Method for inducing and generating outer retinal cells from stem cells and composition for preventing or treating retinal diseases comprising cells generated thereby

Abstract

The present invention relates to a method for differentiating a spherical neural mass (SNM) into retinal outer layer cells and a composition containing the retinal outer layer cells produced by the differentiation method. When using the differentiation method of the present invention, the neural mass can be efficiently differentiated into retinal outer layer cells, which can be usefully used in related research and development and commercialization.

Description

[Technical field]

[0001] The present invention relates to a method for differentiation and generation of retinal outer layer cells from spherical neural mass (SNM).

[0002] This patent application claims priority to Korean Patent Application No. 10-2021-0015038, filed with the Korean Intellectual Property Office on February 2, 2021, the disclosure of which is incorporated herein by reference. [Background technology]

[0003] Blindness, medically defined as the inability to sense light, is a disease that currently affects tens of millions of people, amounting to 0.2-0.5% of the world's population, and is a disease that brings about great personal, social and economic losses. One of the biggest causes of blindness worldwide is the degeneration of retinal visual cells (photoreceptor cells) and retinal pigment epithelial cells, which can occur due to congenital factors or various other causes. Many diseases fall under this category, including retinal aplasia, retinal degeneration, aged macular degeneration, diabetic retinopathy, retinitis pigmentosa, congenital retinal atrophy, Leber congenital amaurosis, retinal detachment, glaucoma, optic nerve disease and trauma. At present, there is no drug therapy for the fundamental treatment of these diseases, and the most promising treatment is to regenerate dysfunctional visual cells and retinal pigment epithelial cells, which are the cause and result of these diseases, into new functional cells and transplant them. Visual cell and retinal pigment epithelial cell transplantation methods are believed to be capable of preventing blindness or restoring damaged vision by slowing or inhibiting retinal degeneration and improving retinal function after regeneration of a degenerated or degenerated retina.

[0004] The possibility of using bone marrow stem cells (BMS), retinal stem cells (RSC), embryonic stem cells (ESC), induced pluripotent stem cells (iPSC), and somatic cell nuclear transfer cells (SCNT) to treat ocular diseases is emerging. However, there are few research results on the differentiation of stem cells into retinal cells and cell therapy using these. If it were possible to differentiate these stem cells into retinal cells, it would be possible to 1) obtain an unlimited supply of cells for effective cell therapy, 2) elucidate the previously unknown mechanism of differentiation of embryonic cells and retinal progenitors into retinal cells, 3) discover retinal differentiation-related genes and molecules and investigate the pathology associated with them, 4) understand the mechanism of development of retinal degenerative diseases, and 5) develop drugs to prevent retinal degeneration and protect retinal neuron.

[0005] Since the first human embryonic stem cell line was established, human embryonic stem cells have the ability to differentiate into a wide variety of cell types, and it has been proposed that the differentiated cells can be applied to human cell therapy to treat diseases occurring in various organs. That is, human embryonic stem cells can be precisely characterized, and are considered to be a strong candidate for providing a large supply of cells to replace dysfunctional cells with new cells during clinical treatment. It has been hypothesized that differentiated cells that constitute various organs induced from human embryonic stem cells can have the same properties and functions as the corresponding cells formed by normal differentiation processes. Based on this possibility, differentiation induction methods that provide an environment similar to the developmental stage have been attempted in the differentiation of pancreatic hormone-expressing endocrine cells, nerve cells, muscle cells, vascular endothelial cells, etc. In the case of retinal diseases, many attempts have been made to produce visual cells and retinal pigment epithelial cells, which are representative cells of retinal cells, differentiated from human embryonic stem cells, but the results have been very poor.

[0006] One of the greatest successes reported to date is the successful induction of retinal origin cells from human embryonic stem cells, although the differentiation of the induced retinal origin cells into visual cells failed (differentiation rate less than 0.01%) (Lamba, Proc. Natl. Acad. Sci. USA, 2006;103:12769-74).

[0007] Therefore, there is a need to develop clinically applicable methods for differentiation of visual cells and retinal pigment epithelial cells. Summary of the Invention [Problem to be solved by the invention]

[0008] The present inventors have made extensive research efforts to develop a method for differentiation of clinically applicable outer retinal cells, i.e., visual cells and retinal pigment epithelial cells. As a result, they have found that when single neural precursor sphere cells and sac-like structures of neural precursor spheres are cultured together in a single medium, differentiation efficiency into visual cells and retinal pigment epithelial cells is high, and have completed the present invention.

