed HLA-Class I molecules. Additionally, it was confirmed that OCT4 and Nanog were not expressed at all. In the case of SOX2 and PAX6, it was found that almost all retinal progenitor cells expressed them. However, ICC results showed that the expression of SOX2 and PAX6 in the retinal progenitor cell population was heterogenic, exhibiting a high and low expression pattern rather than uniform protein expression levels, which is consistent with the patterns observed in the early developmental stages of various ocular tissues. In this regard, scRNA seq analysis results showed that among the total retinal progenitor cell population analyzed, 49.28% of the cells expressed SOX2, 42.14% of the cells expressed PAX6, and 23.68% of the cells expressed both SOX2 and PAX6 (see Example 3.3).

[0036] The aforementioned "photoreceptor cell" refers to the most important cell in vision that converts light energy entering through the cornea and lens into electrical energy and transmits it to the brain, enabling the perception of shape and color. The photoreceptor cell layer consists of cone photoreceptor cells, which receive bright light, and rod photoreceptor cells, which receive weak light. Cone cells are abundant near the macula, the central part of the retina, and play a role in perceiving shape and color, whereas rod cells are mainly distributed in the periphery of the retina and play a role in perceiving shape and brightness.

[0037] In one embodiment, the photoreceptor cell generating factor may be one or more selected from the group consisting of SYNPR, PENK, FRZB, MAL, GRIA4, CBLN2, RORB, SOX6, GAP43, NTRK2, NES, HMX1, THRB, and PAX2. Specifically, the photoreceptor cell generating factor may be one or more selected from the group consisting of FRZB, MAL, GRIA4, CBLN2, RORB, GAP43, NTRK2, and PAX2, and more specifically, the photoreceptor cell generating factor may be FRZB, MAL, GRIA4, CBLN2, RORB, GAP43, NTRK2, and PAX2.

[0038] The aforementioned “Lens Epithelial Cell (LEC)” is a progenitor cell responsible for the growth and development of the eye's lens.

[0039] In one embodiment, the lens epithelial cell (LEC) development factor may be one or more selected from the group consisting of CRYBB1, CRYBB2, MIP, AQP5, CDH1, CRYBA4, CRYBA2, CRYBA1, CRYGS, GRIA4, MAFB, SOX6, FRZB, HMX1, PAX2, SFRP2, FOXE3, LIM2, PITX3, MAF, and SOX2. Specifically, the lens epithelial cell development factor may be one or more selected from the group consisting of CRYBB1, CRYBB2, MIP, AQP5, CDH1, and SOX2, and more specifically, the lens epithelial cell development factor may be CRYBB1, CRYBB2, MIP, AQP5, CDH1, and SOX2.

[0040] In one embodiment, the retinal progenitor cells may express one or more selected from the group consisting of CD44, CD73, CD166, and HLA-Class I, which are part of the mesenchymal stem cell characteristic markers, and specifically, the retinal progenitor cells may express all of CD44, CD73, CD166, and HLA-Class I.

[0041] In one embodiment, the retinal progenitor cells may not express one or more selected from the group consisting of CD14, CD34, CD45, which are some of the hematopoietic stem cell-specific markers; HLA-DR, which is an immunogenicity-related marker; and OCT4 and Nanog, which are some of the pluripotent cell markers. Specifically, the retinal progenitor cells may not express all of CD14, CD34, CD45, HLA-DR, OCT4, and Nanog.

[0042] In one embodiment, the number of retinal progenitor cells expressing CD44 may be 50 to 90%, 50 to 85%, 50 to 80%, 55 to 90%, 55 to 85%, 55 to 80%, 60 to 90%, 60 to 85%, or 60 to 80% relative to the total number of retinal progenitor cells.

[0043] In one embodiment, the number of retinal progenitor cells expressing CD73 may be 40 to 70%, 40 to 65%, 40 to 60%, 45 to 70%, 45 to 65%, 45 to 60%, 50 to 70%, 50 to 65%, or 50 to 60% relative to the total number of retinal progenitor cells.

[0044] In one embodiment, the number of retinal progenitor cells expressing CD166 may be 90 to 100%, 90 to 99%, 900 to 98%, 93 to 100%, 93 to 99%, 93 to 98%, 97 to 100%, 97 to 99%, or 97 to 98% relative to the total number of retinal progenitor cells.

[0045] In one embodiment, the number of retinal progenitor cells expressing HLA-Class I may be 50 to 80%, 50 to 75%, 50 to 70%, 55 to 80%, 55 to 75%, 55 to 70%, 60 to 80%, 60 to 75%, or 60 to 70% relative to the total number of retinal progenitor cells.

[0046] It was confirmed that the retinal progenitor cells according to one embodiment possess the ability to differentiate into retinal pigment epithelial cells, photoreceptor cells, and lens epithelial cells. Therefore, the retinal progenitor cells can be utilized as a multifunctional therapeutic agent capable of treating various retinal diseases at once.

[0047] Another aspect provides retinal pigment epithelium (RPE) differentiated from the above retinal progenitor cells.

[0048] The above terms “retinal progenitor cell,” “differentiation,” and “retinal pigment epithelial cell” are as described above.

[0049] In one embodiment, the retinal pigment epithelial cells may express one or more proteins selected from the group consisting of ZO1, RPE65, and PAX6, and the retinal pigment epithelial cells may express all of ZO1, RPE65, and PAX6.

