Application of Retinal Pigment Epithelial Cells as Corneal Endothelial Substitute

Abstract

The present invention discloses the application of retinal pigment epithelial cells for replacing corneal endothelial cells, preventing and treating diseases or symptoms such as corneal endothelial functional decompensation. The retinal pigment epithelial cell suspension provided by the present invention can restore corneal transparency, reduce corneal thickness, reconstruct corneal endothelial barrier function, effectively treat corneal endothelial functional decompensation, and has a wide range of application values and positive social benefits for the treatment or recovery of people with visual impairment due to corneal injury.

Claims

1. A pharmaceutical composition comprising retinal pigment epithelial cells.

2. The pharmaceutical composition according to claim 1, further comprising a DMEM low-sugar culture medium, wherein the ratio of the retinal pigment epithelial cells to the DMEM low-sugar culture medium is 310.sup.5-1.210.sup.6: 200-300 microliters.

3. The pharmaceutical composition according to claim 2, wherein the ratio of the retinal pigment epithelial cells to the DMEM low-sugar culture medium is 510.sup.5-110.sup.6: 200-300 microliters.

4. The pharmaceutical composition according to claim 2, further comprising one or more specific inhibitors selected from Y27632, nicotinamide, and TGF- inhibitor SB431542.

5. The pharmaceutical composition according to claim 1, wherein the retinal pigment epithelial cells were obtained by differentiating stem cells selected from human embryonic stem cells or human-induced pluripotent stem cells.

6. The pharmaceutical composition according to claim 5, wherein the pigment-producing gene Tyrosinase in the stem cells are knocked out.

7. The pharmaceutical composition of claim 1, wherein the retinal pigment epithelial cells are provided in the form of a cell suspension, a sheet of cells, or a kit.

8. A method for relieving or treating a corneal thickness abnormality, corneal transparency decline, a corneal edema, a corneal endothelial injury, a corneal endothelial lesion, corneal endothelial cell dysfunction, corneal endothelial functional decompensation, vision decline, vision loss, eye dryness, or eye pain in a subject, which comprises administering to the subject retinal pigment epithelial cells.

9. The method according to claim 8, wherein the retinal pigment epithelial cells are administered in the form of a composition comprising a DMEM low-sugar culture medium, wherein the ratio of the retinal pigment epithelial cells to the DMEM low-sugar culture medium is 310.sup.5-1.210.sup.6: 200-300 microliters.

10. The method according to claim 9, wherein the ratio of the retinal pigment epithelial cells to the DMEM low-sugar culture medium is 510.sup.5-110.sup.6: 200-300 microliters.

11. The method according to claim 8, which further comprises administering to the subject one or more specific inhibitors selected from Y27632, nicotinamide, and TGF- inhibitor SB431542.

12. The method according to claim 8, wherein the subject suffers from corneal endothelial function decompensation.

13. The method according to claim 8, wherein the retinal pigment epithelial cells are administered to the subject's eye.

14. The method according to claim 8, wherein the retinal pigment epithelial cells are injected into the anterior chamber of the subject's eye.

15. The method according to claim 8, wherein the retinal pigment epithelial cells were obtained by differentiating stem cells selected from human embryonic stem cells or human-induced pluripotent stem cells.

16. The method according to claim 15, wherein the pigment-producing gene Tyrosinase in the stem cells are knocked out.

17. The method according to claim 8, wherein the retinal pigment epithelial cells were obtained by differentiating stem cells selected from human embryonic stem cells or human-induced pluripotent stem cells and the retinal pigment epithelial cells are injected into the anterior chamber of the subject's eye.

18. A method for relieving or treating a corneal edema, a corneal thickness abnormality, or corneal transparency decline in a subject, which comprises administering to the subject a pharmaceutical composition comprising retinal pigment epithelial cells and a DMEM low-sugar culture medium, wherein the ratio of the retinal pigment epithelial cells to the DMEM low-sugar culture medium is 310.sup.5-1.210.sup.6: 200-300 microliters.

