KR20130048468A - The method for inducing differentiation from cystic structure to retinal pigment epithelial cells - Google Patents

Abstract

PURPOSE: A method for inducing differentiation of a cystic structure to retinal pigment epithelial cells is provided to obtain the retinal pigment epithelial cells by differentiation of the cystic structure, and to treat various degenerative retinal diseases. CONSTITUTION: A method for inducing differentiation of a cystic structure to retinal pigment epithelial cells comprises: a step of culturing pluripotent stem cells and forming embryoid body(EB); a step of culturing and proliferating the EB; a step of cleaving neural rosettes and a neural tube-like structure; a step of isolating spherical neural masses(SNMs) formed from the neural tube-like structure and sub-culturing SNMs; a step of SNM-derived cystic structure; and a step of culturing the cystic structure.

Description

The method for inducing differentiation from Cystic Structure to Retinal Pigment Epithelial Cells}

The present invention relates to a method of inducing differentiation from pluripotent stem cells to retinal pigment epithelial cells. More specifically, the present invention relates to a stem cell therapy research that induces differentiation of cystic structures derived from embryonic stem cells and induced pluripotent stem cells into retinal pigment epithelial cells and to treat retinal degenerative diseases. .

Blindness refers to the absence of medically wide angles, a disease currently suffered by tens of millions of patients worldwide, 0.2-0.5% of the population, causing significant personal, social and economic losses. One of the biggest causes of blindness worldwide is the degeneration of photoreceptor cells and retinal pigment epithelial cells in the retina, which are caused by congenital factors or various other causes. Retinal degeneration, retinal degeneration, aged macular degeneration, diabetic retinopathy, retinitis pigmentosa, congenital atrophy, Leber congenital amaurosis ), Many diseases include retinal detachment, glaucoma, optic neuropathy and trauma. To date, there is no pharmacotherapy for the fundamental treatment of these diseases, and the most likely treatment is to regenerate and transplant the photoreceptor cells and retinal pigment epithelial cells of the dysfunction and resulting dysfunction into new functional cells. have. Photoreceptor cells and retinal pigment epithelial cell transplantation have been evaluated to be able to delay or inhibit retinal degeneration, regenerate degenerated or degenerated retinas, and improve retinal function to prevent blindness or regenerate damaged vision. .

Bone Marrow Stem Cells (BMS), Retinal Stem Cells (RSC), Embryonic Stem Cells (ESC), Induced Ppluripotent Stem Cells (iPSCs) and Somatic Nuclei Treatment potential using somatic cell nuclear transfer cells (SCNT) is emerging. However, research on the differentiation of stem cells from retinal cells and cell therapy using the same has been insufficient. When differentiation from these stem cells to retinal cells is possible, 1) infinite cell supply for effective cell therapy, and 2) retinal cells from embryonic cells and retinal progenitors, which have not been known so far. Mechanism of differentiation of the retina, 3) discovery of genes and molecules related to retinal differentiation and pathology, 4) identification of the mechanism of development of retinal degeneration diseases, and 5) development of drugs for the prevention of retinal degeneration and retinal neuroprotection. Will also be available.

Since the establishment of the first human embryonic stem cell line, human embryonic stem cells have the ability to differentiate into a wide variety of cell types, and human cell therapies that can treat diseases that occur in various organs. It has been proposed to be applicable to. In other words, human embryonic stem cells can be accurately identified, and is considered to be a strong candidate for abundantly supplying cells to replace dysfunctional cells with new cells during clinical treatment. Differentiated cells constituting various organs derived from human embryonic stem cells have been assumed to have the same properties and functions as those of cells formed through normal differentiation. Based on this possibility, differentiation induction methods that provide an environment similar to the developmental stage may be used for pancreatic hormone-expressing endocrine cells (D'Amour, et al., Nat. Biotechnol., 2006; 24: 1392-401), neurons (Pankratz). , et al., Stem Cells 2007; 25: 1511-20), muscle cells (Barberi et al., Nat. Med., 2007; 13: 642-8), vascular endothelial cells (Wang, et al., Nat. Biotechnol., 2007; 25: 317-8). In retinal disease, many attempts have been made to produce photoreceptor cells and retinal pigment epithelial cells, which are representative cells of retinal cells, differentiated from human embryonic stem cells, but the results are very poor.

One of the most successful studies reported so far is that retinal progenitor cells are effectively induced from human embryonic stem cells, but differentiation of photoreceptor cells from induced retinal progenitor cells failed (less than 0.01% differentiation) (Lamba Proc. Natl. Acad. Sci. USA, 2006; 103: 12769-74). Another reported successful differentiation of photoreceptor cells from human embryonic stem cells, but since the total differentiation induction period takes more than 200 days and the differentiation rate is very small (8%), it is applied to clinical treatment of blindness. This is a realistically impossible grade below (Osakada et al., Nat. Biotechnol., 2008; 26: 215-24).