[0009] Therefore, an object of the present invention is to provide a method for differentiation of spherical neural mass (SNM) into retinal outer layer cells, comprising the following steps:

[0010] Culturing the neural precursor sphere single cells and the neural precursor sphere sac-like structures together in a single medium; and The stage where neural precursor spheres are differentiated into retinal outer layer cells.

[0011] It is yet another object of the present invention to provide a pharmaceutical composition for preventing or treating retinal diseases, comprising outer retinal cells generated by the above differentiation method.

[0012] It is yet another object of the present invention to provide a method for treating a retinal disease, comprising administering to an individual outer retinal cells generated by the above differentiation method.

[0013] It is yet another object of the present invention to provide a use of the outer retinal cells generated by the above differentiation method for treating retinal diseases. [Means for solving the problem]

[0014] According to one aspect of the present invention, there is provided a method for differentiation of spherical neural mass (SNM) into retinal outer layer cells, comprising the steps of: Culturing the neural precursor sphere single cells and the neural precursor sphere sac-like structures together in a single medium; and The stage where neural precursor spheres are differentiated into retinal outer layer cells.

[0015] As used herein, the term "outer retinal cells" refers to photoreceptor cells and retinal pigmented epithelium cells present in the outer layer of the cells that make up the multi-layered retina.

[0016] As used herein, the term "visual cells" refers to rod cells and cone cells that reside in the outer layer of the retina. Visual cells may also be referred to as photoreceptor cells, photoreceptor cells, etc.

[0017] As used herein, the term "retinal pigment epithelial cells" refers to cells that constitute the retinal pigment epithelium, which is located in the outermost layer of the retina and is in contact with the choroid.

[0018] As used herein, the term "neural precursor sphere" refers to a spherical aggregate of neural precursor cells generated from omnipotent stem cells such as human embryonic stem cells or induced pluripotent stem cells, or neural precursor cells isolated from neural tissue.

[0019] The neural precursor spheres used in the present invention may have a size of 300-700 μm. According to an embodiment of the present invention, the neural precursor spheres may have a size of 450-550 μm.

[0020] The neural precursor sphere single cells may be isolated by a step of disaggregating the neural precursor sphere into single cells. The disaggregation may include disaggregating the neural precursor sphere using a fine dissection tool. In this case, since disaggregation using a cell separation enzyme causes a lot of cell damage and a low recovery rate, a physical method may be used. As a disaggregation tool, any tool capable of cutting aggregates with a diameter of 500 μm or less under a dissection microscope may be used, and specifically, a thinly drawn glass Pasteur pipette, a metal wire with a diameter of 100 μm or less, and a metal net with holes of micrometer (μm) size may be used. According to an embodiment of the present invention, the disaggregation was performed using a tungsten wire.

[0021] In addition, the dissected neural precursor spheres can be singulated using enzymes well known in the art, such as trypsin-EDTA, dispase, actase, collagenase, etc., as cell separation enzymes. According to an embodiment of the present invention, the singulation of cells was performed using actase.

[0022] According to an embodiment of the present invention, the dissected neural precursor spheres can be cultured adherently in a culture medium containing bFGF and N2 additives in a 60 mm diameter culture dish coated with CellStart for 12 to 24 hours, and then the medium is removed and 3 to 5 ml of actase is added and treated in a 37°C incubator for 1 to 3 hours to dissociate them into single cells.

[0023] The sac-like structure of neural precursor spheres refers to a blister-shaped transparent sac present in the neural precursor spheres, and may be present as a blister-shaped transparent sac protruding from the neural precursor spheres or as a sac entirely surrounded by the transparent sac. The sac-like structure of neural precursor spheres may be isolated using a metal wire, a metal net having micrometer (μm)-sized holes, etc. According to an embodiment of the present invention, the sac-like structure of neural precursor spheres was isolated using a tungsten wire.

[0024] The neural precursor sphere sac-like structures may be sectioned.

[0025] Culturing the neural precursor sphere single cells and the neural precursor sphere sac-like structures together in a single medium This step involves culturing single neural precursor sphere cells and sac-like structures of neural precursor spheres together in a single medium.

[0026] In one embodiment of the present invention, the culturing step is a step of placing a single neural precursor sphere cell on the upper part of a space separated above and below by a porous structure, and placing a sac-like structure of neural precursor spheres on the lower part, and culturing the cells.