[0050] According to one embodiment, the retinal pigment epithelial cells were prepared by differentiating from the retinal progenitor cells, and it was confirmed that they express a retinal pigment epithelial cell marker protein, which indicates that the retinal progenitor cells actually exhibit excellent differentiation ability.

[0051] Another aspect provides a photoreceptor cell differentiated from the above-mentioned retinal progenitor cell.

[0052] The terms "retinal progenitor cell" and "photoreceptor cell" above are as described above.

[0053] The aforementioned "differentiation" refers to the phenomenon in which structures or functions become specialized among cells during their growth through division and proliferation; in other words, it signifies the changes in form or function of biological cells, tissues, etc., as they perform the specific tasks assigned to them.

[0054] In one embodiment, the photoreceptor cell may express one or more proteins selected from the group consisting of GNAT1, Recoverin, Arrestin and Rhodopsin, NRL, NR2E3, PNR, CRX and CD73, specifically, the photoreceptor cell may express one or more proteins selected from the group consisting of GNAT1, Recoverin, Arrestin and Rhodopsin, and more specifically, the photoreceptor cell may express all of GNAT1, Recoverin, Arrestin and Rhodopsin.

[0055] According to one embodiment, the photoreceptor cell was prepared by differentiating from the retinal progenitor cell, and it was confirmed that it expresses a photoreceptor cell marker protein, so it can be seen that the retinal progenitor cell actually exhibits excellent differentiation ability.

[0056] Another aspect provides lens epithelial cells (LECs) differentiated from the aforementioned retinal progenitor cells.

[0057] The above terms "retinal progenitor cell," "differentiation," and "lens epithelial cell" are as described above.

[0058] In one embodiment, the lens epithelial cell may express one or more proteins selected from the group consisting of αA-crystallin, γ-crystallin, FOXE3, ANXA4, MCM4, AQP4, LAMA1, and β-crystallin, specifically, the lens epithelial cell may express one or more proteins selected from the group consisting of AQP4, LAMA1, and β-crystallin, and more specifically, the lens epithelial cell may express all of AQP4, LAMA1, and β-crystallin.

[0059] According to one embodiment, the lens epithelial cells were prepared by differentiating from the retinal progenitor cells, and it was confirmed that they express a lens epithelial cell marker protein, which indicates that the retinal progenitor cells actually exhibit excellent differentiation ability.

[0060] Another aspect provides a method for proliferating retinal progenitor / precursor cells (RPCs), comprising the step of culturing isolated retinal progenitor / precursor cells (RPCs) in a medium containing an EMT (Epithelial-Mesenchymal Transition) inhibitor.

[0061] In one embodiment, the method may further include the step of treating retinal tissue separated from an individual with a proteolytic enzyme to obtain the separated retinal progenitor cells.

[0062] In one embodiment, the individual may be a mammal such as a human, mouse, rat, rabbit, pig, or monkey; an amphibian; a bird, or a reptile.

[0063] In one embodiment, the separated retinal tissue may be retinal tissue derived from a normal embryo, an ectopic embryo, a fetus, or an adult, and specifically, the separated retinal tissue may be retinal tissue derived from an ectopic embryo. The ectopic embryo is an embryo in the early stages of pregnancy, that is, an embryo in the first or second trimester of pregnancy, i.e., an embryo of about 4 to 12 weeks of age, and specifically, the ectopic embryo may be an embryo of 6 to 8 weeks of pregnancy, and more specifically, the ectopic embryo may be an embryo of 6.5 weeks of pregnancy.

[0064] In one embodiment, the protein-degrading enzyme may be collagenase, dispase, trypsin, or elastase, and specifically, the protein-degrading enzyme may be collagenase.

[0065] In one embodiment, the processing step may be performed for 5 to 60 minutes, and specifically, the processing step may be performed for 5 to 60 minutes, 5 to 50 minutes, 5 to 40 minutes, 5 to 30 minutes, 5 to 20 minutes, 10 to 60 minutes, 10 to 50 minutes, 10 to 40 minutes, 10 to 30 minutes, or 10 to 20 minutes.

[0066] In one embodiment, the method may further include the step of separating retinal tissue from the ectopic embryo to obtain the separated retinal tissue. Specifically, the separation may be mechanically separated using, for example, micro-tweezers, micro-scissors, or micro-needles; may be separated by treatment with an enzyme such as a collagenase or protease; or may be separated using a UV, IR, or VIS laser. In a method according to one aspect, the retinal tissue may be mechanically separated from the ectopic embryo.

[0067] In one embodiment, stillborn fetal tissue was isolated from excised ectopic fetus from the first trimester of pregnancy, and skin tissue was removed from developing ocular tissue, after which single cells were isolated by treating with a proteolytic enzyme such as collagenase. The isolated single cells were transferred to an RPC culture medium and cultured (see Example 1).

[0068] In one embodiment, the EMT inhibitor is Y-27632, RepSox, Rapamycin, 17-AAG, LY294002, salinomycin, curcumin, mocetinostat, low Ca 2+ It may be one or more selected from the group consisting of αSMA blockers, LiCl, and metformin, and specifically, the EMT inhibitor may be one or more selected from the group consisting of Y-27632 and RepSox.