It is an object of the present invention to provide a method for obtaining neural precursor spheres from pluripotent stem cells and inducing differentiation from cystic structures formed therefrom into retinal pigment epithelial cells. By using the method of inducing differentiation into retinal pigment epithelial cells of the present invention, a target retinal pigment epithelial cell can be obtained in a relatively short time compared with the conventional technique.

In order to achieve the above object, the present invention provides a method for inducing differentiation of retinal pigment epithelial cells (RPE) from the neural precursor sphere-derived cystic structure obtained by culturing an embryonic stem cell line (Retinal Pigment Epithelial Cells, RPE) do.

In another aspect, the present invention provides a retinal pigment epithelial cell differentiation induced through the above method and its progeny, and provides a composition for treating retinal degeneration disease comprising the same.

According to the present invention, it is possible to differentiate the cystic structure that has not been used conventionally to obtain retinal pigment epithelial cells and to differentiate them into desired retinal pigment epithelial cells in a relatively short time. Can be used.

1 schematically illustrates the process of differentiation of human embryonic stem cells (hESCs) into Retinal Epithelial Cells (RPE). It takes about 14 days for RPE cells to form from the cystic vesicles of the spherical neural masses (SNM). Characteristic forms appearing in the differentiation stage are shown from A to G. A and B represent neuronal rosettes and neural tube-like structures, respectively. C represents the initial SNM composed of neural tube-like structures. D is a purely isolated and expanded open neural tube-like structure by mechanical passage culture, E is a cystic vesicle form. F is a 3D cultured cystic vesicle with pigments on the surface of RPE cells, and G is a cystic vesicle-derived RPE cell after fragmentation and monolayer culture.
2A and 2B show evidence for the differentiation step. A is the embryonic body (EB), B is the neurological rosette, C is the neural precursor mass (SNM), D is the neural progenitor (NP) selection process, E is the NP proliferation process Shows. F to H are expression of neuroepithelial markers in SNM, G is expression of Sox1 and Sox2 in EB (Embryo body), J is expression of Pax6 and βIII-tubulin in SNM including cystic vesicles after neuronal differentiation To show.
3A and 3B identify ES and iPS derived RPE cells. A, B are RPE cells and pigments in cystic vesicles, C is RPE cells after monolayer culture of cystic vesicles, D is remodeling of vesicles after monolayer culture of ESC-derived RPE cells, E and F are derived from cystic EB Unknown cells are shown. G depicts molecular marker expression of functional RPE cells. H shows the results confirming that RPE is produced through the same process from induced pluripotent stem cells. I shows that Ki67, a marker of dividing cells, also expresses during monolayer culture of ESC derived RPE cells. J to M show the results of confirming the expression of RPE specific markers via immunochemistry.
4 shows transmission electron micrographs (TEM) of cystic SNM-derived RPE cells (A, B) and shows confirmation of phagocytosis (C-H). TEM shows cubic epithelial cells with melanin granules (box A), apical microvilli (B), and tight junctions between cells in the apical part of the cell. Confocal microscopy shows the internalization of latex beads (C through E) and the outer segment (F through H) of FITC labeled photoreceptor cells. z-scans were performed to obtain optical cross sections (E, H).
5 shows the functional activity and appearance of the grafts of ES derived RPE cells. A and B represent the secretion of neurotrophic factors PEDF and VEGF by ELISA test. The Y axis shows the relative% concentration of neurotrophic factors compared to the differentiation medium. (ES is the culture of ESC, RPE is the culture of ES-derived RPE cells. ARPE means the culture of adult RPE cells, (2W) for 2 weeks culture, (4W) for 4 weeks culture. RA treatment is 2 weeks) Lasted.)
C and D are compared by staining the retinal tissue, C is the result of H & E staining of normal retinal tissue, degenerative retinal tissue after D sodium-iodate inoculation. E to G depict survival, in situ, and expression of RPE markers of ESC derived RPE cells confirmed by immunochemistry after transplantation.
6 shows RPE maturation and marker expression during sustained culture. The pigment (A) of cystic vesicles disappeared early in monolayer culture (B) during active differentiation, and expression of Mitf (C) and Pax6 (D) was decreased in the mature RPE group but increased in expression of ZO-1. It was.

The present invention relates to a method for inducing differentiation of retinal pigment epithelial cells (RPE) from the cystic structure of the neural precursor sphere obtained by culturing pluripotent stem cell lines.

As used herein, “Retinal Pigment Epithelial Cells” (RPE) means monolayered pigment cells that form part of an external vascular retinal barrier. These cells are characterized by playing an important role in maintaining the function of the retina, absorbing light energy, interacting with photoreceptors to maintain the visual cycle, and maintaining the binding structure between the photoreceptor and the choriocapillary layer. It is a cell that secretes various growth factors, transfers ions, water, and metabolites from the subretinal space to the blood, and performs phagocytosis of the outer segment of the photoreceptor.

