Using retinal pigment epithelial cells to replace the corneal endothelium

Abstract

The present invention discloses the use of retinal pigment epithelial cells to replace corneal endothelial cells and prevent and treat diseases or conditions such as corneal endothelial decompensation. The retinal pigment epithelial cell suspension provided by the present invention can restore corneal transparency, reduce corneal thickness, and reconstruct corneal endothelial barrier function, effectively treat corneal endothelial decompensation, and has extensive application value and positive social effects in treating or restoring visual impairment caused by corneal damage.

Description

[Technical field]

[0001] The present invention is in the field of medicine and relates to the use of retinal pigment epithelial cells to alleviate or treat corneal endothelial decompensation. [Background technology]

[0002] The cornea is a transparent membrane in the anterior wall of the eyeball, and is divided into five layers, which are composed of the epithelial cell layer, Bowman's membrane, stromal layer, Descemet's layer, and endothelial cell layer from the front to the back. High transparency and optical properties of the cornea are one of the prerequisites for normal physiological function, and corneal endothelial cells play an important role in maintaining the normal physiological function of the cornea. Corneal endothelial cells are a single layer of cells in the inner layer of the cornea, which constitute a physical barrier between the Descemet's layer and aqueous humor, and regulate the ion concentration and water content in the cornea through the ion "pump" function, maintaining the semi-dehydrated state of the cornea and ensuring the normal thickness and transparency of the cornea. Corneal endothelial cell dysfunction tends to cause corneal edema, which can lead to partial or complete corneal blindness.

[0003] Normal human corneal endothelial cells have a very limited proliferation capacity in vivo. Damage and loss of endothelial cells due to trauma, inflammation, cataract surgery, etc. can only be compensated for by the expansion and migration of surrounding cells. When the density of human corneal endothelial cells reaches its physiological limit (approximately 400-500 cells / mm), 2), corneal edema occurs, and in severe cases, vision loss occurs. Currently, there are approximately 4 million patients with corneal blindness nationwide, of which nearly 1 million are endothelial blindness patients. Corneal transplantation is the only clinical means of treating corneal endothelial decompensation. Due to the shortage of donor corneas in our country, fewer than 10,000 patients recover their vision through corneal transplantation surgery each year, which is far from meeting clinical needs. To solve the problem of the shortage of donor corneas, the current main research strategies for corneal endothelial replacement seed cells include cultured human corneal endothelial cells, adult stem cells such as skin progenitor cells, and corneal endothelial-like cells derived from human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC).

[0004] Although corneal endothelial function can be improved using primary cultured human corneal endothelial cells, corneal endothelial-like cells derived from adult stem cells or pluripotent stem cells, the effect is limited. Until now, there has been no ideal corneal endothelial replacement seed cells that can maintain corneal transparency for a long period of time and be widely applied clinically. A team led by Japanese scientist Professor Kinoshita conducted clinical treatment for 11 patients with corneal endothelial dystrophy by transplanting cultured human corneal endothelial cells via intracameral injection, restoring corneal transparency, but a 5-year follow-up study showed that pathological "dark spot" structures appeared again in the corneal endothelium of some patients. Cultured human corneal endothelial cells still depend on high-quality donor corneas, and adult corneal endothelial seed cells cannot be expanded in large quantities in vitro, so the source and number of cells are limited. In addition, replacement cells derived from adult stem cells, such as corneal endothelial-like cells derived from skin progenitor cells, have poor purity, are difficult to prepare industrially, and have limited therapeutic effects. hESCs / hiPSCs have the ability to proliferate indefinitely and the potential to differentiate in multiple directions, and it has been reported that hESCs / hiPSCs can be differentiated into neural crest, corneal endothelial progenitor cells, and mature corneal endothelial-like cells, and some experiments have demonstrated that corneal endothelial progenitor cells and mature corneal endothelial-like cells can be applied to animal models to restore corneal transparency. However, currently there is no standardized method for the directed differentiation of hESCs / hiPSCs into corneal endothelial-like cells, and the long-term efficacy and safety of the application of hESCs / hiPSC-derived corneal endothelial-like cells in in vivo therapy have not yet been investigated. Therefore, the search for an ideal seed cell for replacing corneal endothelial cells remains an urgent task in the field of corneal endothelial therapy. Summary of the Invention [Problem to be solved by the invention]