[0027] In one embodiment of the present invention, the porous structure is a porous mesh.

[0028] In one embodiment of the present invention, the neural precursor sphere single cell is separated from the non-capsular structure of the neural precursor sphere.

[0029] In one embodiment of the present invention, the neural precursor sacculus-like structure is separated from a neural precursor sacculus derived from a neural precursor sacculus.

[0030] The sac-like structure of the neural precursor sphere can be sliced ​​and cultured. There is no limit to the slice size of the neural precursor sphere sac-like structure.

[0031] The sac-like structure of the neural precursor spheres can be cultured in a culture dish coated with CellStart, with 15 to 20 pieces of the sac-like structure per culture dish, in a DMEM / F12 culture medium containing N2 and B27 additives, but is not limited thereto. The sac-like structure of the neural precursor spheres is located on the lower part of the space separated from the upper and lower parts by the porous structure.

[0032] Single neural precursor spheres were plated on a CellStart-coated culture dish, and 0.5 × 10 cells were cultured at 4 °C. 5 ~1.5×10 5 After placing the single cells in the neural precursor sphere, the single cells can be cultured together with the sac-like structure of the neural precursor sphere for 1 to 3 weeks, but is not limited thereto. The single neural precursor sphere is located in the upper part of the space separated from the upper and lower parts by the porous structure.

[0033] The coating material used in each step is cell starter, but is not limited thereto, and may further include extracellular matrix (ECM) for cell attachment, because undifferentiated stem cells and neural cells cannot attach and grow on a culture dish by themselves, and can be maintained and cultured with the help of extracellular matrix or other supporting cells.

[0034] In addition to Cellstart, other substances can be used as the extracellular matrix, either alone or in combination.

[0035] In one embodiment of the present invention, the neural precursor spheres are differentiated from stem cells.

[0036] In one embodiment of the present invention, the stem cells are totipotent stem cells.

[0037] In one embodiment of the present invention, the stem cells are selected from the group consisting of embryonic stem cells, induced pluripotent stem cells (iPSCs), adult stem cells, somatic cell nuclear transfer embryonic stem cells, and stem cells generated by direct reprogramming. According to an embodiment of the present invention, the stem cells are embryonic stem cells or induced pluripotent stem cells.

[0038] In one embodiment of the present invention, the neural progenitor spheres are differentiated from stem cells through the following steps: Formation of embryoid bodies from stem cells; forming neural rosettes and neural tube-like structures from said embryoid bodies; and forming neural precursor spheres from said neural rosettes and neural tube-like structures;

[0039] The step of forming embryoid bodies from stem cells may be carried out for 4 to 6 days, but is not limited thereto.

[0040] The step of forming embryoid bodies from stem cells may be performed using, but is not limited to, Essential 6 cell culture medium.

[0041] As used herein, the term "embryoid body" refers to a three-dimensional aggregate of omnipotent stem cells, such as embryonic stem cells, which are capable of recapitulating the differentiation process of early embryonic development and are capable of differentiating into all three germ layers of cells: endoderm, mesoderm, and ectoderm.

[0042] The step of forming neural rosettes and neural tube-like structures from the embryoid bodies may be carried out for 4 to 6 days, but is not limited thereto.

[0043] The step of forming neural rosettes and neural tube-like structures from the embryoid bodies may be performed using a culture dish coated with cell starters, but is not limited thereto.

[0044] The step of forming neural rosettes and neural tube-like structures from the embryoid bodies may be performed by culturing the embryoid bodies formed in the embryoid body formation process in a DMEM / F12 minimal culture medium supplemented with only 1 / 2 content of N2 additive in a cell start-coated culture dish for 4 to 6 days, and then replacing the 1 / 2 N2-supplemented DMEM / F12 minimal culture medium with a DMEM / F12 culture medium supplemented with bFGF and N2 for 5 to 10 days for proliferation, but is not limited thereto.

[0045] The culture medium used at this stage may be DMEM / F12 cell culture medium supplemented with bFGF and N2 additive, but any culture medium capable of forming / maintaining neural rosettes can be used without restriction.

[0046] Differentiation of neural precursor spheres into retinal outer layer cells This step is a step in which the single neural precursor sphere cells and the sac-like structures of the neural precursor spheres are cultured together in a single medium, and then the single neural precursor sphere cells and the sac-like structures are differentiated into outer retinal cells.