19. The method according to claim 18, wherein the ratio of the retinal pigment epithelial cells to the DMEM low-sugar culture medium is 510.sup.5-110.sup.6: 200-300 microliters.

20. The method according to claim 18, which further comprises administering to the subject one or more specific inhibitors selected from Y27632, nicotinamide, and TGF- inhibitor SB431542.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0044] FIG. 1 shows the cell morphology diagram of the hESC-derived retinal pigment epithelial cells and the staining diagram of symbolic gene in Example 1.

[0045] FIG. 2 shows the general image and OCT photos of the cornea at 1st day, 3rd day, 7th day and 14th day after retinal pigment epithelial cell transplantation in Example 1, showing corneal transparency and thickness.

[0046] FIG. 3 shows the general image and OCT photos of the cornea at 1st day, 3rd day, 7th day and 14th day after retinal pigment epithelial cell transplantation in Example 2, showing corneal transparency and thickness.

[0047] FIGS. 4A, 4B, and 4C show the identification of the knockout of the pigment-producing gene Tyrosinase in retinal pigment epithelial cells and corneal endothelial repair function in vivo in Example 3. FIG. 4A: Gene expression of retinal pigment epithelial cell before and after Tyrosinase knockout; iRPE: normal induced retinal pigment epithelial cells, shtyro-iRPE: induced retinal pigment epithelial cells with Tyrosinase knockout. FIG. 4B: General image and OCT photographs of the cornea at 1st day, 3rd day, and 7th day after shtyro-iRPE transplantation. FIG. 4C: General image of the corneal 1 month after transplantation of normal cells and knockout cells.

[0048] FIGS. 5A and 5B show the repair effect of rabbit primary retinal pigment epithelial cells on corneal endothelial functional decompensation in Example 4. FIG. 5A: Photos of isolated and cultured retinal pigment epithelial cells from New Zealand white rabbits and grey rabbits. FIG. 5B: General image of the cornea from New Zealand white rabbits and grey rabbits at 7th day after retinal pigment epithelial cell transplantation.

[0049] FIG. 6 general image of the cornea at 7th day after transplantation in Comparative Example 2.

[0050] FIG. 7 general image of the cornea at 7th day after transplantation in Comparative Example 3.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0051] The present invention is further illustrated by the following embodiments explaining the present invention, the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. Unless otherwise indicated, the technical and scientific terms used herein are generally understood by those of ordinary skill in the art to which the invention belongs. If the specific conditions are not indicated in the embodiment, the conditions recommended by the manufacturer shall be carried out in accordance with the general conditions or the conditions recommended by the manufacturer. The reagents or instruments used are conventional products that are commercially available if the manufacturer is not identified.

[0052] In the embodiments of the present invention, the hESC cell line H1 was donated by Professor Yin Zhengqin's laboratory; The hiPSC cell line DY0100 was purchased from the Chinese Academy of Sciences Cell Bank/Stem Cell Bank; Tryosinase-specific knockout hESC H1 cell line: Tryosinase-specific knockout virus were purchased from Shanghai GK Gene Medical Technology Co., Ltd. and the Tryosinase-specific knockout hESC H1 cell line was prepared according to its instruction; New Zealand white rabbits and grey rabbits were purchased from Xilingjiao Breeding Center in Jinan.

Example 1 (hESC Cell Line H1)

[0053] (1) Directed Differentiation

[0054] Based on the differentiation method priorly disclosed (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 till fusion rate reached approximately 80% using mTeSR1 medium, then digested with 5 mg/ml type IV collagenase for 15 mins and seeded into 1% Matrigel-coated dish; cell differentiation medium 1 (DMEM/F12 and Neuralbasal medium (1:1), 2 mM glutamine, 0.1 mM non-essential amino acids, 0.1 mM -mercaptoethanol and 1% N2 supplement) mixed with 2% Matrigel was utilized to culture for 2 days, then changed to Matrigel-free medium for 5 days; using differentiation medium 2 (DMEM/F12 medium, 10% serum substitute, 2 mM glutamine, 0.1 mM non-essential amino acid, 0.1 mM -mercaptoethanol) to culture for 3 weeks; mechanical separation of retinal pigment epithelial cells and cell expansion.