[0005] The present inventors have conducted many studies and found that although retinal pigment epithelial cells are significantly different from corneal endothelial cells in terms of their origin of tissue differentiation, anatomical location, and cell function in somatic tissues, they have a regular hexagonal morphology and express tight junction proteins, suggesting the possibility that retinal pigment epithelial cells can provide a barrier function in place of corneal endothelial cells and treat corneal endothelial decompensation. In order to solve the shortcomings of the prior art, the present invention aims to provide a corneal endothelial substitute cell. Furthermore, the present invention aims to provide a source of seed cells that can be supplied in unlimited quantities and can be safely applied clinically by selecting retinal pigment epithelial cells derived from hESC / hiPSC as seed cells. Furthermore, the present invention aims to enhance the function of transplanted cells, optimize the preparation process of the cell suspension, and ensure the normal function of transplanted cells. [Means for solving the problem]

[0006] In order to achieve the above object, the present invention provides a retinal pigment epithelial cell suspension comprising retinal pigment epithelial cells and a DMEM low glucose medium, the blending ratio of the retinal pigment epithelial cells to the DMEM low glucose medium being 3×10 5 ~1.2×10 6 The retinal pigment epithelial cell suspension is provided at a concentration of 200 to 300 μL. Preferably, the mixing ratio of the retinal pigment epithelial cells to the DMEM low glucose medium is 5×10 5 ~1×10 6 Individual volume: 200-300 μL.

[0007] In one preferred embodiment of the present invention, the retinal pigment epithelial cells are obtained by differentiation from human embryonic stem cells or human induced pluripotent stem cells.

[0008] In one preferred embodiment of the present invention, the human embryonic stem cells or human induced pluripotent stem cells have a knockout of the pigment-producing gene, tyrosinase.

[0009] In one preferred embodiment of the invention, the cell suspension further comprises one or more specific inhibitors, said specific inhibitors comprising Y27632, nicotinamide and / or the TGF-β inhibitor SB431542.

[0010] The present invention relates to Differentiation induction: Step 1 to obtain hESC / hiPSC-derived retinal pigment epithelial cells using differentiation medium; Enzymatic digestion: Step 1 is to treat the retinal pigment epithelial cells derived from hESCs / hiPSCs with cell digestion enzymes, and step 2 is to stop the enzymatic reaction with complete medium; Recovery of single cells: Slowly pipetting the cells in step 2 with a pipette to make single cells, recovering them in a centrifuge tube, discarding the supernatant after centrifugation, and retaining the cell pellet in step 3; Preparation of cell suspension: Resuspend the cells from step 3 in DMEM basal medium to 3 x 10 cells per 200-300 µl of DMEM basal medium. 5 ~1.2×10 6 step 4, lysing the cells to obtain a cell suspension; The present invention further provides a method for preparing a retinal pigment epithelial cell suspension, comprising:

[0011] In one preferred embodiment of the present invention, the differentiation medium in step 1 contains a 1:1 ratio of DMEM / F12 and Neuralbasal medium, 1 to 4 mM glutamine, 0.1 to 1.3 mM non-essential amino acids, 0.1 to 1.3 mM β-mercaptoethanol, and 1% N2 additive.

[0012] In one preferred embodiment of the present invention, the differentiation medium in step 1 contains DMEM / F12 medium, 5% to 15% serum replacement, 1 to 4 mM glutamine, 0.1 to 1.3 mM non-essential amino acids, and 0.1 to 1.3 mM β-mercaptoethanol.

