Patent Application
20240016852
The present invention belongs to the field of medicine, and relates to an application of retinal pigment epithelial cells in relieving or treating corneal endothelial functional decompensation.
The cornea is a transparent tissue located on the anterior of the eyeball, and is divided into five layers, which sequentially comprises epithelial cell layer, bowman membrane, stromal layer, descemet's membrane, and endothelial cell layer from front to back. The highly transparent and optical properties of the cornea are one of the prerequisites that normally exert physiological functions, while corneal endothelial cells play an important role in maintaining the normal physiological function of the cornea. Corneal endothelial cells are single-layer cells in corneal inner layer, which forms a physical barrier between descemet's membrane and aqueous humor, adjusts the concentration and moisture of ions in the cornea by the ion “pump” function to maintain the semi-dehydration state of the cornea, and ensures the normal thickness and transparency of the cornea. Once the function of corneal endothelial cells is disordered, it often leads to corneal edema, causing partial or even complete corneal blindness.
Normal human corneal endothelial cells have extremely limited proliferation capacity in vivo. Endothelial cell damage and loss caused by trauma, inflammation, cataract surgery, etc., can only be filled by the enlargement and migration of surrounding cells. When the density of human corneal endothelial cells drops to its physiological critical value (about 400-500 cells/mm2), corneal edema occurs and vision loss in severe cases occurs. At present, there are about 4 million patients with corneal blindness in China, including nearly 1 million patients with endothelial blindness. Corneal transplantation is the only clinical therapeutic strategy for corneal endothelial decompensation. Due to the lack of corneal donors in China, only less than 10000 patients regain their sight through corneal transplantation every year, which is far from covering all clinical needs. In order to solve the problem of the corneal donor shortage, the current main corneal endothelial alternative seed cell research comprises cultured adult stem cells such as human corneal endothelial cells and skin progenitor cells, and corneal endothelial cells derived from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs).
Although the transplantation of primary corneal endothelial cells, adult stem cells, or pluripotent stem cells-derived cells can improve corneal endothelial function, the effect is limited. Up to now, there are no ideal corneal endothelial alternative seed cells to achieve long-term corneal transparency and wide clinical application. The team of Japanese scientist Professor Kinoshita transplanted human corneal endothelial cells by anterior chamber injection to clinically treat 11 patients with corneal endothelial dystrophy, and they restored corneal transparency, but the results of 5-year follow-up showed that the corneal endothelium of some patients reappeared pathological “guttae” structure. Cultured human corneal endothelial cells still rely on high-quality donor cornea, and adult corneal endothelial seed cells cannot be expanded in vitro in large quantities, so the source and number of these cells is limited; Adult stem cell-derived alternative cells, such as skin progenitor cells, have poor purity, difficult industrial preparation and limited therapeutic effect; hESC/hiPSC has unlimited proliferative ability and pluripotency, it has been reported that hESC/hiPSC differentiated into neural crest, corneal endothelial precursor and mature corneal endothelioid cells, some experiments have proved that corneal endothelial precursor cells and mature corneal endothelioid cells can be applied to animal models to restore corneal transparency, however, there is currently no standardized method for directed differentiation of hESC/hiPSC to corneal endothelial cells, the long-term efficacy and safety of hESC/hiPSC-derived corneal endothelial cells in vivo remains to be studied. Therefore, to find the ideal corneal endothelial alternative seed cells is still an urgent problem to be solved in the field of corneal endothelial treatment.
After a large number of studies, the inventors find that although retinal pigment epithelial cells are greatly different from corneal endothelial cells in terms of tissue differentiation source, anatomical location, and somatic tissue cell function, they have a homologous regular hexagonal morphology and express tight junction proteins, suggesting that retinal pigment epithelial cells have the possibility of providing barrier function in substitute of corneal endothelial cells and treating corneal endothelial functional decompensation. In order to solve the shortcomings of the prior art, the object of the present invention is to provide corneal endothelial alternative cells; the further objective is to select hESC/hiPSC-derived retinal pigment epithelial cells as seed cells, providing a seed cell source that could be supplied indefinitely and used clinically safely; more further purpose is to improve the function of transplanted cells, optimize the preparation process of cell suspension, and ensure the normal function of transplanted cells.