[0069] In one embodiment, the culture may be performed under culture conditions coated with an extracellular matrix or under culture conditions not coated with an extracellular matrix, and specifically, the culture may be performed in a medium coated with an extracellular matrix.

[0070] In one embodiment, the extracellular matrix may be one or more selected from the group consisting of collagen, fibronectin, laminin, vitronectin, and gelatin, and specifically, the extracellular matrix may be fibronectin.

[0071] In one embodiment, the medium may further include one or more substances selected from the group consisting of MEMα, NEAA, GlutaMAX, N2 supplement, fetal bovine serum (FBS), THT solution, and antibiotics, and specifically, the medium may further include MEMα, NEAA, GlutaMAX, N2 supplement, fetal bovine serum (FBS), THT solution, and antibiotics.

[0072] In another embodiment, isolated retinal progenitor cells were subcultured in culture medium 1 supplemented with MEMα, NEAA, GlutaMAX, N2 supplement, fetal bovine serum (FBS), THT solution, antibiotics, bFGF, and EGF, and culture medium 2 supplemented with MEMα, NEAA, GlutaMAX, N2 supplement, fetal bovine serum (FBS), THT solution, antibiotics, and the EMT inhibitors Y-27632 and RepSOX. As a result of culture, it was found that the retinal progenitor cells cultured in the medium supplemented with bFGF and EGF transformed into mesenchymal stem cells and did not proliferate further after 10 passages. On the other hand, when proliferated in the medium supplemented with EMT inhibitors, it was confirmed that active proliferation occurred while maintaining the epithelial cell form up to 30 passages.

[0073] In addition, regarding proliferation conditions, culturing early retinal progenitor cells as single cells or in the form of cell colonies did not significantly affect the degree of proliferation. However, regarding the presence or absence of fibronectin coating, it was confirmed that the initial engraftment and proliferation of retinal progenitor cells were superior in the fibronectin-coated medium (see Example 2).

[0074] When retinal progenitor cells are cultured according to the method of one embodiment, it was confirmed that the retinal progenitor cells do not change into a stem cell form during the middle of passage culture, and actively proliferate while maintaining the form of epithelial cells, which is the characteristic of the original cells, up to 30 passages. Therefore, when retinal progenitor cells are cultured according to the above method, a large amount of retinal progenitor cells can be obtained in a short period of time, and the obtained retinal progenitor cells do not lose their ability to differentiate into various cells, including retinal pigment epithelial cells, even if they are passage cultured for a long period, so they can be used in various ways for retinal transplantation research and neuroregeneration therapy.

[0075] Another aspect provides a pharmaceutical composition for the prevention or treatment of retinal disease comprising the above-mentioned retinal progenitor cells.

[0076] The above terms "retinal progenitor cell" and "retinal pigment epithelial cell" may be within the aforementioned range.

[0077] In one embodiment, the cell may be a single cell, a cell cluster formed by two or more cells, or a cell population formed by aggregating multiple cells. Additionally, the cell may be in the form of a cell aggregate, a cell mass, or a tissue-like structure composed of cells. Furthermore, the cell may include a homogeneous cell population or a heterogeneous cell population, and the cell may be a naturally derived cell, a transformed cell, or a genetically modified cell.

[0078] In one embodiment, the retinal progenitor cell may be a retinal progenitor cell isolated from an individual, and specifically, the retinal progenitor cell may be a retinal progenitor cell derived from an ectopic embryo. The ectopic embryo may be an ectopic embryo in the first or second trimester of pregnancy at 6 to 8 weeks of gestation, and specifically, the ectopic embryo may be an ectopic embryo at 6.5 weeks of gestation.

[0079] In one embodiment, the retinal progenitor cells may be proliferated by a method of proliferation of retinal progenitor cells comprising the step of culturing in a medium containing an EMT (Epithelial-Mesenchymal Transition) inhibitor.

[0080] The above "EMT inhibitor" and "culture," etc., may be within the aforementioned range.

[0081] The above "retinal disease" refers to a disease resulting from damage to the retina caused by reasons such as accidents of unknown cause, aging, or disease gene abnormalities, and the above retinal disease may be one or more selected from the group consisting of Stargardt disease, cataract, pediatric cataract, diabetic retinopathy, choroidal neovascularization, macular degeneration, macular degeneration, retinal degeneration, macular edema, retinal edema, macular swelling, Retinitis Pigmentosa, retinal vascular occlusion, retinal detachment, and hereditary retinal disease, and more specifically, the above retinal disease may be macular degeneration.

[0082] In one embodiment, the "retinal disease" may be induced by oxidative stress. Specifically, the "retinal disease" may be induced as retinal pigment epithelial cells are damaged by oxidative stress through various pathways, including necrosis or apoptosis.

[0083] The above term "prevention" may refer to any act of suppressing or delaying retinal disease in an individual by administering a pharmaceutical composition according to one aspect.

[0084] The term "treatment" above may refer to any act in which the symptoms of an individual's retinal disease are improved or beneficially altered by the administration of a pharmaceutical composition according to one aspect.

[0085] The term "administration" above means introducing a specific substance to an individual by an appropriate method.

[0086] In one embodiment, the pharmaceutical composition may further include a suitable carrier, excipient, or diluent commonly used in the manufacture of pharmaceutical compositions.