[0047] In one embodiment of the present invention, the neural precursor sphere is a single neural precursor sphere cell, a sac-like structure of neural precursor spheres, or a single neural precursor sphere cell and a sac-like structure of neural precursor spheres.

[0048] In one embodiment of the present invention, the outer retinal cells are photoreceptors, retinal pigment epithelium cells, or photoreceptors and retinal pigment epithelium cells.

[0049] In one embodiment of the present invention, the outer retinal cells have increased expression of rhodopsin and βIII-tubulin, or increased expression of RPE65 and bestrophin.

[0050] In one embodiment of the present invention, the outer retinal cells ameliorate or prevent blindness.

[0051] In another aspect, the present invention provides a pharmaceutical composition for preventing or treating a retinal disease, comprising outer retinal cells generated by the above differentiation method.

[0052] In one embodiment of the present invention, the retinal disease is selected from the group consisting of retinal aplasia, retinal degeneration, aged macular degeneration, diabetic retinopathy, retinitis pigmentosa, glaucoma, optic neuropathy, trauma, retinal detachment, Leber's congenital amaurosis, and congenital retinal atrophy.

[0053] The outer retinal cells are the same as the outer retinal cells described in the differentiation method, and therefore a description of the outer retinal cells will be omitted to avoid overcomplicating the specification.

[0054] In yet another aspect, the present invention relates to a method for treating a retinal disease, comprising administering to an individual outer retinal cells generated by the above differentiation method.

[0055] The term "individual" refers to a subject in need of treatment for a disease, and more specifically refers to mammals such as human or non-human primates, mice, dogs, cats, horses, and cows.

[0056] According to yet another aspect, the present invention relates to use of outer retinal cells generated by the above differentiation method for treating retinal diseases.

[0057] The outer retinal cells are the same as the outer retinal cells described in the differentiation method described above, so a description of the outer retinal cells will be omitted to avoid overcomplicating the specification. Effect of the Invention

[0058] The features and advantages of the present invention can be summarized as follows: The present invention provides a method for differentiation of a spherical neural mass (SNM) into retinal outer layer cells, and a composition comprising the retinal outer layer cells produced by the differentiation method.

[0059] When using the differentiation method of the present invention, neural precursor cells can be efficiently differentiated into outer retinal cells using only neural precursor spheres without any other chemical components or other supporting cells other than the culture medium and basic culture additives, which can be usefully used in related research and development and commercialization. [Brief description of the drawings]

[0060] [Figure 1A] 1 shows cell morphology at each stage of induction of differentiation of outer retinal cells and a culture dish used for isolation and culture according to one embodiment of the present invention. [Figure 1B] 1 shows cell morphology at each stage of induction of differentiation of outer retinal cells and a culture dish used for isolation and culture according to one embodiment of the present invention. [Diagram 2] 1 shows the results of analyzing the characteristics of SNM according to one embodiment of the present invention. [Figure 3A]The results of confirming the optimal isolation and culture method for differentiation of outer retinal cells are shown (culture of single neural precursor sphere cells only). [Figure 3B] We present the results of confirming the optimal isolation and culture method for differentiation of outer retinal cells (mixed culture of single neural precursor sphere cells and capsule-like slices). [Figure 3C] The results of confirming the optimal isolation and culture method for differentiation of outer retinal cells are shown below (capsular slices on cell culture inserts, and single neural precursor sphere cells on 6-well cell culture dishes, cultured for one day, then isolated and cultured). [Figure 3D] The results of confirming the optimal isolation and culture method for differentiation of outer retinal cells are shown below (single neural precursor sphere cells were cultured on cell culture inserts, and capsule-like slices were cultured on 6-well cell culture dishes for one day before isolation and culture). [Figure 3E] The results of confirming the optimal isolation and culture method for differentiation of outer retinal cells are shown below (single neural precursor sphere cells on the underside of a cell culture insert, and capsule-like slices on a 6-well cell culture dish were cultured for one day before isolation and culture). [Figure 4A] 1 shows the results of confirming whether or not visual cells differentiated from embryonic stem cells are differentiated based on the expression of markers according to one embodiment of the present invention. [Figure 4B] 1 shows the results of confirming whether or not a marker was expressed to determine whether or not visual cells differentiated from induced pluripotent stem cells according to one embodiment of the present invention. [Figure 4C] 1 shows the results of confirming whether or not visual cells were differentiated by measuring the concentration of cyclic GMP according to one embodiment of the present invention. [Figure 4D] According to one embodiment of the present invention, the differentiation of visual cells differentiated from embryonic stem cells was confirmed by the expression of rhodopsin, βIII-tubulin, and nestin genes. [Figure 4E] According to one embodiment of the present invention, the results of confirming the presence or absence of differentiation of retinal epithelial cells differentiated from embryonic stem cells using RPE65, bestrophin, and ZO-1 markers are shown. [Figure 4F]According to one embodiment of the present invention, the results of confirming the presence or absence of differentiation of retinal epithelial cells differentiated from induced pluripotent stem cells using RPE65, bestrophin, and ZO-1 markers are shown. [Figure 5A] According to one embodiment of the present invention, the results of an animal experiment confirming the in vivo engraftment of differentiated visual cells and retinal epithelial cells are shown. [Figure 5B] FIG. 1 shows an electroretinogram measurement performed to measure the efficacy of differentiated visual cells and retinal epithelial cells according to one embodiment of the present invention. [Figure 5C] 1 shows the results of confirming the efficacy of differentiated visual cells and retinal epithelial cells by electroretinogram measurement according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0061] The present invention will be described in more detail below with reference to examples. It will be apparent to those skilled in the art that these examples are merely intended to more specifically illustrate the present invention, and that the scope of the present invention is not limited to these examples according to the gist of the present invention. EXAMPLES