[0055] (2) Enzymatic Digestion of Cells

[0056] hESC-derived retinal pigment epithelial cells were treated at 37 C. by Accutase enzyme for about 10-20 mins, and the enzyme reaction was terminated by complete medium; cells were gently pipetted into single cells and collected into 15 ml centrifuge tubes, then centrifuged at 1000 rpm for 3 mins; the supernatant was discarded and the precipitate was retained.

[0057] (3) Preparation of Cell Suspension

[0058] Cells were resuspended using DMEM low-glucose basal medium, the number of cells were counted by cell counter for distribution. About 510.sup.5-110.sup.6 cells were dissolved in about 200-300 l DMEM basal medium in addition of 10 M Y27632 for transplantation.

[0059] (4) Injection of Cell Suspension into the Anterior Chamber

[0060] Ketamine hydrochloride (40 mg/kg) and chlorpromazine hydrochloride (20 mg/kg) was intramuscularly administrated to anesthetize 10 New Zealand white rabbits. The right eye was washed after the eyelids were opened by eye speculum. A lateral incision of about 2 mm was made at 10 o'clock spot at the corneal limbus, and carbacholine was injected into the anterior chamber to shrink the pupil. Sodium hyaluronate was injected from the lateral incision to stabilize the anterior chamber. Autologous corneal endothelial cells within a diameter of about 7-9 mm in the center of the eye were scraped with a 20-gauge silicone needle. The scraped cell fragments and the residual sodium hyaluronate in the anterior chamber were washed with normal saline. 1:10 heparin sodium injection was injected to prevent anterior chamber exudation, and 10-0 nylon thread was intermittently sutured to the limbal lateral incision.

[0061] The cell suspension was injected into the anterior chamber of the right eye from the limbus into the anterior chamber using a 1 ml syringe and tobramycin dexamethasone eye ointment was applied to cover the eye. The rabbit was held in the side-lying position under anesthesia for 3 hours to keep the right eye downward in order to facilitate rapid attachment of transplanted cells. 10 mM Y-27632 was given four times daily postoperatively. After one week change to 1 mM Y-27632 four times daily, while tobramycin dexamethasone eye drops four times daily and cyclosporine eye drops twice daily were given.

[0062] (5) Functional Evaluation

[0063] After surgery, the recovery of corneal transparency was observed by slit lamp microscopy, the morphology and density of transplanted corneal endothelial cells were evaluated by living confocal corneal microscopy, and the change of corneal thickness was measured by ultrasound corneal thickness gauge.

[0064] Results & Analysis:

[0065] Based on the present example, the differentiation from the hESC cell line H1 into retinal pigment epithelial cells can be induced (FIG. 1); Transplantation of hESC-derived retinal pigment epithelial cells restored corneal clarity and corneal thickness within 7 days and remained corneal transparency till 14 days (FIG. 2). The results suggest that hESC-derived retinal pigment epithelial cells can replace the function of corneal endothelial cells and quickly restore corneal transparency.

Example 2 (hiPSC Cell Line DY0100)

[0066] This example used the hiPSC cell line DY0100 to induce retinal pigment epithelial cells.

[0067] (1) Directed Differentiation

[0068] The induction differentiation method was the same as Example 1, using mTeSR1 medium to culture hiPSC cell line DY0100 till cell fusion reach approximately 80%, then using 5 mg/ml type IV collagenase to digest for 15 minutes, inoculating into 1% Matrigel-coated dishes, and using cell differentiation medium 1 mixed with 2% Matrigel to culture for 2 days, changing to Matrigel-free medium for 5 days; using differentiation medium 2 to culture for 3 weeks; mechanical separation of retinal pigment epithelial cells and cell expansion.

[0069] (3) Preparation of Cell Suspension

[0070] Cells were resuspended using DMEM low-glucose basal medium, the number of cells were counted by cell counter for distribution. About 810.sup.5-110.sup.6 cells were dissolved in about 200-300 l DMEM basal medium with addition of 10 M Y27632 and 5 mM nicotinamide for transplantation.