[0013] In a more preferred embodiment of the present invention, the differentiation medium in step 1 comprises differentiation medium 1 and differentiation medium 2, differentiation medium 1 comprises DMEM / F12 and Neuralbasal medium (1:1), 2 mM glutamine, 0.1 mM non-essential amino acids, 0.1 mM β-mercaptoethanol and 1% N2 additive, and differentiation medium 2 comprises DMEM / F12 medium, 10% serum replacement, 2 mM glutamine, 0.1 mM non-essential amino acids and 0.1 mM β-mercaptoethanol. That is, the differentiation induction method involves culturing the cells for 2 days in cell differentiation medium 1 (DMEM / F12 and Neuralbasal medium (1:1), 2 mM glutamine, 0.1 mM non-essential amino acids, 0.1 mM β-mercaptoethanol and 1% N2 additive) mixed with 2% Matrigel, then exchanging the medium for 5 days without Matrigel and culturing, and then exchanging the medium for differentiation medium 2 (DMEM / F12 medium, 10% serum replacement, 2 mM glutamine, 0.1 mM non-essential amino acids, 0.1 mM β-mercaptoethanol) and culturing for 3 weeks, and then mechanically isolating and amplifying the retinal pigment epithelial cells.

[0014] In a further preferred embodiment of the present invention, the cell digestive enzyme in step 2 is actase enzyme, the treatment temperature is 37° C., and in step 4, 5 to 15 μM Y27632 is further added.

[0015] The present invention provides the use of retinal pigment epithelial cells as corneal endothelial replacement cells, ie, to replace damaged, diseased or missing corneal endothelial cells.

[0016] The present invention further provides use of retinal pigment epithelial cells in the preparation of a pharmaceutical composition for alleviating or treating corneal endothelial damage, corneal endothelial lesions, corneal endothelial cell dysfunction, or corneal endothelial decompensation.

[0017] The invention further provides the use of retinal pigment epithelial cells in the preparation of a pharmaceutical composition for reducing or treating associated symptoms such as abnormal corneal thickness, loss of corneal transparency, corneal edema, reduced or lost vision, dry eyes, eye pain, etc. in patients suffering from corneal endothelial decompensation.

[0018] The retinal pigment epithelial cells provided by the present invention can be provided in any convenient dosage form, with preferred dosage forms including injections, cell sheets, or kits. Regardless of the dosage form in which the retinal pigment epithelial cells are provided, they are administered to the anterior chamber of the patient's eye.