[0062] Throughout this specification, "%" used to indicate the concentration of a particular substance is, unless otherwise specified, % (w / w) for solid / solid, % (w / vol) for solid / liquid, and % (vol / vol) for liquid / liquid.

[0063] Human Embryonic stem cells (hESC) culture Undifferentiated hESCs (SNUhES32) for differentiation into outer retinal cells were cultured in Vitronectin (GIBCO, A27940)-coated culture dishes using Essential 8 (GIBCO, A15169-01) medium.

[0064] At this time, the undifferentiated stem cells were cultured in the form of colonies. In principle, they were cultured for 7 days, but when the center of the colony was about to be induced to differentiate, they were cut into a grid of about 20 to 30 pieces using a tool with a hook-shaped tip of a glass pipette, and 100 pieces were transferred to a new culture dish coated with Vitronectin for subculture, and maintained by changing the culture medium every 24 hours from the 3rd to the 7th day.

[0065] Induced pluripotent stem cells (iPSCs) culture Undifferentiated iPSCs (hFSiPS1) for visual cell differentiation were cultured using the same method as the hESC culture method described above.

[0066] Immunocytochemistry analysis The cells were fixed in 4% paraformaldehyde solution for 10 minutes.

[0067] To ensure that each antibody was fully absorbed into the cytoplasm, the cells were reacted with a 0.1% tryptophan X-100 (in PBS) solution for 15 minutes, and then with a 2% bovine serum albumin (BSA, in PBS) solution at room temperature for 1 hour.

[0068] Then, the cells were bound with the primary antibodies at 4° C. To confirm the cells bound to the primary antibodies, secondary antibodies that matched the species of each primary antibody were used (see Table 1 below).

[0069] Finally, to confirm the cell nuclei, the cells were stained with PBS containing 4',6-diamino-2-phenylindole (DAPI) for 10 minutes, and images were obtained using a fluorescent microscope to confirm and analyze important markers.

[0070] [Table 1]

[0071] Differentiation of human total-potential stem cells into outer retinal cells Undifferentiated human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs), which are omnipotent stem cell lines, were stabilized by two subcultures from the time of thawing, and maintained in culture for 7 days after the third subculture.

[0072] The culture medium of human embryonic stem cells (hESC) or induced pluripotent stem cells (iPSC) (all differentiation potential stem cells) cultured for 7 days was removed, and the cells were washed once with DPBS(-), and then 1 ml of dispase was added and incubated for 3 minutes in a 37℃ incubator to separate the cells. The separated cells were cultured in EB medium (Essential 6, 0.5% penicillin streptomycin) in a Petri culture dish for 5 days to form embryoid bodies (EBs).

[0073] On day 5 of EBs, EBs were transferred to Cell Start-coated dishes so that only neural cells could grow, and cultured for 5 days in neural selection medium DMEM / F12 medium (containing 0.5% N2 substrate, 2 mM L-glutamine, 1% non-essential amino acids (NEAA), 0.5% penicillin-streptomycin, and 0.1 mM beta-mercaptoethanol). Furthermore, for the formation and proliferation of neural rosettes and neural tube-like structures, EBs were cultured and expanded for 4 to 7 days in proliferation medium DMEM / F12 medium (containing 0.5% N2 substrate, 2 mM L-glutamine, 20 ng / ml bFGF (basic fibroblast growth factor), 1% non-essential amino acids (NEAA), 0.5% penicillin-streptomycin, and 0.1 mM beta-mercaptoethanol).