The term “pluripotent stem cells” of the present invention includes “embryonic stem cells” and “induced pluripotent stem cells”, and “embryonic stem cells” are obtained from the inner cell mass of the blastocyst stage during the development of fertilized eggs and given a specific differentiation environment. A cell that is capable of continuing to divide into an undifferentiated state and that can differentiate into cells of almost all tissues under conditions, and "induced pluripotent stem cells" refers to artificially injecting pluripotency-related factors into somatic cells. By means of cells that have the same proliferation and differentiation capacity as embryonic stem cells.

In the present invention, a method of inducing differentiation into retinal pigment epithelial cells comprises the steps of (a) culturing pluripotent stem cell lines to form an embryoid body (EBryoid body, EB); (b) propagating the embryoid body; (c) dividing and cleaving the neural rosette and neural tube-like structures that appear during embryoid body culture; (d) selective separation and passage of neural precursor spheres (SNMs) formed from neural tube-like structures; (e) dividing the SNM derived cystic construct; And (f) culturing the divided cystic structure; it may be a method of inducing differentiation into Retinal Pigment Epithelial Cells (RPE). In addition, the pluripotent stem cell line may be preferably characterized in that the embryonic stem cells or induced pluripotent stem cells.

In the present invention, the segmentation method separates only neurospecific constructs from neural precursor spheres, which can be done by various known methods. In a specific implementation of the invention, the preferred method of ablation is a mechanical method, in particular a mechanical separation using a glass Pasteur pipette. The mechanical method using the glass Pasteur pipette is a method of pulling a thin portion of pasteur pipet by applying heat to a thin portion of the pasteur pipet and cutting one end to a thickness of hair to cut a desired portion under a microscope.

In order to achieve the object of the present invention, pluripotent stem cell line may be cultured in an appropriate embryonic stem cell culture medium to induce differentiation into retinal pigment epithelial cells, but preferably mitomycin-C (mitomycin-C) is It is preferable to incubate on treated support cells and incubate using DMEM / F12 medium. The composition of DMEM / F12 medium was 20% knockout serum replacement (SR), 2 mM L-glutamine, 0.4 ng / ml basic fibroblast growth factor (bFGF), 1% non essential amino acids , NEAA), 0.5% penicillin-reptomycin, 0.1 mM beta-mercaptoethanol.

The embryoid body formed by differentiation from embryonic stem cells sufficiently cultured for a certain period of time can be cultured by attaching to a culture plate coated with one of Matrigel, laminin, or L-polyornithine, and by this medium, neural progenitor cells can be selectively survived. can do.

The embryoid body of the present invention can be incubated for 3 to 7 days in NP selective medium, and the desired neuroprecursor sphere can be obtained by proliferating in proliferation medium for 4 to 7 days again.

The goblet-derived neuronal progenitor cells produced in the present invention may form a neurospecific composition, and these structures may exhibit morphological characteristics of neural rosettes or neural tubes. In order to induce preferred differentiation into retinal pigment epithelial cells, the generated neural precursor spheres may be characterized by having an open neural tube structure.

The cystic structure of the present invention is formed from an open neural tube structure, and may preferably be characterized by showing pigments on the outside after separation by mechanical cleavage. More preferably, the pigment may be formed within 5 to 9 days after separation.

The isolated cystic constructs may be attached to a culture dish coated with Matrigel (Matrigel, BD, Franklin lakes, NJ, USA).

According to the present invention, it can be additionally confirmed whether the differentiated cells are properly differentiated into retinal pigment epithelial cells, and for this purpose, the morphological and functional characteristics of the retinal pigment epithelial cells can be confirmed without limitation. As a preferred embodiment, the present invention provides a method for examining the expression of mRNA or protein of a retinal epithelial cell-specific gene, and more specifically, the expression of one or more genes selected from Mitf, Pax6, RPE65, and Bestrophin can be confirmed. . In another preferred embodiment, the present invention provides a method for evaluating phagocytosis of differentiated retinal pigment epithelial cells. The expression or phagocytosis of such genes can be assessed using assay methods for identifying general gene expression, such as PT-PCR, which measures mRNA levels, and confirms general phagocytosis. This can be done through biotechnological methods.

In order to promote the differentiation of retinal pigment epithelial cells of the present invention, differentiation promoters known in the art for promoting the differentiation of cells may be used without limitation, and preferably, differentiation may be performed by treating retinoic acid (RA). Can be promoted, and more preferably retinoic acid can be treated for 1 to 3 weeks.

The present invention can also provide a retinal pigment epithelial cell or progeny thereof differentiated by the above invention and a composition for treating retinal degeneration disease, including the same.

"Retinal degeneration disease" refers collectively to abnormalities caused by structural, biochemical and physiological causes in the retina. In other words, abnormalities in the retina usually include retinal detachment, macular degeneration, monoretinopathy, and central chorioretinopathy. Representative examples include retinal detachment and macular degeneration.

The therapeutic composition of the present invention may be prepared in a suitable formulation including an acceptable carrier depending on the mode of administration. Formulations suitable for the mode of administration are known and may comprise agents that facilitate movement, typically through the membrane.