[0087] The above pharmaceutical composition may have any one dosage form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, liquids, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories, and may be various dosage forms for oral or parenteral administration. When formulating, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

[0088] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc., with one or more compounds. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

[0089] Liquid preparations for oral administration include suspensions, oral liquids, emulsions, and syrups; in addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients, such as humectants, sweeteners, flavorings, and preservatives. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleates may be used as non-aqueous solvents and suspension solvents. Witepsol, Macrogol, Tween 61, cocoa paste, laurin paste, and glycerogelatin may be used as bases for suppositories.

[0090] In one embodiment, the pharmaceutical composition may also be formulated as an ophthalmic composition, such as eye drops and an eye ointment. Such forms may include any ophthalmic preparation for topical administration to the eye used in the field of ophthalmology. Eye drops are prepared by dissolving an active ingredient in a sterile aqueous solution, such as saline and buffer solution. Eye drops may be provided as a powder composition to dissolve before use, or may be provided by combining with a powder composition to dissolve before use. An eye ointment may be prepared by mixing an active ingredient into an ointment base.

[0091] The above pharmaceutical composition is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to prevent or treat cancer with a reasonable benefit / risk ratio applicable to medical use, and the effective dose level may be determined based on factors including individual type and severity, age, gender, drug activity, weight, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. In this case, the content of the active ingredient included in the above pharmaceutical composition may be 0.0001 weight % to 10 weight %, specifically 0.001 weight % to 1 weight %, based on the total weight of the composition.

[0092] In another embodiment, when retinal progenitor cells were administered subretinally to a mouse model with induced macular degeneration, the effect of regenerating the damaged retina was confirmed. In addition, it was confirmed that the light response of photoreceptor cells increased, and the retinal thickness and the thickness of retinal layer cells also increased simultaneously (see Example 9).

[0093] In one embodiment, the pharmaceutical composition may further include retinal progenitor / precursor cell (RPC)-derived exosomes (EVs) and functional substances, and the pharmaceutical composition may further include other pharmaceutical compositions for the prevention or treatment of retinal diseases in addition to the retinal progenitor / precursor cells.

[0094] The above pharmaceutical composition may be provided mixed with a pharmaceutical composition for the prevention or treatment of other retinal diseases, and the pharmaceutical composition for the prevention or treatment of other retinal diseases may be a conventionally known pharmaceutical composition or a newly developed pharmaceutical composition.

[0095] If the above pharmaceutical composition further comprises a pharmaceutical composition for the prevention or treatment of other retinal diseases, it is important that the amounts mixed allow for maximum effect to be obtained with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

[0096] When the above pharmaceutical composition further includes other pharmaceutical compositions for the prevention or treatment of retinal diseases, a synergistic effect may occur in which the preventive or therapeutic effects of retinal diseases, such as the regenerative effect of damaged retina or the effect of increasing retinal thickness and retinal layer cell thickness, become more pronounced than when only the pharmaceutical composition containing the above retinal progenitor cells is included as an active ingredient.

[0097] In one embodiment, the pharmaceutical composition may be administered alone or in combination with other pharmaceutical compositions for the prevention or treatment of retinal diseases. That is, the pharmaceutical composition may be administered in conjunction with other known pharmaceutical compositions having a preventive or therapeutic effect on retinal diseases, and may be administered simultaneously, separately, or sequentially, and may be administered as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects, taking all of the above factors into consideration, and this can be easily determined by a person skilled in the art.

[0098] The above term “concurrent administration” means administering two or more drugs simultaneously or during the same treatment period.

[0099] When the above pharmaceutical composition is administered in combination with another pharmaceutical composition for the prevention or treatment of retinal diseases, a synergistic effect may occur in which the preventive or therapeutic effects of retinal diseases, such as the regenerative effect of damaged retina or the effect of increasing retinal thickness and retinal layer cell thickness, become more pronounced than when the above pharmaceutical composition is administered alone.

[0100] A pharmaceutical composition according to one aspect may exhibit a synergistic effect when administered in combination with another pharmaceutical composition for the prevention or treatment of retinal diseases, compared to when the pharmaceutical composition is administered alone.

[0101] In one embodiment, the retinal progenitor cells may be administered to an individual in any amount or number, e.g., an effective amount, that causes a detectable therapeutic benefit to the individual. The cells may be administered to the individual in an absolute or relative number of cells, and specifically, the retinal progenitor cells are about, at least about, or up to about 1 x 10⁶ 5 , 5 x 10 5 , 1 x 10 6 , 5 x 10 6 , 1 x 10 7 , 5 x 10 7 , 1 x 10 8 , 5 x 10 8 , 1 x 10 9 , 5 x 10 9 , 1 x 10 10 , 5 x 10 10 , or 1 x 10 11 It may be administered to a dog, or the retinal progenitor cell-derived exosomes may be about, at least about, or up to about 1 x 10⁶ 5 , 5 x 10 5 , 1 x 10 6 , 5 x 10 6 , 1 x 10 7 , 5 x 10 7 , 1 x 10 8 , 5 x 10 8 , 1 x 10 9 , 5 x 10 9 , 1 x 10 10 , 5 x 10 10 , or 1 x 10 11 It can be administered to individual dogs.