[0071] (2) Enzymatic digestion of cells, (4) Injection of cell suspension into the anterior chamber and (5) Functional evaluation were the same as Example 1.

[0072] Results & Analysis:

[0073] Based on the present example, the differentiation of the hiPSC cell line DY0100 into retinal pigment epithelial cells can be induced; Transplantation of hiPSC-derived retinal pigment epithelial cells restored corneal clarity and corneal thickness within 7 days and remained corneal transparent till 14 days (FIG. 3).

Example 3 (Knockout of the Chromogenic Gene Tyrosinase)

[0074] In order to reduce pigmentation, the present example knocked out the chromogenic gene Tyrosinase, and prepared unpigmented hESC/hiPSC-RPE cells, which can also maintain corneal transparency after transplantation. The present embodiment used CRISPR-Cas9 technology to specifically knock out the Tryosinase gene to prepare pigment-free retinal pigment epithelial cells.

[0075] In some embodiments, the hES cell line H1 was used; In some other embodiments, the hiPS cell line DY0100 was used.

[0076] (1) Construction of Tryosinase Knockout Cells:

[0077] After digestion of the hES cell line H1 or hiPS cell line DY0100 which reached about 80% cell fusion, the cells were inoculated according to the ratio of about 1:20-30, and transfection reagent mixed with siRNA was added in the next day, transfection was performed when the confluence reached about 50-60% after 16-24 hours culture, and the amount of the added virus=(MOInumber of cells)/virus titers. After 12-20 hours of transfection, change to mTeSR1 complete medium for 72-96 hours culture, and the transfection performance was evaluated according to fluorescence intensity. The sets with most fluorescent signals were selected for flow cytometry sorting, culture, and expansion to establish Tryosinase knockout cell lines.

[0078] (2) Induction of differentiation, (3) Enzymatic digestion of cells, (4) Preparation of cell suspension, (5) Injection of cell suspension into the anterior chamber and (6) Functional evaluation were the same as Example 1.

[0079] Results & Analysis:

[0080] Knockout of the chromogenesis-related gene Tyrosinase in hESC/hiPSC-derived retinal pigment epithelial cells was achieved by the method described in the present example (FIG. 4A); After transplantation, genetically modified retinal pigment epithelial cells can quickly restore corneal transparency and reduce corneal thickness (FIG. 4B); Corneal transparency and corneal thickness were maintained one month after transplantation with no pigmentation (FIG. 4C).

Example 4 (Isolation and Culture of Primary Retinal Pigment Epithelial Cells Derived from New Zealand White Rabbits and Gray Rabbits)

[0081] In some embodiments, primary unpigmented retinal pigment epithelial cells of New Zealand white rabbit cultured in vitro were used; In some other embodiments, pigmented retinal pigment epithelial cells of gray rabbit cultured in vitro were used.

[0082] (1) Isolation and Culture

[0083] 2-4 weeks old New Zealand white rabbits and gray rabbits were used, sacrificed by air embolization method; eyeballs were removed under sterile conditions, soaked in 1000 u gentamicin saline at 4 C. for 30 minutes, and then replaced in normal saline for 3 hours. Cut the anterior segment and neuroretinal epithelium under a dissecting microscope, put the posterior eye cup into a 12-well plate Petri dish, added 0.25% pancreatic enzyme to about eye cup, put it into a 37 C. incubator for 30 min for digestion, added a stop solution to terminate digestion, gently pipetted to detach RPE cells, then collected, centrifuged and inoculated; DMEM/F12 medium containing 10% fetal bovine serum was used to culture, and the solution was refreshed every two days for approximately 2 weeks, and identification was performed by morphology, PCR, and staining.

[0084] (3) Preparation of Cell Suspension

[0085] Cells were resuspended using DMEM low-glucose basal medium, the number of cells was counted by cell counter for distribution. About 510.sup.5-810.sup.6 cells were dissolved in about 200-300 l DMEM basal medium in addition of 10 M Y27632 for transplantation.

[0086] (2) Enzymatic digestion of cells, (4) Injection of cell suspension into the anterior chamber and (5) Functional evaluation were the same as Example 1.