In addition, the therapeutic composition of the present invention can be used in the form of a general pharmaceutical formulation. Parenteral preparations may be prepared in the form of sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions or lyophilized preparations, oral administration in the form of tablets, troches, capsules, elixirs, suspensions, syrups or wafers. It can be prepared in unit dosage ampoules or in multiple dosage forms. In addition, the therapeutic composition of the present invention may be administered with a pharmaceutically acceptable carrier. For example, in the case of oral administration, a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a coloring agent, or a flavoring agent may be used, and in the case of an injection, a buffer, a preservative, a painless agent, a soluble agent may be used. Topical agents, enteric agents, stabilizers and the like can be mixed and used, and for topical administration, bases, excipients, lubricants, preservatives and the like can be used.

In addition, the pharmaceutical composition may be administered by any device capable of transferring the active substance to the target cell. Preferred modes of administration and preparations are intravenous, subcutaneous, intradermal, intramuscular or injectable. Injections include non-aqueous solvents such as aqueous solvents such as physiological saline solution or ring gel solution, vegetable oils, higher fatty acid esters (e.g., oleic acid, etc.), and alcohols (e.g., ethanol, benzyl alcohol, propylene glycol or glycerin). Stabilizers (e.g., ascorbic acid, sodium bisulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers for pH control, to prevent microbial development Pharmaceutical carriers such as preservatives (eg, mercury nitrate, chimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, and the like). Preferably, the method for treating brain or nervous system diseases using the therapeutic composition of the present invention comprises administering a therapeutically effective amount of the therapeutic composition of the present invention. The pharmaceutically effective amount is well known in the medical field such as the type of disease, the age, weight, health, sex of the patient, sensitivity to the drug of the patient, the route of administration, the method of administration, the frequency of administration, the duration of treatment, the combination or drug used It can be easily determined by those skilled in the art depending on the factors.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

< Example >

1. a person Pluripotent stem cells  origin SNM from RPE Eruption

Mitomycin-C (Mitomycin-C) is used for the passage and maintenance of undifferentiated human embryonic stem cell line (SNUhES3) and pluripotent stem cell line (human iPS cell line set, System Bio). ) Were treated on treated cells (mouse embryonic fibroblast cell line, STO or human foreskin fibroblast, HFF) treated with 20% knockout serum replacement (SR), 2 mM L-glutamin, 0.4 ng. DMEM / F12 medium containing / ml basic fibroblast growth factor (bFGF), 1% non essential amino acids (NEAA), 0.5% penicillinstreptomycin, and 0.1 mM beta-mercaptoethanol was used. After 7 days of incubation, the propagated embryonic stem cells were mechanically divided into agglomerates having 200 to 300 cells, and then cultured again on support cells.

A schematic process of Retinal Pigment Epithelial Cells (RPE) differentiation is shown in FIG. 1.

For embryonic body (EB) formation, undifferentiated pluripotent stem cell colonies were mechanically isolated and incubated for 7 days in bacterial culture dishes. Spherical neural masses (SNMs) were formed from EBs. EB is 5 days in DMEM / F12 medium, which contains NP (neural progenitor) selection medium (0.5% N2 substrate, 2 mM L-glutamin, 1% non essential amino acids (NEAA), 0.5% penicillinstreptomycin, 0.1 mM beta-mercaptoethanol) DMEM / F12 medium ( 0.5% N2 substrate, 2 mM L-glutamin, 20ng / ml basic fibroblast growth factor (bFGF), 1% non essential amino acids (NEAA)) 0.5% penicillinstreptomycin, including 0.1 mM beta-mercaptoethanol).

Neural rosettes and neural tube like structures observed during neuronal growth were mechanically cut using a glass pasteur pipette and then cultured in bacterial culture dishes containing NP growth media. It became. During the initial passage of SNMs (passage 1-4), some SNMs contained open neural tube structures (FIG. 1D) and these SNMs were selected, isolated and passaged. (Figure 1E). The cystic structure appeared from the neural tube structure opened through passage culture, which was mechanically separated and suspended in the growth medium.

These cystic structures are mechanically partitioned and attached to culture dishes coated with Matrigel (BD, Franklin lakes, NJ, USA) and then 1 x 10 ^-6 M of all-trans-retinoic acid for 1 to 4 weeks. (RA, SIGMA, St. Louis, MO, USA) was incubated in conditions with or without. BFGF (Invitrogen, Carlsbad, CA, USA) was replaced with B27 (2%, Invitrogen) derived from NP growth medium for differentiation and proliferation of RPE.

2. RPE  Feature analysis

2.1 Electron Microscopy Analysis

After immobilization of 2.5% glutaraldehyde, a post fixation step of cells was performed in a 0.04 M phosphate buffer with 1% osmium tetroxide. After successively performing dehydrogenation with ethanol and infiltration with epoxy resin, the cells were transferred to the beam capsule for polymerization. Ultra-thin slices were obtained using an ultramicrotome, and the slices were observed at 80 kV using a transmission electron microscope (JEM-1400; JEOL Ltd., Japan).