[0019] The present invention also relates to any one of the following items 1 to 13. [Item 1] Use of retinal pigment epithelial cells as corneal endothelial replacement cells. [Item 2] Use of retinal pigment epithelial cells in the preparation of a pharmaceutical composition for alleviating or treating corneal endothelial damage, corneal endothelial lesion, corneal endothelial cell dysfunction, or corneal endothelial decompensation. [Item 3] Use of retinal pigment epithelial cells in the preparation of a pharmaceutical composition for reducing or treating corneal thickness abnormalities, loss of corneal transparency, corneal edema, reduced or lost vision, dry eyes, and pain in patients suffering from corneal endothelial decompensation. [Item 4] The retinal pigment epithelial cells are administered to the anterior chamber of the patient's eyeball, and the pharmaceutical composition comprises the retinal pigment epithelial cells and a DMEM low glucose medium, and the mixing ratio of the retinal pigment epithelial cells to the DMEM low glucose medium is (3×10 5 ~1.2×10 6 Item 4. The use according to items 2 to 3, wherein the total amount of the solution is 200 to 300 μL. [Item 5] The mixing ratio of the retinal pigment epithelial cells to the DMEM low glucose medium is (5 × 10 5 ~1×10 6 Item 5. The use according to item 4, characterized in that the amount of the solution is (200 to 300 μL). [Item 6] The use described in Item 4, characterized in that the cell suspension further contains one or more specific inhibitors, and the specific inhibitors include Y27632, nicotinamide and / or the TGF-β inhibitor SB431542. [Item 7] The use according to any one of Items 2 to 3, wherein the retinal pigment epithelial cells are obtained by differentiation from human embryonic stem cells or human induced pluripotent stem cells. [Item 8] The use according to Item 7, wherein the human embryonic stem cells or human induced pluripotent stem cells are those in which tyrosinase, a pigment-producing gene, has been knocked out. [Item 9] The use according to any one of Items 2 to 3, wherein the pharmaceutical composition is in the form of an injection, a cell sheet, or a kit. [Item 10] Differentiation induction: Step 1 of obtaining retinal pigment epithelial cells derived from hESC / hiPSC using a differentiation medium; Enzymatic digestion: Step 1 is to treat the retinal pigment epithelial cells derived from hESCs / hiPSCs with cell digestion enzymes, and step 2 is to stop the enzymatic reaction with complete medium; Recovery of single cells: Slowly pipetting the cells in step 2 with a pipette to make single cells, recovering them in a centrifuge tube, discarding the supernatant after centrifugation, and retaining the cell pellet in step 3; Preparation of cell suspension: Resuspend the cells from step 3 in DMEM basal medium to 3 x 10 cells per 200-300 µl of DMEM basal medium. 5 ~1.2×10 6 step 4, lysing the cells to obtain a cell suspension; A method for preparing a retinal pigment epithelial cell suspension, comprising: [Item 11] The preparation method according to Item 10, characterized in that the differentiation medium in step 1 comprises differentiation medium 1 and differentiation medium 2, differentiation medium 1 comprises DMEM / F12 medium, Neuralbasal medium medium, glutamine, non-essential amino acids, β-mercaptoethanol, and N2 additive, and differentiation medium 2 comprises DMEM / F12 medium, serum replacement, glutamine, non-essential amino acids, and β-mercaptoethanol, and the DMEM / F12 medium and the Neuralbasal medium medium in the differentiation medium 1 are mixed in a 1:1 ratio. [Item 12] The preparation method according to Item 10, characterized in that step 1 comprises culturing in cell differentiation medium 1 mixed with 2% Matrigel for 2 days, then exchanging the medium for 5 days without Matrigel, and exchanging the medium for differentiation medium 2 for 3 weeks to mechanically isolate and amplify retinal pigment epithelial cells, wherein differentiation medium 1 comprises a 1:1 mixture of DMEM / F12 and Neuralbasal medium, 2 mM glutamine, 0.1 mM non-essential amino acids, 0.1 mM β-mercaptoethanol, and 1% N2 additive, and differentiation medium 2 comprises DMEM / F12 medium, 10% serum replacement, 2 mM glutamine, 0.1 mM non-essential amino acids, and 0.1 mM β-mercaptoethanol. [Item 13] The preparation method according to any one of Items 10 to 12, characterized in that the cell digestion enzyme in step 2 is a lactase enzyme, the treatment temperature is 37°C, and the cell suspension in step 4 further contains 5 to 15 μM Y27632. Effect of the Invention

[0020] The beneficial technical effects of the present invention are as follows: The present invention is the first to use retinal pigment epithelial cells as seed cells to replace corneal endothelium. The cell suspension and preparation method thereof provided by the present invention can ensure cell viability and effectively replace corneal endothelial function to restore corneal transparency and thickness. In addition, the seed cells to replace corneal endothelium provided by the present invention can be obtained by differentiation induction from hES / hiPS cells and are in unlimited supply, and the safety of their application has been reported in clinical trials. The preparation method and transplantation method provided by the present invention do not require highly specialized equipment, reagents, or techniques and can be easily operated by researchers and medical professionals, so they have broad application value and positive social benefits. [Brief description of the drawings]