[0087] Results & Analysis:

[0088] The preparation of primary retinal pigment epithelial cells of New Zealand white rabbits and gray rabbits was achieved by the method described in the present example, which exhibited a regular cell morphology (FIG. 5A); Corneal transparency can be quickly restored 7 days after cell transplantation (FIG. 5B).

Comparative Example 1 (Pancreatin/Collagenase)

[0089] (1) Induction of Differentiation: Steps were the Same as Example 1.

[0090] (2) Enzymatic Digestion of Cells

[0091] hESC-derived retinal pigment epithelial cells were treated at 37 C. by 0.25% pancreatin for about 3-10 mins, or hESC-derived retinal pigment epithelial cells were treated at 37 C. by 5 mg/ml IV collagenase for about 5-15 mins, and the enzyme reaction was terminated by complete medium; cells were gently pipetted into single cells and collected into 15 ml centrifuge tubes, then centrifuged at 1000 rpm for 3 mins; the supernatant was discarded and the precipitate was retained.

[0092] (3) Cell Viability and Size Detection

[0093] Trypan blue staining was performed and cell viability, size, etc. were counted by cell counter.

[0094] Results & Analysis:

[0095] The mortality rate of cells obtained by pancreatic digestion was high, and that by collagenase digestion was not easy to dissociate to obtain single cells, and the digestion time was too long, so pancreatase and collagenase can be used for enzymatic digestion of cells, but Accutase is preferred.

Comparative Example 2 (DMEM High Sugar Medium)

[0096] In this example, cell suspension was prepared using DMEM high sugar medium (containing 4.5 g/ml glucose).

[0097] (1) Induction of differentiation, (2) Enzymatic digestion of cells, (4) Injection of cell suspension into the anterior chamber and (5) Functional evaluation were the same as Example 1.

[0098] (3) Preparation of Cell Suspension

[0099] Cells were resuspended using DMEM high sugar medium (containing 4.5 g/ml glucose), the number of cells were counted by cell counter for distribution. About 510.sup.5-110.sup.6 cells were dissolved in about 200-300 l DMEM high sugar medium for transplantation.

[0100] Results & Analysis:

[0101] After transplantation of cell suspension resuspended in DMEM high-glucose medium as a solvent, the anterior chamber exudation was severe, and cornea edema lasted, and corneal transparency did not restore (FIG. 6).

Comparative Example 3

[0102] The present example prepares cell suspensions with different cell volumes.

[0103] (1) Induction of differentiation, (2) Enzymatic digestion of cells, (4) Injection of cell suspension into the anterior chamber and (5) Functional evaluation were the same as Example 1.

[0104] (3) Preparation of Cell Suspensions

[0105] Resuspend cells using DMEM low-glucose basal medium, count the number of cells by cell counter for distribution. Dissolve about 1.510.sup.6-110.sup.6, about 110.sup.6-510.sup.5, about 510.sup.5-110.sup.5 cells in about 200-300 l DMEM basal medium respectively for transplantation.

[0106] Results & Analysis:

[0107] Postoperative evaluation found that severe exudation of the anterior chamber and persistent cornea edema occurred after transplantation with over 1.210.sup.6 cells; while persistent cornea edema occurred and cornea transparency recovery failed after transplantation with less than 310.sup.5 cells (FIG. 7).