2.2 immunochemical assay Immunocytochemistry )

Cells were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde. The blocking solution was treated with 3% bovine serum albumin solution in PBS containing 0.1% Triton X100 for one day, after which the samples were incubated with the primary antibody for one day. Primary antibodies used for immunocytochemical analysis are as follows:

mouse- and rabbit anti-human nestin (1: 100, Chemicon, Temecula, CA, USA), mouse anti-musashi (1: 200, Chemicon), mouse anti-paired box gene 6 (Pax6, 1: 200, Chemicon) , rabbit anti-tight junction protein ZO-1 (1: 200, Zymed, South San Francisco CA, USA), mouse anti-microphthalmia-associated transcription factor (Mitf, 1: 200) (Abcam, Cambridge, CB, UK), mouse anti-retinal pigment epithelium-specific 65 kDa protein (RPE65, 1: 200), mouse anti-bestrophin (1: 200) (Novus biological, Littleton, CO, USA), mouse anti-αvβ5 integrin (1: 200, Milipore , Billerica, MA, USA).

The primary antibody for detecting primary antigen is Alexa Fluor? 488 donkey anti-mouse and anti-rabbit IgG, Alexa Fluor? 594 donkey anti-mouse, anti-rabbit IgG (1: 200, Molecular Probes, Eugene, OR, USA) was used and the cell nuclei were counterstained with DAPI.

3. Reverse transcription  Polymerase Amplification Reaction ( RT - PCR )

In order to confirm the differentiation into RPE, RT-PCR analysis of expression at the transcription level of Mitf, Pax6, RPE65, and Bestrophin, which are markers expressed in RPE, was performed as follows.

RNA was obtained from RPE cells, ARPE-19 cells (American Type Culture Collection, Rockville, MD, USA) differentiated from hESCs, EBs, SNMs, cystic SNM using TRIzol Reagent ((Invitrogen, Carlsbad, CA, USA) , CDNA synthesis was performed using oligo-dT as a primer according to the manufacturer's instructions (AccuPower RT PreMix; Bioneer, Taejeon, Korea) using AMV reverse transcriptase (RT).

PCR amplification was performed using Taq Polymerase (HiPi Plus 5x PCR Premix; Elpis biotech, Taejeon, Korea), and the standard method was used. PCR conditions were repeated for 30 minutes at 94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 10 minutes after denaturation at 94 ℃ for 5 minutes. To analyze the relative expression with other mRNAs, the amount of cDNA was normalized based on the signal of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA.

As a result of repeated experiments with various annealing temperatures and cycle times, proportionally amplified sections were determined for each primer pair and PCR conditions were optimized.

The length of the primer sequence and the amplification product is as follows.

Gene name Gene direction Base sequence Size of output GAPDH Forward 5'-ACCACAGTCCATGCCATCAC-3 ' 450 bp
Reverse 5'-TCCACCACCCTGTTGCTGTA-3 ' Mitf Forward 5'-TTCACGAGCGTCCTGTATGCAGAT-3 ' 106bp Reverse 5'-TTGCAAAGCAGGATCCATCAAGCC-3 ' Pax6 Forward 5'-GGCAACCTACGCAAGATGGC-3 ' 459 bp
Reverse 5'-TGAGGGCTGTGTCTGTTCGG-3 ' RPE65 Forward 5'-GCCCTCCTGCACAAGTTTGACTTT-3 ' 259 bp Reverse 5'-TGAGGGCTGTGTCTGTTCGG-3 ' Bestrophin Forward 5'- GCCCTCCTGCACAAGTTTGACTTT-3 ' 359 bp

4. In vitro phagocytosis assays

To investigate the specific / nonspecific phagocytosis of RPE, cells isolated from bovine retinas were incubated with FITC-labeled POS and FITC-labeled latex beads. One of the main functions of RPE is phagocytosis of photoreceptor outer segments (POS). POS is digested by the RPE, reused and returned to the photoreceptor to reconstruct the photosensitive outer segment from the photoreceptor base. To this end, nonspecific phagocytosis by RPE cells derived from cystic SNMs was assessed by latex bead ingestion.

Carboxylate-modified polysterene FITC-labeled latex beads (1 μm diameter, Sigma) were washed several times to remove reagents for stabilization and cells were exposed to 1 × 10 ⁵ to 1 × 10 ⁶ beads at 37 ° C. for one day.

After incubation, the beads bound to the surface were removed by washing with PBS and 0.4% Trypan Blue was added to remove extracellular fluorescence. After immobilization and blocking, cells were stained with anti-ZO-1 antibody (1: 200, Zymed, South San Francisco, CA). Alexa Fluor? 594 donkey anti-rabbit IgG was used to detect primary antibodies and cell nuclei were counterstained with DAPI. Evaluation of intracellular uptake of beads was assessed using confocal microscopy (LSM 510, Carl Zeiss).