[0074] After that, they were cultured in N2bF medium (DMEM / F12 medium containing 1% N2 substrate, 2 mM L-glutamine, 20 ng / ml bFGF (basic fibroblast growth factor), 1% essential amino acids (NEAA), 0.5% penicillin-streptomycin, and 0.1 mM beta-mercaptoethanol) for 10 to 14 days to form and proliferate neural rosettes and neural tube-like structures.

[0075] When neural rosettes and neural tube-like structures were observed during the culture period, the glass Pasteur pipette was cut away from the base using a thin drawn tool, and then transferred to a Petri dish and cultured in N2bF medium to form spherical neural masses (SNMs).

[0076] When the SNM reached a diameter of approximately 500 μm, they were mechanically cut using a 0.1 mm tungsten wire, and could be passaged up to 10 times without any change in their characteristics.

[0077] One day before the isolation culture for differentiation into outer retinal cells, neural precursor sphere-derived capsule-like structures were cultured as adherent cells on a Cellstart-coated 6-well culture dish, and single cells (neural precursor sphere single cells) from neural precursor sphere non-capsular structures were cultured as adherent cells on a Cellstart-coated cell culture insert. SNM single cell isolation was performed by culturing sliced ​​SNM as adherent cells on a Cellstart-coated culture dish, and one day later, they were treated with actase to isolate them. N2B27 cell culture medium was used for cell isolation culture.

[0078] The isolation culture was performed by placing a cell culture insert in which single-celled neural precursor spheres were cultured into a 6-well culture dish in which slices of the sac-like structures were cultured. N2B27 cell culture medium was used for cell culture, and the medium was changed every two days for 3 weeks of isolation culture until differentiation was complete.

[0079] 1A and 1B are diagrams showing a simple daily scheme of the above process and cell morphology at each stage.

[0080] Optimization of outer retinal cell differentiation Using cells that had completed differentiation to SNM, we performed culture under the following conditions in order to find the optimal culture conditions for differentiation of outer retinal cells.

[0081] a) SNM cells only were cultured. b) Mixed culture of SNM cells and capsule-like slices; c) Capsular slices were placed on cell culture inserts, and SNM single cells were cultured on 6-well cell culture dishes for 1 day, after which they were isolated and cultured. d) SNM single cells were cultured on cell culture inserts, and capsule-like slices were cultured on 6-well cell culture dishes for 1 day before dissociation. e) SNM single cells on the underside of cell culture inserts and capsule-like slices on 6-well cell culture dishes after 1 day of culture.

[0082] The above conditions were performed in a CellStart-coated culture dish, and the cells were cultured using N2B27 medium.

[0083] Reverse transcription polymerase chain reaction (RT-PCR) In order to confirm whether or not differentiation into outer retinal cells occurred depending on the isolation culture time, RT-PCR analysis was performed as follows for the expression at the transcription level of rhodopsin, a marker expressed in visual cells among outer retinal cells.

[0084] RNA from differentiated visual cells was obtained from the SNM using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA), and cDNA synthesis was performed using AMV RT (reverse transcriptase) according to the manufacturer's instructions (AccuPower RT PreMix; Bioneer, Taejeon, Korea) with oligo-dT as a primer.

[0085] PCR amplification was performed using Taq polymerase (HiPi Plus 5x PCR Premix; Elpis biotech, Taejeon, Korea) and standard procedures. PCR conditions were denaturation at 94°C for 5 min, followed by 30 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 10 s. To analyze the relative expression with other mRNAs, the amount of cDNA was normalized based on the signal of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) mRNA.

[0086] After repeated experiments with various annealing temperatures and cycle numbers, the proportionally amplified interval was determined for each primer pair, and the PCR conditions were optimized.

[0087] The sequences of the primers used for gene expression analysis are shown in Table 2 below.

[0088] [Table 2]

[0089] Measurement of phototransduction pathway metabolite concentrations A cGMP enzyme immunoassay kit (GE Healthcare) was used according to the manufacturer's protocol to measure the concentration of phototransduction pathway metabolites from isolated outer retinal cells on day 7 of culture. A phosphodiesterase inhibitor (IBMX, 50 mM) was added 48 hours before cGMP measurement.