[0102] In one embodiment, the dosage of the pharmaceutical composition may be 0.01 mg to 10,000 mg, 0.1 mg to 1,000 mg, 1 mg to 100 mg, 0.01 mg to 1,000 mg, 0.01 mg to 100 mg, 0.01 mg to 10 mg, or 0.01 mg to 1 mg. However, the dosage may vary depending on factors such as the formulation method, administration method, patient's age, body weight, gender, pathological condition, food, time of administration, route of administration, excretion rate, and response sensitivity, and a person skilled in the art may appropriately adjust the dosage by considering these factors. The number of administrations may be one or two or more within the range of clinically acceptable side effects, and regarding the administration site, it may be administered at one or two or more sites. For animals other than humans, the above dosage may be administered at the same amount per kg as for humans, or calculated based on, for example, the ratio of organ volumes (such as the heart) between the target animal and humans (e.g., average value).

[0103] The above pharmaceutical composition may be administered orally or parenterally, and when the above pharmaceutical composition is administered parenterally, it may be administered as a topical application to the skin or as an intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, subretinal injection, intramuscular injection, arterial injection, bone marrow injection, cardiac injection, intradural injection, transdermal injection, nasal injection, enteral injection, local injection, sublingual injection, rectal injection, ventricular injection, intracerebral micro-injection, control injection, intrathecal injection, facet joint injection, or thoracic injection, and specifically, as an intravenous injection or subretinal injection.

[0104] A pharmaceutical composition according to one embodiment includes the retinal progenitor cells as an active ingredient, and the effect of the pharmaceutical composition in restoring a damaged retina has been confirmed through actual examples. Therefore, the pharmaceutical composition according to one aspect can be used to prevent or treat various retinal diseases by performing the role of replacing or protecting damaged retinal cells.

[0105]

[0106] When retinal progenitor cells are isolated and cultured using a single method, they can be produced in large quantities within a short period, making it economical. Furthermore, the isolated retinal progenitor cells exhibit differentiation characteristics for retinal pigment epithelial cells, photoreceptor cells, and lens epithelial cells. Therefore, this method can be usefully applied in the research of retinal diseases such as macular degeneration and in the research and development of therapeutic agents.

[0107]

[0108] Figure 1 is a diagram showing the process of retinal progenitor cells obtained from ectopic embryonic tissue differentiating into various cell types of the eye.

[0109] Figure 2 is a figure showing various factors related to the development process of the eye.

[0110] Figures 3a and 3b show the karyotype analysis results and chromosome microarray results of retinal progenitor cells obtained from ectopic embryonic tissue.

[0111] Figure 4 is a figure showing the degree of cell proliferation and cell morphology according to the composition of the culture medium and retinal progenitor cells obtained from ectopic embryonic tissue.

[0112] Figure 5 shows the results of EE-RPC proliferation in a medium containing growth factors and a medium containing an EMT inhibitor.

[0113] Figure 6 shows the EE-RPC proliferation results depending on the presence or absence of fibronectin coating.

[0114] Figure 7 shows the results of RNAseq analysis for characterization of retinal progenitor cells.

[0115] Figure 8 shows the results of single-cell RNAseq analysis for characterization of retinal progenitor cells.

[0116] Figures 9a to 9e show the results of a gene ontology (GO) test for characterizing retinal progenitor cells.

[0117] Figure 10 shows the experimental results for identifying surface proteins expressed by retinal progenitor cells.

[0118] Figure 11 shows the expression levels of SOX2 and PAX6 in retinal progenitor cells.

[0119] Figure 12 is a figure showing the direction of differentiation of ocular tissue cells according to the expression levels of SOX2 and PAX6, etc., during the development of the eye.

[0120] Figure 13 is a figure showing the differentiation process of retinal progenitor cells into retinal pigment epithelial cells.

[0121] Figure 14 is a figure showing the change in cell shape during the process of retinal progenitor cells differentiating into retinal pigment epithelial cells.

[0122] Figure 15 is a figure showing the results of confirming whether marker proteins are expressed in differentiated retinal pigment epithelial cells.

[0123] Figure 16 is for confirming the function of differentiated retinal pigment epithelial cells

[0124] Figure 17 is a figure showing the results of confirming whether specific genes related to the function of differentiated retinal pigment epithelial cells are expressed.

[0125] Figure 18 is a figure comparing the morphology of cells during the differentiation process of retinal pigment epithelial cells according to the parent cell.

[0126] Figure 19 shows the results of comparing marker proteins expressed according to the parent cells of differentiated retinal pigment epithelial cells.

[0127] Figure 20 is a figure showing the results of a phagocytosis analysis to confirm the cell function according to the parent cell of differentiated retinal pigment epithelial cells.

[0128] Figure 21 is a figure showing the results of comparing whether genes related to cell function are expressed according to the parent cells of differentiated retinal pigment epithelial cells.

[0129] Figure 22 is a figure showing the differentiation process of retinal progenitor cells into photoreceptor cells.

[0130] Figure 23 shows the results of confirming, through flow cytometry, that retinal progenitor cells express a specific protein marker during the process of differentiation into photoreceptor cells.

[0131] Figure 24 shows the results of confirming, through immunohistochemical staining, that retinal progenitor cells express a specific protein marker during the process of differentiation into photoreceptor cells.

[0132] Figure 25 shows the results of confirming whether the differentiated photoreceptor cells are functionally intact using the Single cell patch clamp method.

[0133] Figure 26 is a figure showing the differentiation process of retinal progenitor cells into lens epithelial cells.