Assessment of phagocytosis was performed by bovine photoreceptor outer segments (POS) ((Molday RS, Hicks D, Molday L. Peripherin. A rim-specific membrane protein of rod outer segment discs.Invest Ophthalmol Vis Sci. 1987 Jan; 28 (1): 50-61.), Lin H, Clegg DO.Integrin alphavbeta5 participates in the binding of photoreceptor rod outer segments during phagocytosis by cultured human retinal pigment epithelium.Invest Ophthalmol Vis Sci 1998; 39: 1703-1712.

More specifically, the retina was obtained from the eye of a slaughtered cow and in the red light source, the retina contained 20% (wt / vol) sucrose, 20 mM Tris HCl, pH 7.4, 10 mM glucose, 5 mM taurine, 2 mM MgCl ₂ . Transfer to 10 ml homogeneous solution. The retinal suspension is placed in sucrose consisting of 5 concentrations (27% -60% wt / vol) containing 20 mM Tris HCl, 10 mM glucose and 10 mM taurine, followed by ultracentrifugation at 25,000 rpm at 4 ° C. for 1 hour. Isolated (XL-90, Beckman).

Purified cow POS was labeled according to the manufacturer's protocol using the FluoReporter FITC protein labeling kit (Invitrogen). FITC-labeled POS was washed and mixed back free in cell culture medium. It was then incubated with RPE cells at 5% carbon dioxide, 37 ℃.

After 4 hours of incubation, surface-attached beads were washed by PBS and 0.4% Trypan Blue was added to remove extracellular fluorescence.

After immobilization and blocking, cells were stained with anti-ZO-1 antibody (1: 200, Zymed, South San Francisco, CA). Alexa Fluor? 594 donkey anti-rabbit IgG was used to detect primary antibodies and cell nuclei were counterstained with DAPI. Evaluation of intracellular uptake of beads was assessed using confocal microscopy (LSM 510, Carl Zeiss). For evaluating the internalization of POS, the y-axis projection through the cells was examined.

5. Enzyme Immunoassay PEDF  And VEGF Secretion measurement)

RPE cells and adult RPE cells isolated from cystic structure-derived SNM were incubated for 2 to 4 weeks with 1 x 10 ^-6 M of retinoic acid (RA) and without control, and the culture supernatant was secreted. The secreted pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor (VEGF) were isolated for detection.

ChemiKine ^™ PEDF sandwich ELISA kit (Chemicon) and humanVEGF ELISA kit (Thermo scientific, Rockford, IL, USA) were used to detect PEDF and VEGF, respectively, according to the manufacturer's protocol.

6. Retinal Degeneration in a Mouse Model cystic SNM -Of derived RPEs Subretinal  transplantation

The following examples were carried out in accordance with the regulations of the ARVO Declaration, which regulates the use of animals for Ophthalmic and Vision Research.

Six week-old SD rats were injected once intravenously (70 mg / kg) with sterile 1% NaIO ₃ saline solution. Four days after intravenous injection, animals were anesthetized via intramuscular injection of tiletamine / zolazepam (Zoletil?) And xylazine.

The pupil was expanded with 0.5% tropicamide and 2.5% phenylephrine HCl eye drops (Mydrin-P?) And treated with proparacine HCl 0.5% (Alcaine?) Local anesthetic.

Prior to transplantation, cystic SNM derived RPE was incubated with DAPI (10 μg / ml) for 30 minutes. Several washing procedures were performed to remove DAPI from the medium. Isolation by incubation at 37 ° C., 0.05% trypsin / 0.1% EDTA for 5 minutes.

Visualized through a surgical microscope and isolated RPE cells (10 ⁵ cells / 5μl) were injected into the subretinal space through the sclera using a 33-gauge needle attached to a Hamilton syringe.

Cyclosporine A (210 mg / l, Cipol-N ?, Chong Kun Dang, Seoul, Korea) was administered to drinking water the day before transplantation before denuclearization. One week after transplantation, the eyes were denuclearized and rapidly frozen using an embedding compound (FSC 22 ?, Leica microsystems, IL).

The cooling piece was washed with PBS and incubated in 3% bovine serum albumin solution of PBS solution containing 0.1% Triton X100. After incubation, the cells were incubated overnight with the primary antibody. Anti-ZO-1, anti-RPE65 and anti-Bestrophin antibodies were used for immunohistochemistry. Antibodies for detecting primary antibodies include Alexa Fluor 488 donkey anti-mouse IgG, Alexa Fluor? 594 donkey anti-rabbit IgG was used.

7. Results

7.1 hESC  Derived neuroepithelial cells RPE  Differentiation into cells

To produce functional RPE cells, hESC-derived SNM was used. hESC is known to be able to differentiate into neuroepithelial cells through the differentiation process of SNM is formed in EB through extracellular reaction (A-C of Figure 2a). Initial SNM (passage culture 1-4) can be confirmed that the neuronal rosette (FH of Fig. 2b) and neural tube (Fig. 1C), it was confirmed that various markers for neuroepithelial cells are expressed (FH of Fig. 2b) ).