[0090] Transplantation of SNM-derived outer retinal cells in a rodent model of retinal degeneration Six-week-old SD rats were given a single intravenous injection of sterile 1% NaIO3 saline solution (70 mg / kg). Four days after intravenous injection, the animals were anesthetized with an intramuscular injection of tiletamine / zolazepam (Zoletil®) and xylazine.

[0091] Pupils were dilated with tropicamide 0.5% and phenylephrine HCl 2.5% eye drops (Mydrin-P®) and treated with proparacine HCl 0.5% (Alcaine®) topical eye anesthetic.

[0092] Before transplantation, SNM-derived outer retinal cells were incubated with DAPI (10 μg / ml) for 30 min, washed several times with medium to remove DAPI, and then dissociated by incubation with 0.05% trypsin / 0.1% EDTA for 5 min at 37°C.

[0093] Each outer retinal cell (1 × 10 5 cells / 2 μl) were injected into the subretinal space or vitreous cavity using a 26-gauge needle attached to a Hamilton syringe.

[0094] Cyclosporine A (210 mg / l, Cipol-N®, Chong Kun Dang, Seoul, Korea) was administered in drinking water 1 day before transplantation until the enucleation process. One week after transplantation, the eyes were enucleated and flash frozen using embedding compound (FSC 22®, Leica microsystems, IL).

[0095] The cooled sections were washed with PBS and blocked with 3% bovine serum albumin in PBS containing 0.1% trypanosamine X100. After blocking, they were reacted with the primary antibodies for 1 day. For immunohistochemical examination, the antibodies used were anti-bШ-tub antibody and mixed human antibodies, RPE65 and ZO-1 antibodies.

[0096] Electroretinograms (b-waves) were measured 4, 12, and 24 weeks after cell transplantation.

[0097] result Differentiation of outer retinal cells from human omnipotent stem cells 1A and 1B are schematic diagrams illustrating a series of steps of the present invention, showing cell morphology at each stage.

[0098] In order to differentiate pluripotent stem cells (including embryonic stem cells and induced pluripotent stem cells) into outer retinal cells, they must be induced to differentiate into neural cells. From the early stages, we induced efficient differentiation into neural progenitor cells by using a culture structure and cell culture medium through the stages of neural progenitor cell selection culture, proliferation culture, neural-specific structure induction, and neural progenitor sphere formation, and we found that the neural progenitor spheres maintained the properties of neural progenitor cells even at the early stage and after repeated passages unless they were given a separate differentiation signal (Figure 2).

[0099] Ultimately, the researchers isolated and cultured only two types of neural precursor sphere structures, and confirmed that they were efficiently differentiated into visual cells and retinal epithelial cells, which is the objective of the present invention.

[0100] The present invention has confirmed that differentiation efficiency can be dramatically increased in the differentiation of visual cells and retinal epithelial cells using all-potential stem cells even by a culture method without media supplements such as external cell signaling activators and inhibitors.

[0101] Optimization of outer retinal cell differentiation In the case of differentiation condition a) in the above "Optimization of Outer Retinal Cell Differentiation" section, the expression of rhodopsin, a marker for visual cells, was hardly observed (Figure 3A), and in the case of culture condition b), only cells derived from the sac-like structure survived, and single neural precursor sphere cells hardly survived (Figure 3B). In culture condition c), rhodopsin expression was observed in single neural precursor sphere cells (Figure 3C), but it tended to be lower than in culture condition d), and it was confirmed that rhodopsin expression was highest in culture condition d) (Figure 3D). In culture condition e), the expression of rhodopsin in single neural precursor sphere cells showed a similar tendency to culture condition d) (Figure 3E), but since single neural precursor sphere cells were attached to the underside of the cell culture insert, it was not possible to completely eliminate the mixture derived from the sac-like structure, and it was not easy to recover each, so culture condition d) was found to be optimal for differentiation of outer retinal cells (visual cells).

[0102] Functional characterization of pan-potent stem cell-derived outer retinal cells We used immunocytochemistry to confirm the presence of specific molecular markers related to the cellular functions of mature visual cells. As can be seen in Figure 4A and Figure 4B, cells that had completed differentiation from embryonic stem cells or induced pluripotent stem cells expressed blueopsin, redopsin, PDE6b, recoverin, and rhodopsin, as well as βIII-tubulin, a marker for neural cells.