[0134] Figure 27 is a figure showing the results of confirming that retinal progenitor cells take on a lens organoid form during the process of differentiating into lens epithelial cells.

[0135] Figure 28 shows the results of confirming that differentiated cells express protein markers of lens epithelial cells.

[0136] Figure 29 shows the experimental process of confirming the retinal regeneration effect by administering retinal pigment epithelial cells to a mouse model of macular degeneration.

[0137] Figure 30 shows the results of administering retinal pigment epithelial cells to a mouse model of macular degeneration, which increased the light response of photoreceptor cells, retinal thickness, and retinal layer cell thickness.

[0138] Figure 31 shows the results of confirming, by immunohistochemical staining, that cells transplanted under the retina have engrafted into the retinal layer and differentiated into retinal pigment epithelial cells.

[0139]

[0140] The present invention will be explained in more detail below through examples. However, these examples are intended to illustrate the invention and the scope of the invention is not limited to these examples.

[0141]

[0142] Examples

[0143] Example 1. Isolation of ectopic embryonic-derived retinal progenitor cells (RPCs) and confirmation of karyotype stability

[0144] Human ocular tissue develops from the second week of pregnancy and develops into pigment layer retina, neural layer retina, and lens epithelial cells around the sixth week (Fig. 2). Accordingly, in this embodiment, 'Ectopic Embryonic Retinal Progenitor Cells (EE-RPC)' were isolated from the ocular tissue of an ectopic fetus in the first or second trimester of pregnancy between the 6th and 8th weeks of pregnancy, which is the Optic cup & Lens maturation stage.

[0145] After isolating stillborn fetal tissue from the excreted ectopic fetus of the first trimester of pregnancy and removing skin tissue from the developing ocular tissue, single cells were isolated by treating with proteolytic enzymes such as collagenase at 37°C for 10 to 20 minutes and washed with a sufficient amount of PBS or HBSS solution. After collecting the cells by centrifugation, they were transferred to RPC culture medium and cultured in a CO2 incubator (5% CO2, 37°C, > 80% humidity) (Fig. 1).

[0146] The isolated single cells were subjected to karyotype analysis and chromosome microarray to determine the copy number and karyotype stability (Fig. 3).

[0147]

[0148] Example 2. Confirmation of a method for mass production of retinal progenitor cells (RPCs)

[0149] Among the single cells isolated above, EE-RPC cells isolated from 6.5-week-old fetal ocular tissue were cultured in different media compositions to compare the degree of cell proliferation according to adult retinal-derived retinal pigment epithelial growth factor and EMT inhibitor.

[0150] In a culture medium supplemented with RPC culture media such as MEMα, NEAA, GlutaMAX, N2 supplement, fetal bovine serum (FBS), THT solution, and antibiotics, as well as adult retinal pigment epithelial growth factors bFGF and EGF, it was confirmed that the single cells transformed into mesenchymal stem cell morphology and did not proliferate after 10 passages. On the other hand, in a culture medium supplemented with epithelial mesenchymal transition inhibitors (EMT inhibitors) Y-27632 and RepSOX instead of growth factors, it was confirmed that the epithelial cells maintained their morphology well and proliferated up to about 30 passages.

[0151] In other words, it was confirmed that EE-RPC maintained the characteristics of epithelial cells and cultured excellently in a medium containing an EMT inhibitor (Figs. 4 and 5).

[0152] In addition, during the process of subculturing EE-RPC, there was no significant difference in the subsequent proliferation process whether the cells were subcultured as single cells or in a cell colony form to maintain gap junctions. Meanwhile, when EE-RPC was cultured in a medium containing the EMT inhibitor, the initial engraftment and proliferation of the cells were found to be superior in the fibronectin-coated medium (Fig. 6).

[0153]

[0154] Example 3. Characterization of produced retinal progenitor cells (RPCs)

[0155] Example 3.1. RNAseq Analysis

[0156] To confirm the characteristics of the isolated retinal progenitor cells (RPCs) mentioned above, gene expression analysis was performed using RNAseq.

[0157] Analysis of additional genes expressed in RPCs revealed that genes specific to photoreceptor cells as well as retinal pigment epithelial cells were identified. Specifically, it was confirmed that EE-RPC cells possess distinctly different characteristics, such as specifically expressing RPE development factors like 'SOX2, PAX6, and PMEL', which are not expressed at all in adult retinal-derived retinal pigment epithelial cells (RPE); photoreceptor development factors like 'FRZB, MAL, GRIA4, CBLN2, RORB, GAP43, and NTRK2'; and factors important for lens epithelial cell development like 'CRYBB1, CRYBB2, MIP, AQP5, CDH1, and SOX2' (Fig. 7).

[0158]

[0159] Example 3.2. Single-cell RNAseq analysis

[0160] Single-cell RNAseq analysis was performed to confirm the characteristics of the isolated single cell (RPC) above.

[0161] Upon verification, it was confirmed that EE-RPCs consist of nine major cell groups as shown in Fig. 8, and upon checking the Gene Ontology (GO), it was confirmed that EE-RPCs express genes specific to early neuroretinal progenitor cells that differentiate into RPE progenitor cells and rod and cone photoreceptors (Figs. 9a to 9e).

[0162]

[0163] Example 3.3. Analysis of Expression Markers

[0164] To confirm the characteristics of the above-mentioned isolated single cells (RPCs), the expression of surface markers and pluripotent stem cell-specific markers was checked.