ADDITIONAL EMBODIMENTS

[0108] Embodiment 1. Application of retinal pigment epithelial cells in being substitution of corneal endothelial cells. [0109] Embodiment 2. Application of retinal pigment epithelial cells in the preparation of pharmaceutical composition for relieving or treating corneal endothelial injury, corneal endothelial lesion, corneal endothelial cell dysfunction, and corneal endothelial functional decompensation. [0110] Embodiment 3. Application of retinal pigment epithelial cells in the preparation of pharmaceutical composition for relieving or treating corneal thickness abnormality, corneal transparency decline, corneal edema, vision decline or loss, eye dryness, eye pain of an individual suffering from corneal endothelial function decompensation. [0111] Embodiment 4. The application according to Embodiment 2 or 3, wherein said retinal pigment epithelial cells are administered to the anterior chamber of an individual's eyeballs; said pharmaceutical composition comprises cell retinal pigment epithelial cells and DMEM low-sugar culture medium, wherein the ratio of said cell retinal pigment epithelial cells and DMEM low-sugar culture medium is 310.sup.5-1.210.sup.6: 200-300 micro liters. [0112] Embodiment 5. The application according to Embodiment 4, wherein the ratio of said retinal pigment epithelial cells and DMEM low-sugar culture medium is 510.sup.5-110.sup.6: 200-300 micro liters. [0113] Embodiment 6. The application according to Embodiment 4, wherein said pharmaceutical composition also comprises one or more specific inhibitors, and said specific inhibitor comprises Y27632, nicotinamide and/or TGF- inhibitor SB431542. [0114] Embodiment 7. The application according to Embodiment 2 or 3, wherein said retinal pigment epithelial cells are obtained by differentiation of human embryonic stem cells or human-induced pluripotent stem cells. [0115] Embodiment 8. The application according to Embodiment 7, wherein the pigment-producing gene Tyrosinase of said human embryonic stem cells or human-induced pluripotent stem cells are knocked out. [0116] Embodiment 9. The application according to Embodiment 2 or 3, the dosage form of said pharmaceutical composition comprises injection, cell sheet or kit. [0117] Embodiment 10. A method for preparing retinal pigment epithelial cell suspension, comprising the following steps: [0118] Step 1. directed differentiation: obtaining hESC/hiPSC-derived retinal pigment epithelial cells by using differentiation medium; [0119] Step 2. enzymatic dissociation: treating the hESC/hiPSC-derived retinal pigment epithelial cells in step 1 with cell digestive enzyme, and using complete culture medium to terminate the enzyme reaction; [0120] Step 3. single cell collection: using a pipettor to gently blow cells in step 2 into single cells, collecting the cells into a centrifuge tube, centrifuging and discarding supernatant to retain the cell precipitate; [0121] Step 4. cell suspension preparation: resuspending the cells in step 3 by using a DMEM basal culture medium, and dissolving about 310.sup.5-1.210.sup.6 cells in about 200-300 L of DMEM basal culture medium to obtain cell suspension. [0122] Embodiment 11. The method according to Embodiment 10, wherein said differentiation medium in step 1 comprises differentiation medium 1 and differentiation medium 2, wherein said differentiation medium 1 comprises DMEM/F12, Neuralbasal Medium, glutamine, non-essential amino acids, -mercaptoethanol, and N2 additives; said differentiation medium 2 comprises DMEM/F12 medium, serum substitute, glutamine, non-essential amino acid, and -mercaptoethanol; said differentiation culture medium 1 is mixed with the Neuralbasal Medium culture medium in a 1:1 proportion. [0123] Embodiment 12. The method according to Embodiment 10, wherein step 1 comprises using cell differentiation medium 1 mixed with 2% Matrigel to culture for 2 days, then changing to Matrigel-free medium for 5 days; using differentiation medium 2 to culture for 3 weeks; mechanical separation of retinal pigment epithelial cells and cell expansion; said differentiation medium 1 comprises DMEM/F12 mixed with Neuralbasal medium in a ratio of 1:1, 2 mM glutamine, 0.1 mM non-essential amino acids, 0.1 mM -mercaptoethanol and 1% N2 additive; said differentiation medium 2 comprises DMEM/F12 medium, 10% serum substitute, 2 mM glutamine, 0.1 mM non-essential amino acid, 0.1 mM -mercaptoethanol. [0124] Embodiment 13. The method according to any one of Embodiments 10-12, wherein said cell digestive enzyme in step 2 is Accutase, and the processing temperature is 37 C.; and said cell suspension in step 4 also comprises 5-15 m Y27632.

[0125] Although the specific embodiments of the present invention have been described in detail, those skilled in the art would understand that according to all the teachings that have been disclosed, various modifications and substitutions may be made to those details and doses, which are within the scope of protection of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.