During neural precursor selection and proliferation, cells expressed Pax6, which is very important for eye development. During mammalian development, the initial neural tube becomes an organ that contains organs that can swell into the retina. In early SNM, several neural tube structures swelled into organs (FIG. 1D) and formed a cystic portion of SNM (FIG. 1E). These cystic parts were easily separated by mechanical separation under a stereomicroscope and formed cystic vesicles (FIG. 1F). Interestingly, the cystic region of SNM was found to express Pax6 even when there was adhesion for neuronal differentiation (FIG. 2B).

Cystic vesicles showed typical RPE morphology, such as polygonal epithelial monolayer. In addition, one week after the separation was confirmed that the pigment begins to form (A, B of Fig. 3a), through which the SNM-derived cystic vesicles cultured and isolated from the embryonic stem cell line was confirmed that the differentiation was induced by RPE.

Previous studies have used media supplements such as Wnt, Nodal antagonists, nicotinamide, activin A, transforming growth factor beta (TGF-β) to enhance or promote RPE formation from embryonic stem cells. However, such differentiation is known that only about 70% of the cell population shows pigmentation after 8 weeks after differentiation. Therefore, in the case of the present invention, the cystic vesicles of the SNM showed pigmentation only one week after separation from the SNM, and thus, it was confirmed that the efficiency of inducing differentiation of RPE was significantly improved compared to the previous studies.

The sectioned cystic vesicles were cultured in a monolayer in a culture dish and the cells showed RPE morphology. On the other hand, the cystic structure of EB showed various forms (E, F of FIG. 3A). This confirms that the morphological characteristics of RPE cells only appear from SNM including cystic vesicles. The attached cystic vesicles were capable of differentiation until fusion and showed pigmentation (C in FIG. 3A). The vesicular layer of cells (FIG. 3D) also reappeared in the fusion environment. Expression of Pax6 was maintained until monolayer culture (J in FIG. 3B). However, it was confirmed that the expression level gradually decreases as maturation occurs.

7.2 Cystic  Parcel origin RPE Characteristic Analysis of

Characterization of monolayer cells cultured from SNM-derived cystic vesicles was analyzed to confirm that the cells were differentiated into RPE.

Expression of specific molecular markers related to cellular function of mature RPE was confirmed by RT-PCR and immunocytochemistry. MRNA expression of Pax6, Mitf, RPE65, bestrophin was confirmed in hESC-derived RPE cells (G in FIG. 3A). Pax6 and Mitf are helix-loop-helix leucine zipper transcription factors involved in melanin formation, and RPE65 is a required factor for retinol conversion during retinoid regeneration for phototransduction and visual regeneration. Bestrophin is also known to act as an ion channel as an RPE-specific protein located on the basolateral plasma membrane.

Expression of early RPC cell markers Pax6 and Mitf, as well as mature RPE cell markers bestrophin (L in FIG. 3B) and RPE65 (M in FIG. 3B), was confirmed by immunostaining (J, K in FIG. 3B). After incubation, most of the cells (90% or more) expressed such RPE markers. In addition, the presence of Ki67-positive cells, which can confirm the presence of rapidly proliferating cells, was also confirmed (I of FIG. 3B).

7.3 Extracellular hESCs  origin RPE  Functional Characterization of Cells

Observation of the cells with an electron microscope confirmed the characteristics of mature RPE. TEM was used to identify pigmented square epithelial cells with features such as tight junctions of microvilli apical, melanin granules and apical cells of the apical part (Fig. 4A, B).

After suppressing the extracellular fluorescent substance with Trypan Blue, the internalization of the latex beads through confocal microscopy confirmed the intrinsic fluorescent substance as shown in Figs. 4C and D, through which the differentiated cells matured RPE. It was confirmed that the phagocytic action is characterized.

To confirm that labeled beads were internalized, y-axis projection was performed. Immunostaining confocal microscopy of cells to identify surface marker ZO-1 of RPE cells confirmed the incorporation of FITC-labeled latex beads in these cells (FIG. 4E). After 4 hours of incubation, confocal microscopy confirmed the internalization of the FITC-labeled POS (Fig. 4F, G). FITC-labeled POS was observed inside the cells near the DAPI-stained nucleus (FIG. 4H). Therefore, it was confirmed that differentiation-induced RPE from cystic structures exhibited the function of mature RPE cells that actively exhibit phagocytosis.

7.4 Enzyme Immunoassay: PEDF  And VEGF Secretion of

RPE is known to secrete various growth factors such as PEDF and VEGF. To determine if differentiated RPE secreted these RPE growth factors, a conditioned medium (CM) was obtained from the culture supernatant of hESC-derived RPE and adult RPE cells.

As a result of relative quantification, it was confirmed that the nutritional factors secreted from hESC-derived RPE are similar to those secreted from adult RPE. The concentration of nutrients secreted was higher as the incubation time continued.