[0021] [Figure 1] FIG. 1 shows cell morphology and marker gene staining of hESC-derived retinal pigment epithelial cells in Example 1. [Diagram 2] 1 shows macroscopic images and OCT photographs of the cornea 1, 3, 7, and 14 days after transplantation of retinal pigment epithelial cells in Example 1, showing the transparency and thickness of the cornea. [Diagram 3] 1 shows macroscopic images and OCT photographs of the cornea 1 day, 7 days, and 14 days after transplantation of retinal pigment epithelial cells in Example 2, showing the transparency and thickness of the cornea. [Figure 4] In Example 3, knockout of pigment-producing genes in retinal pigment epithelial cells and identification of corneal endothelial repair function in vivo are shown. (A) Tyrosinase gene expression in retinal pigment epithelial cells before and after knockout is shown. iRPE: normal induced retinal pigment epithelial cells; shtyro-iRPE: induced retinal pigment epithelial cells with tyrosinase gene knockout. (B) Macroscopic images and OCT photographs of the cornea on days 1, 3, and 7 after shtyro-iRPE transplantation are shown. (C) Macroscopic images of the cornea one month after transplantation of normal cells and knockout cells are shown. [Diagram 5] 1 shows the repair effect of rabbit primary retinal pigment epithelial cells on corneal endothelial decompensation in Example 4. (A) Photographs of isolated and cultured retinal pigment epithelial cells derived from New Zealand white rabbits and gray rabbits; (B) Macroscopic images of rabbit corneas 7 days after transplantation of retinal pigment epithelial cells derived from New Zealand white rabbits and gray rabbits. [Figure 6] 1 shows a macroscopic image of the cornea 7 days after transplantation in Comparative Example 2. [Figure 7] 1 shows a macroscopic image of the cornea 7 days after transplantation in Comparative Example 3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] The present invention is further illustrated by the following examples which illustrate the present invention, but the following examples are only used to explain the present invention and should not be considered as limiting the scope of the present invention. Unless otherwise specified, technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. If no specific conditions are described in the examples, conventional conditions or conditions proposed by the manufacturer shall be followed. Any reagents or equipment used without a manufacturer's name are commercially available conventional products.

[0023] In the present embodiment, hESC cell line H1 was gifted from Professor Yin Zhengqin's laboratory. hiPSC cell line DY0100 was purchased from the Cell Bank / Stem Cell Bank of the Chinese Academy of Sciences. For the tyrosinase-specific knockout hESC H1 cell line, tyrosinase-specific knockout virus was purchased from Shanghai Jikai Genetic Medical Technology Co., Ltd., and the tyrosinase-specific knockout hESC H1 cell line was prepared according to the specifications. New Zealand white rabbits and gray rabbits were purchased from Jinan Xilingjiao Aquaculture and Breeding Center. EXAMPLES

[0024] Example 1 (hESC cell line H1) (1) Differentiation induction Referring to a previously published differentiation method (Rapid Differentiation of Multi-Zone Ocular Cells from Human Induced Pluripotent Stem Cells and Generation of Corneal Epithelial and Endothelial Cells, Stem Cells Dev. 2019 Apr 1;28(7):454-463), hESC cell line H1 cells were cultured in mTeSR1 medium and grown to about 80% confluence, digested with 5mg / ml type IV collagenase for 15 minutes, inoculated into a culture dish coated with 1% Matrigel, and cultured for 2 days in cell differentiation medium 1 (DMEM / F12 and Neurolbasal medium (1:1), 2mM glutamine, 0.1mM non-essential amino acids, 0.1mM β-mercaptoethanol and 1% N2 additive) mixed with 2% Matrigel, and then replaced with a medium without Matrigel and cultured for 5 days. The medium was then replaced with differentiation medium 2 (DMEM / F12 medium, 10% serum replacement, 2 mM glutamine, 0.1 mM non-essential amino acids, 0.1 mM β-mercaptoethanol) and cultured for 3 weeks. Pigmented retinal pigment epithelial cells were mechanically isolated and expanded.

[0025] (2) Enzymatic digestion of cells hESC-derived retinal pigment epithelial cells were treated with Accutase enzyme at 37°C for 10-20 minutes, and the enzyme reaction was stopped with complete medium. The cells were then slowly pipetted to separate them into single cells, which were then collected in a 15 ml centrifuge tube and centrifuged at 1000 rpm for 3 minutes. The supernatant was discarded and the pellet was retained.

[0026] (3) Preparation of cell suspension The cells were resuspended in DMEM low glucose basal medium, counted with a cell counter, and aliquoted at 5 × 10 cells per 200–300 μL of DMEM basal medium. 5 ~1×10 6 The cells were lysed and supplemented with 10 μM Y27632 in preparation for transplantation.