[0165] EE-RPCs were found to express almost no hematopoietic stem cell (HSC) markers, but some mesenchymal stem cell (MSC) markers, such as CD44 at 70.9%, CD73 at 50.9%, and CD166 at 97.7% (Fig. 10). Immunogenicity-related HLA-DR markers were not expressed at all, and HLA-Class I markers were partially expressed, similar to fetal-derived cells.

[0166] Next, the expression levels of Oct4 and Nanog, specific markers for pluripotent stem cells, were checked. Since the pluripotency factors Oct4 and Nanog were not expressed at all, it was found that there was no pluripotency.

[0167] Single-cell RNAseq was performed to determine the expression levels of SOX2 / PAX6. The total number of cells used was 11,938, of which 5,883 cells expressed SOX2, accounting for 49.28% of the total cells. Additionally, 5,031 cells expressed PAX6, accounting for 42.14% of the total cells. The number of cells expressing both SOX2 and PAX6 was 2,827, accounting for 23.68% of the total cells (Fig. 11a).

[0168] As a result of performing immunocytochemistry (ICC), it was confirmed that SOX2 was expressed in all EE-RPCs and simultaneously, PAX6 was also expressed in almost all EE-RPCs (Fig. 11b). RT-PCR confirmed that the expression level of SOX2 was high enough to reach 8.7% of the expression level in embryonic stem cells.

[0169]

[0170] Example 4. Confirmation of the differentiation ability of produced retinal progenitor cells into retinal pigment epithelial cells

[0171] Example 4.1. Induction of differentiation into retinal pigment epithelial cells

[0172] Differentiation into retinal pigment epithelium (RPE) was induced from the above-described isolated single cells (RPC). Differentiation was carried out in an RPE differentiation medium for about 2 to 3 weeks, and the overall differentiation induction process is shown in Fig. 13.

[0173] During the differentiation process into RPE, RPCs changed their cell morphology by forming tight junctions, which are characteristic of epithelial cells, and after 15 to 20 days of differentiation induction, they exhibited a cobblestone shape, which is characteristic of RPE cells (Fig. 14).

[0174]

[0175] Example 4.2. Analysis of Characteristics of Differentiated Retinal Pigment Epithelial Cells

[0176] To confirm the characteristics of differentiated RPE cells, expressed proteins and expressed genes were identified.

[0177] Immunocytochemistry (ICC) was used to identify the expression of proteins in differentiated RPE cells, and it was confirmed that they expressed the marker proteins ZO1, RPE65, and PAX6 of RPE cells (Fig. 15). In addition, it was confirmed that the expression of specific genes related to the function of mature RPE cells, such as ZO1, RPE65, PAX6, CRALBP, and MITF, increased during the RPE differentiation process (Fig. 17).

[0178]

[0179] Example 4.3. Comparative analysis of characteristics of retinal pigment epithelial cells according to blast cells

[0180] In addition, the cell morphology, expression markers, function, and expression genes of RPE cells were compared and analyzed for EE-RPC-derived RPE, adult retina-derived aRPE, and iPSC-derived RPE.

[0181] Upon verification, it was confirmed that EE-RPC-derived RPE exhibited the most distinct cobblestone structure, which is one of the characteristics of RPE cells, in terms of cell morphology (Fig. 18). Furthermore, analysis of expression markers via ICC revealed that EE-RPC-derived RPE expressed ZO1 and RPE65—RPE cell marker proteins—superiorly compared to aRPE (Fig. 19). In addition, analysis of cell function via phagocytosis analysis showed that EE-RPC-derived RPE exhibited the highest phagocytic activity, indicating that the function of EE-RPC-derived RPE was the most superior (Fig. 20). Finally, analysis of cell expression genes via RT-PCR revealed that EE-RPC-derived RPE expressed SOX2, RPE65, and PAX6—specific genes related to RPE cell function—at significantly high levels (Fig. 21).

[0182]

[0183] Example 5. Confirmation of the differentiation ability of produced retinal progenitor cells into photoreceptor cells

[0184] Differentiation into photoreceptor cells was induced from the above-described isolated single cells (RPCs). The differentiation induction process was carried out according to the method shown in Fig. 22. The EE-RPCs of the single cells underwent a total of three differentiation stages and differentiated into photoreceptor cells, retinal ganglion cells, and Muller glial cells.

[0185] Retinal progenitor cells were cultured in a photoreceptor cell differentiation medium for about 28 days, and the overall differentiation process is as shown in Figure 22.

[0186] Through FACS analysis, it was confirmed that specific markers expressed during the differentiation process into photoreceptor cells, namely Chx10, CRX, and Rho proteins, were expressed (Fig. 23).

[0187] In addition, through ICC analysis, the expression of GNAT1, Recoverin, Arrestin, and Rodopsin, markers of matured rod photoreceptors; Recoverin and Arrestin, markers of cone photoreceptors; ISL1, marker of retinal ganglion cells; and markers of Müller glial cells was confirmed (Fig. 24).

[0188] Finally, the degree of ion channel activity, changes in membrane potential, and visual signal transmission ability of photoreceptor cells differentiated by the single cell patch clamp method were confirmed, and it was found that they fully functioned as photoreceptor cells (Fig. 25).