[0103] The function of visual cells can be evaluated in vitro by measuring the difference in the concentration of phototransduction pathway metabolites in light and darkness. When a functional phototransduction pathway is present, there is a difference in the concentration of cyclic GMP. When differentiated visual cells were cultured in light and darkness for the same period, the concentration of cyclic GMP in cells cultured in light was reduced compared to that in darkness, and the same phenomenon was confirmed when treated with a phosphodiesterase inhibitor (IBMX). This means that the visual cells differentiated by the present invention have an internal phototransduction pathway, and therefore have been differentiated into visual cells (Figure 4C).

[0104] The expression of rhodopsin, a protein specific to visual cells, was confirmed by RT-PCR during the isolation culture period, and it was confirmed that rhodopsin mRNA expression was highest on day 7 of isolation culture. These results indicate that differentiation of visual cells was already occurring at a high level on day 7 of isolation culture, and that the majority of cells had completed differentiation, as nestin, a marker for neural precursor cells, was barely expressed on day 21 (Figure 4D).

[0105] The differentiation of retinal pigment epithelial cells was confirmed using immunocytochemistry for specific molecular markers. As can be seen in Figure 4E and Figure 4F, retinal pigment epithelial cells that had completed differentiation from embryonic stem cells or induced pluripotent stem cells were confirmed to express ZO-1, RPE65, and bestrophin.

[0106] Efficacy of SNM-derived outer retinal cell transplants in a rodent model of retinal degeneration The visual cells and retinal pigment epithelial cells differentiated according to the present invention were transplanted into retinal degenerated rats, and the engraftment ability was confirmed after 12 weeks. As a result, it was confirmed that human antibodies and the nerve cell marker βIII-tubulin were expressed together with DAPI. In addition, it was confirmed that the retinal epithelial cell markers RPE65 and bestrophin were expressed together with the epithelial cell marker ZO-1 (Figure 5A). All of the transplanted cells were observed from the outer retina, indicating that the transplanted cells had high engraftment ability.

[0107] The electroretinogram measurements confirmed that b-wave amplitude was maintained in all transplant groups (Figure 5B), and visual function showed a slight decrease over time after transplantation, but was significantly increased compared to the control group. Furthermore, visual cell transplantation showed a greater improvement in visual function than RPE transplantation (Figure 5C). [Industrial Applicability]

[0108] The present invention relates to a method for differentiation and generation of retinal outer layer cells from spherical neural mass (SNM).

Claims

1. A method for differentiating spherical neural mass (SNM) into retinal outer layer cells, the method comprising: Culturing the neural precursor sphere single cells and the neural precursor sphere sac-like structures together in a single medium; and Differentiating the neural precursor spheres into retinal outer layer cells; Including, A method, wherein in the culturing step, the single neural precursor sphere cell and the sac-like structure of the neural precursor sphere are separated by a porous structure.

2. The method according to claim 1, wherein the culturing step is a step of placing a single neural precursor sphere cell on the upper part of a space separated above and below by a porous structure and culturing the single neural precursor sphere cell on the lower part of the space.

3. The method of claim 2 , wherein the porous structure is a porous mesh.

4. The method of claim 1, wherein the neural precursor sphere single cells are separated from neural precursor sphere-derived non-capsular structures.

5. The method of claim 1, wherein the neural precursor sphere sac-like structure is separated from a neural precursor sphere-derived clear sac.

6. The method of claim 1 , wherein the neural progenitor spheres are differentiated from stem cells.

7. 7. The method of claim 6, wherein the stem cells are selected from the group consisting of embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), adult stem cells, somatic cell nuclear transfer embryonic stem cells, and stem cells generated by direct reprogramming.

8. The method of claim 1, wherein the neural precursor spheres are differentiated from stem cells by the following steps: forming an embryoid body from the stem cells; forming neural rosettes and neural tube-like structures from the embryoid bodies; and forming neural precursor spheres from said neural rosettes and neural tube-like structures;

9. 2. The method of claim 1, wherein the outer retinal cells have increased expression of rhodopsin and βIII-tubulin, or increased expression of RPE65 and bestrophin.

10. 2. The method of claim 1, wherein the outer retinal cells are photoreceptor cells, retinal pigment epithelium cells, or photoreceptor cells and retinal pigment epithelium cells.

11. The method of claim 1 , wherein the outer retinal cells ameliorate or prevent vision loss.

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