[0027] (4) Injection of cell suspension into the anterior chamber Ten New Zealand white rabbits were anesthetized by intramuscular injection of ketamine hydrochloride (40 mg / kg) and chlorpromazine hydrochloride (20 mg / kg). After opening the right eyelid with an eyelid retractor, the eye was washed and a side incision was made about 2 mm from the limbus at 10 o'clock. Carbachol injection solution was injected into the anterior chamber to constrict the pupil. Sodium hyaluronate was injected from the side incision to stabilize the anterior chamber, and autologous corneal endothelial cells within a diameter range of 7 to 9 mm from the center of the eyeball were scraped with a 20-gauge silicone needle. The scraped cell debris and residual sodium hyaluronate in the anterior chamber were washed out with saline, and 1:10 sodium heparin injection solution was injected to prevent leakage of aqueous humor. The side incision at the limbus was intermittently sutured with 10-0 nylon thread.

[0028] A 1 ml syringe was used to puncture the anterior chamber from the limbus, and the cell suspension was injected into the anterior chamber of the right eye, and the eye was wrapped with tobramycin-dexamethasone ophthalmic ointment. To promote rapid attachment of the transplanted cells, the rabbit was kept in a lateral position with the right eye facing down under anesthesia for 3 hours. After surgery, 10 mM Y-27632 was instilled four times a day, and one week later, 1 mM Y-27632 was instilled four times a day. At the same time, after surgery, tobramycin-dexamethasone ophthalmic solution was instilled four times a day and cyclosporine ophthalmic solution was instilled twice a day.

[0029] (5) Functional evaluation After surgery, the recovery of corneal transparency was observed using a slit lamp microscope, the morphology and density of the transplanted corneal endothelial cells were evaluated using a confocal corneal microscope, and the change in corneal thickness was measured using an ultrasonic pachymetry device.

[0030] Results and Analysis: Based on the embodiment of this example, it was possible to induce differentiation of the hESC cell line H1 into retinal pigment epithelial cells (Figure 1). After transplantation of the hESC-derived retinal pigment epithelial cells, the transparency and thickness of the cornea were restored within 7 days, and the cornea remained transparent even on day 14 (Figure 2). This result suggested that the hESC-derived retinal pigment epithelial cells could replace the function of the corneal endothelial cells and rapidly restore the transparency of the cornea.

[0031] Example 2 (hiPSC cell line DY0100) In this example, retinal pigment epithelial cells were induced using the hiPSC cell line DY0100. (1) Differentiation induction The differentiation induction method was the same as in Example 1. The hiPSC cell line DY0100 was cultured in mTeSR1 medium and grown to about 80% confluence, digested with 5 mg / ml type IV collagenase for 15 minutes, inoculated into a culture dish coated with 1% Matrigel, cultured for 2 days in cell differentiation medium 1 mixed with 2% Matrigel, and cultured for 5 days in a medium without Matrigel. In addition, the medium was changed to differentiation medium 2 and cultured for 3 weeks. Pigmented retinal pigment epithelial cells were mechanically isolated and amplified.

[0032] (3) Preparation of cell suspension The cells were resuspended in DMEM low glucose basal medium, counted with a cell counter, and aliquoted. The cells were counted at 8 × 10 per 200–300 μL of DMEM basal medium. 5 ~1×10 6 The cells were lysed and supplemented with 10 μM Y27632 and 5 mM nicotinamide in preparation for transplantation.

[0033] The procedures for (2) enzymatic digestion of the cells, (4) injection of the cell suspension into the anterior chamber, and (5) functional evaluation were the same as in Example 1.

[0034] Results and Analysis: The technical aspects of this example enabled the induction of retinal pigment epithelial cells derived from the hiPSC cell line DY0100, and similarly restored corneal transparency and corneal thickness 7 and 14 days after transplantation of a hiPSC-derived retinal pigment epithelial cell suspension (Figure 3).

[0035] Example 3 (Knockout of the pigment-producing gene tyrosinase) To reduce pigment production, in this example, the pigment-producing gene tyrosinase was knocked out to prepare non-pigmented hESC / hiPSC-RPE cells, which also maintained corneal transparency after transplantation. In this example, the Tyrosinase gene was specifically knocked out using CRISPR-Cas9 technology to prepare non-pigmented retinal pigment epithelial cells.

[0036] In some examples, the hES cell line H1 was used. In other examples, the hiPS cell line DY0100 was used.