[0189]

[0190] Example 6. Confirmation of the differentiation ability of produced retinal progenitor cells into lens epithelial cells

[0191] Differentiation of EE-RPC single cells into lens epithelial cells was induced using the method shown in Fig. 26, and marker expression was confirmed.

[0192] RPCs were differentiated into lens epithelial cells through pellet culture and organoid differentiation. During the differentiation process, the lens organoid morphology was confirmed (Fig. 27), and ICC was performed to confirm the expression of AQP4, LAMA1, and β-crystallin, which are markers of mature lens epithelial cells (Fig. 28).

[0193]

[0194] Example 7. Confirmation of the retinal regenerative effect of retinal pigment progenitor cells

[0195] In a NaIO3-induced macular degeneration mouse model (intravenous administration at 20 mg / kg), EE-RPC cells (10,000 cells, 50,000 cells / eye) were transplanted subretinally to confirm the retinal regeneration effect. The mice were sacrificed 40 days after administration to confirm the retinal regeneration effect, and the overall experimental procedure is shown in Figure 29.

[0196] Starting three days prior to the subretinal administration of the above RPC cells (50,000 cells / eye), an immunosuppressant (Cyclosporione, 201 mg / L water) was added to drinking bottles shielded from light by aluminum foil and administered to all experimental animals. On the day of subretinal cell transplantation (D0), mice were placed under general anesthesia, and after imaging the retinal baseline using OCT, 1 µl of RPC-derived cells (50,000 cells / eye) was subretinally administered to both eyes using an IO kit (33 G needle) on individuals without retinal abnormalities (Group G3). For Group G2, BSS + The solution was administered to both eyes at a volume of 1 µl / eye, and after administration was completed, one drop of antibiotic eye drops was administered to the mouse's eye to prevent infection.

[0197] Afterwards, the success of subretinal administration was determined by performing FP / OCT imaging to check for the formation of blebs and hemorrhage in the retina.

[0198] On day 28 after cell administration, to induce dAMD, NaIO3 was administered bolus-IV at a dose of 20 mg / kg to the G2 and G3 groups via microvenous injection, while PBS was administered bolus-IV to the G1 (control group) via microvenous injection. OCT and ERG were measured on days 36 and 37 after cell administration, and on day 40, the animals were sacrificed, their eyes were enucleated, and tissue staining was performed.

[0199] Upon verification, it was confirmed that in the group (G3) administered EE-RPC cells subretinally, the photoreceptor cell light response (Electroretinography, ERG), retinal thickness, and retinal layer cell (ONL) thickness all increased (Fig. 30).

[0200] In addition, it was confirmed by immunohistochemistry (IHC) that the cells transplanted under the retina engrafted into the retinal layer and successfully differentiated into RPE cells (Fig. 31).

Claims

1. Isolated retinal progenitor / precursor cells (RPCs) expressing one or more selected from the group consisting of retinal pigment epithelial (RPE) development factor, photoreceptor cell development factor, and lens epithelial cell (LEC) development factor.

2. The cell according to Claim 1, wherein the retinal pigment epithelium (RPE) development factor is one or more selected from the group consisting of SOX2 and PAX6.

3. The cell of claim 1, wherein the retinal progenitor cell expresses one or more selected from the group consisting of CD44, CD73, CD166 and HLA-Class I.

4. The cell of claim 1, wherein the retinal progenitor cell does not express one or more selected from the group consisting of CD14, CD34, CD45, HLA-DR, OCT4, and Nanog.

5. Cells according to claim 2, wherein the number of retinal progenitor cells expressing SOX2 or PAX6 is 20 to 90% of the total number of retinal progenitor cells.

6. The cell according to claim 1, wherein the photoreceptor cell generating factor is one or more selected from the group consisting of FRZB, MAL, GRIA4, CBLN2, RORB, GAP43, NTRK2, and PAX2.

7. The cell according to claim 1, wherein the lens epithelial cell (LEC) development factor is one or more selected from the group consisting of CRYBB1, CRYBB2, MIP, AQP5, CDH1, and SOX2.

8. The cell of Claim 1, wherein the retinal progenitor cell has the ability to differentiate into one or more cells from the group consisting of retinal pigment epithelium (RPE), photoreceptor cells, and lens epithelial cells (LEC).

9. Retinal pigment epithelial cells (RPE) differentiated from the retinal progenitor cells of Claim 1.

10. Photoreceptor cell differentiated from the retinal progenitor cell of Claim 1.

11. Lens epithelial cell (LEC) differentiated from the retinal progenitor cell of Claim 1.

12. A pharmaceutical composition for the prevention or treatment of retinal disease comprising the retinal progenitor cells of Claim 1.

13. A pharmaceutical composition according to claim 12, wherein the retinal disease is one or more selected from the group consisting of Stargardt disease, cataract, pediatric cataract, diabetic retinopathy, choroidal neovascularization, macular degeneration, macular degeneration, retinal degeneration, macular edema, retinal edema, macular swelling, Retinitis Pigmentosa, retinal vascular occlusion, retinal detachment, and hereditary retinal disease.

14. A method for proliferating retinal progenitor cells (RPCs), comprising the step of culturing isolated retinal progenitor cells (RPCs) in a medium containing an EMT (Epithelial-Mesenchymal Transition) inhibitor.

15. The method of claim 14, wherein the medium is coated with an extracellular matrix.

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