In addition, the secretion of PEDF and VEGF was compared between the group treated with retinoic acid and the group not treated. Retinoic acid is known to affect the visual cycle, which is known to be involved in the maturation and differentiation of various cells. Interestingly, the secretion of PEDF was markedly increased by treatment of retinoic acid (RA) for 2 weeks. On the other hand, it was confirmed that the secretion of VEGF shows no change (Fig. 5A, B).

It was confirmed that the cystic structure-derived RPE can secrete the same growth factor secreted by RPE, and that retinoic acid can promote the differentiation and maturation of RPE.

7.5 Retinal Degeneration Let  In the model cystic SNM -origin RPE Cell Subretinal  Confirmation of transplant results

Hematoxylin and eosin (H & E) staining of retinal sites isolated from the rats injected with sodium-iodate showed loss of RPE layer and collapse of the outer nuclear layer compared to the control group (FIG. 5C, D).

After injection of RPE cells differentiated into the above retinal debris into the subretinal layer, it was confirmed whether the differentiated RPE cells function properly.

One week after transplantation, the survival and positioning of the transplanted cells were confirmed by immunohistochemistry. Confocal microscopy confirmed the integration of DAPI-prestained cells into the subretinal space. The cells formed the expression of mature RPE markers such as bestrophin, ZO-1, RPE65, etc. with the RPE layer (FIG. 5F, G), whereas the control group did not show this change (FIG. 5E).

Cystic vesicles can be expanded by passage culture, and cystic vesicle-derived RPE also has the advantage that it can be effectively expanded for transplantation. In addition, since most cystic vesicle-containing cells (90% or more) are differentiated into RPE, RPE can be efficiently obtained. According to the present invention, the pigment formation period can also be effectively shortened, so that the efficiency for obtaining RPE cells is effectively improved. Could. In addition, as a result of confirming the function through the retinal transplantation, it was confirmed that the RPE is effectively implanted, thereby confirming the possibility that the RPE derived from the cystic structure of the present invention can be used for the treatment of retinal degeneration diseases.

Claims (18)

A method of inducing differentiation from cystic structures obtained by culturing pluripotent stem cell-derived neural precursor spheres into Retinal Pigment Epithelial Cells (RPE).
The method of claim 1, wherein the method of inducing differentiation is
(a) culturing pluripotent stem cells to form an embryoid body (EB);
(b) propagating the embryoid body;
(c) dividing and cleaving the neural rosette and neural tube-like structures that appear during embryoid body culture;
(d) selective separation and passage of neural precursor spheres (SNMs) formed from neural tube-like structures;
(e) dividing the SNM derived cystic construct; And
(F) culturing the divided cystic structure; a method of inducing differentiation into Retinal Pigment Epithelial Cells (RPE).
The method of claim 2, wherein the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells.
The method of claim 2, wherein the pluripotent stem cells are cultured on the support cells treated with mitomycin-C (mitomycin-C) characterized in that the culture using DMEM / F12 medium.
The method of claim 4, wherein the DMEM / F12 medium is 20% knockout serum replacement (SR), 2 mM L-glutamine, 0.4 ng / ml basic fibroblast growth factor (bFGF), 1% amino acid supplement ( non essential amino acids (NEAA), 0.5% penicillin-straptomycin, and 0.1 mM beta-mercaptoethanol.
The method of claim 2, wherein the dividing in the step (c) or (e) is a mechanical dividing method using a glass pasteur pipette.
The method of claim 2, wherein the SNM has an open neural tube structure.
The method of claim 2, wherein the embryoid body is incubated for 3-7 days in a neural progenitor (NP) selection medium, and then propagated in a growth medium for 4-7 days.
The method of claim 2, wherein the cystic structure is characterized in that the pigment is formed within 5 to 9 days after separation.
The method of any one of claims 2 to 9, wherein in step (f), retinoic acid (RA) is treated for 1 to 3 weeks.
The method of claim 2, wherein the culturing of step (f) is characterized in that it is attached to a culture plate coated with Matrigel.
10. The method of any one of claims 2-9, wherein the method further comprises determining whether the differentiated cells have differentiated to the desired cells.
13. The method of claim 12, wherein said determining identifies whether differentiated cells express mRNA or protein of a retinal pigment epithelial cell specific gene.
The method of claim 13, wherein the specific gene is one or more genes selected from Mitf, Pax6, RPE65, and Bestrophin.
The method of claim 12, wherein said determination is made through assessing phagocytosis of differentiated cells.
Retinal pigment epithelial cells or progeny thereof differentiated by the method of any one of claims 2 to 9.
17. A composition for the treatment of retinal degenerative disease, comprising the retinal epithelial cell of claim 16 or a progeny thereof.
18. The method of claim 17, wherein the retinal degeneration disease is retinal dysplasia, retinal degeneration, aged macular degeneration, diabetic retinopathy, retinitis pigmentosa, glaucoma ( glaucoma), optic neuropathy, trauma, retinal detachment, Lever's congenital melanoma and congenital retinal atrophy that is one or more selected from the group consisting of retinal pigment degeneration disease treatment composition.

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