[0037] (1) Construction of tyrosinase knockout cells: hES cell line H1 or hiPS cell line DY0100, grown to about 80% confluence, were digested and inoculated at a ratio of 1:20 to 1:30. The next day, a transfection reagent mixed with siRNA was added, and the cells were cultured for 16 to 24 hours until the confluence reached about 50 to 60%, and transfection was started. The amount of virus added was = (MOI x number of cells) / virus titer. After transfection for 12 to 20 hours, the medium was replaced with mTeSR1 complete medium and cultured for 72 to 96 hours, after which the transfection effect was evaluated based on the fluorescence intensity. The group with the strongest fluorescent signal was selected and subjected to fluorescence-activated cell sorting, culture, and amplification to establish a Tyrosinase knockout cell line.

[0038] The procedures for (2) differentiation induction, (3) enzymatic digestion of the cells, (4) preparation of the cell suspension, (5) injection of the cell suspension into the anterior chamber, and (6) functional evaluation were the same as those in Example 1.

[0039] Results and Analysis: The method described in this example achieved knockout of tyrosinase, a pigment production-related gene, in hESC / hiPSC-derived retinal pigment epithelial cells (Figure 4A). After transplantation, the genetically modified retinal pigment epithelial cells were able to rapidly restore corneal transparency and reduce corneal thickness (Figure 4B). The genetically modified cells maintained corneal transparency and corneal thickness even one month after transplantation and did not show pigment production (Figure 4C).

[0040] Example 4 (Isolated and cultured primary retinal pigment epithelial cells from New Zealand white and grey rabbits) In some examples, primary non-pigmented retinal pigment epithelial cells from New Zealand White rabbits cultured in vitro were used, while in other examples, pigmented retinal pigment epithelial cells from grey rabbits cultured in vitro were used.

[0041] (1) Isolation culture New Zealand white and gray rabbits aged 2 to 4 weeks were prepared and sacrificed by air embolism. The eyeballs were removed under sterile conditions and immersed in 1000u of saline containing gentamicin at 4°C for 30 minutes, and then left in saline for 3 hours. The anterior eye and neural retinal epithelium were cut out under a dissecting microscope, the posterior eye cup was placed in a 12-well plate culture dish, 0.25% trypsin was added to about 3 / 4 of the eye cup, and the eye cup was placed in a 37°C incubator for 30 minutes to digest, after which a stop solution was added to stop the digestion, and the RPE cells were peeled off by slow pipetting, collected, centrifuged, and inoculated. The RPE cells were cultured in DMEM / F12 medium containing 10% fetal bovine serum, the medium was changed every 2 days, and the culture was continued for about 2 weeks, and identified by morphology, PCR, and staining.

[0042] (3) Preparation of cell suspension The cells were resuspended in DMEM low glucose basal medium, counted with a cell counter, and aliquoted at 5 × 10 cells per 200–300 μL of DMEM basal medium. 5 ~8×10 5The cells were lysed and supplemented with 10 μM Y27632 in preparation for transplantation.

[0043] The procedures for (2) enzymatic digestion of the cells, (4) injection of the cell suspension into the anterior chamber, and (5) functional evaluation were the same as in Example 1.

[0044] Results and Analysis: By the method described in this example, we were able to prepare primary retinal pigment epithelial cells from New Zealand white and grey rabbits, which showed regular cell morphology (Figure 5A). The transparency of the cornea could be rapidly restored 7 days after cell transplantation (Figure 5B).

[0045] Comparative Example 1 (Trypsin / Collagenase) (1) Differentiation induction: The procedure was the same as in Example 1.

[0046] (2) Enzymatic digestion of cells hESC-derived retinal pigment epithelial cells were treated with 0.25% trampsin for 3-10 minutes at 37°C, or with 5mg / ml type IV collagenase for 5-15 minutes at 37°C, and the enzyme reaction was stopped with complete medium. The cells were then slowly pipetted to separate them into single cells, which were then collected in a 15ml centrifuge tube and centrifuged at 1000rpm for 3 minutes, the supernatant was discarded, and the precipitate was retained.

[0047] (3) Detection of cell viability and size Trypan blue staining was performed and cell viability, size, etc. were statistically analyzed using cytometer techniques.

[0048] Results and Analysis: The cells obtained by digestion with trypsin have a high mortality rate, and when collagenase is used, it is difficult to obtain single cells by dissociation and the digestion time is too long. Therefore, although trypsin and collagenase can be used for enzymatic decomposition of cells, it is preferable to use collagenase.

[0049] (Comparative Example 2) (DMEM high glucose medium) In this example, a cell suspension was prepared using DMEM high glucose medium (containing 4.5 g / ml glucose).

[0050] The procedures for (1) differentiation induction, (2) enzymatic digestion of the cells, (4) injection of the cell suspension into the anterior chamber, and (5) functional evaluation were the same as in Example 1.

[0051] (3) Preparation of cell suspension The cells were resuspended in DMEM high glucose basal medium (containing 4.5 g / ml glucose), counted with a cell counter, and aliquoted at 5 × 10 cells per 200–300 μl of DMEM high glucose basal medium. 5 ~1×10 6 The cells were lysed in preparation for transplantation.

[0052] Results and Analysis: After transplantation, the cell suspension resuspended in DMEM high glucose medium as a solvent caused severe leakage of aqueous humor, persistent corneal edema, and was unable to restore corneal transparency (Figure 6).

[0053] Comparative Example 3 In this example, cell suspensions were prepared with different cell amounts. The procedures for (1) differentiation induction, (2) enzymatic digestion of the cells, (4) injection of the cell suspension into the anterior chamber, and (5) functional evaluation were the same as in Example 1.

[0054] (3) Preparation of cell suspension The cells were resuspended in DMEM low glucose basal medium, counted with a cell counter, and dispensed. The cells were then diluted to 1.5 × 10 per 200–300 μL of DMEM low glucose basal medium. 6 ~1×10 6 , 1×10 6 ~5×10 5 , 5×10 5 ~1×10 5 The cells were lysed in preparation for transplantation.

[0055] Results and Analysis: Postoperative evaluation revealed a 1.2 × 10 6 With more than 3 × 10 cells, aqueous humor leakage became severe and corneal edema persisted after transplantation. 5 It was found that with fewer than 10 cells, corneal edema persisted after transplantation and transparency could not be restored (Figure 7).

[0056] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that based on all the teachings disclosed, various changes and substitutions can be made to these details and amounts of use, and all of these changes are within the scope of protection of the present invention. The overall scope of the present invention is based on the appended claims and their equivalents.

Claims

1. Use of retinal pigment epithelial cells in the preparation of a pharmaceutical composition for alleviating or treating corneal endothelial damage, corneal endothelial lesions, corneal endothelial cell dysfunction, or corneal endothelial decompensation.

2. Use of retinal pigment epithelial cells in the preparation of a pharmaceutical composition for reducing or treating corneal thickness abnormalities, loss of corneal transparency, corneal edema, reduced or lost vision, dry eyes, and pain in patients suffering from corneal endothelial decompensation.

3. The retinal pigment epithelial cells are administered to the anterior chamber of the patient's eyeball, and the pharmaceutical composition comprises the retinal pigment epithelial cells and a DMEM low glucose medium, and the mixing ratio of the retinal pigment epithelial cells to the DMEM low glucose medium is (3×10 ５ ~1.2 x 10 ６ The use according to claim 1 or 2, characterized in that the total amount of the solution is 200 to 300 μL.

4. The mixing ratio of the retinal pigment epithelial cells to the DMEM low glucose medium was (5×10 ５ ~1×10 ６ The use according to claim 3, characterized in that the total amount of the solution is 200 to 300 μL.

5. 4. The use according to claim 3, characterized in that the pharmaceutical composition further comprises one or more specific inhibitors, said specific inhibitors comprising Y27632, nicotinamide and / or the TGF-β inhibitor SB431542.

6. The use according to claim 1 or 2, characterized in that the retinal pigment epithelial cells are obtained by differentiation from human embryonic stem cells or human induced pluripotent stem cells.

7. The use according to claim 6, characterized in that the human embryonic stem cells or human induced pluripotent stem cells are knocked out for the pigment-producing gene tyrosinase.

8. The use according to claim 1 or 2, wherein the pharmaceutical composition is in the form of an injection, a cell sheet or a kit.

Images