In order to achieve the above objectives, the present invention provides a retinal pigment epithelial cell suspension, the cell suspension 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 3×105-1.2×106: 200-300 μL; preferably, the ratio of the retinal pigment epithelial cells to the DMEM low-sugar culture medium is 5×105-1×106: 200-300 μL.
In a preferred embodiment of the present invention, the retinal pigment epithelial cells are obtained by differentiation of human embryonic stem cells or human-induced pluripotent stem cells.
In a preferred embodiment of the present invention, the pigment-producing gene Tyrosinase of the human embryonic stem cells or human induced pluripotent stem cells are knocked out.
In a preferred embodiment of the present invention, the cell suspension further comprises one or more specific inhibitors, and the specific inhibitor comprises Y27632, nicotinamide and/or TGF-β inhibitor SB431542.
The present invention also provides a method for preparing retinal pigment epithelial cell suspension, comprising the following steps:
In a preferred embodiment of the present invention, the differentiation medium in step 1 comprises 1: 1 proportion of DMEM/F12 and Neuralbasal Medium, 1-4 mM glutamine, 0.1-1.3 mM non-essential amino acid, 0.1-1.3 mM β-mercaptoethanol, and 1% N2 additive;
In a preferred embodiment of the present invention, the differentiation culture medium in step 1 comprises DMEM/F12 culture medium, 5-15% serum substitute, 1-4 mM glutamine, 0.1-1.3 mM non-essential amino acid, and 0.1-1.3 mM β-mercaptoethanol.
In a more preferred embodiment of the present invention, the differentiation medium in step 1 comprises differentiation medium 1 and differentiation medium 2, wherein the 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 additives; the differentiation medium 2 comprises DMEM/F12 medium, 10% serum substitute, 2 mM glutamine, 0.1 mM non-essential amino acid, and 0.1 mM β-mercaptoethanol. The method of induction differentiation comprises using 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 supplement) mixed with 2% Matrigel to culture for 2 days, then changing 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 β-mercaptoethanol) to culture for 3 weeks; mechanical separation of retinal pigment epithelial cells and cell expansion.
In a more preferred embodiment of the present invention, the cell digestive enzyme in step 2 is Accutase, and the processing temperature is 37° C.; and 5-15 μm Y27632 is added in step 4.
The present invention provides a use of retinal pigment epithelial cells in substitute of corneal endothelial cells, i.e., for replacing damaged, diseased or missing corneal endothelial cells.
The present invention also provides an 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.
The present invention also provides an 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 and related symptoms of an individual suffering from corneal endothelial functional decompensation.
The retinal pigment epithelial cells provided by the present invention can be administered in any convenient dosage form, and the preferred dosage form comprises injection, cell sheet or kit. Regardless of the dosage form, retinal pigment epithelial cells are administered to the anterior chamber of the individual's eyeballs.
The present invention also relates to any of the following 1 to 13 items:
The Beneficial Technical Effects of the Present Invention are as Follows:
According to the invention, retinal pigment epithelial cells are used as alternative seed cells of corneal endothelium for the first time; the cell suspension and the preparation method provided by the invention can effectively replace corneal endothelial function to recover corneal transparency and corneal thickness while ensuring cell viability. In addition, the seed cells for replacing corneal endothelium provided by the present invention can be obtained by inducing differentiation from hESC/hiPSCs, and are infinitely supplied, and the application security thereof has been reported in existing clinical experiments. According to the preparation method and the transplantation method provided by the invention, highly specialized equipment, reagents or skills are not needed, so that researchers and clinical personnel can operate conveniently, and therefore, the method has wide application values and positive social benefits.
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.
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.
(1) Directed Differentiation
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 β-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 β-mercaptoethanol) to culture for 3 weeks; mechanical separation of retinal pigment epithelial cells and cell expansion.
(2) Enzymatic Digestion of Cells
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.
(3) Preparation of Cell Suspension
Cells were resuspended using DMEM low-glucose basal medium, the number of cells were counted by cell counter for distribution. About 5×105-1×106 cells were dissolved in about 200-300 μl DMEM basal medium in addition of 10 μM Y27632 for transplantation.
(4) Injection of Cell Suspension into the Anterior Chamber
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.
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.
(5) Functional Evaluation
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.
Results & Analysis:
Based on the present example, the differentiation from the hESC cell line H1 into retinal pigment epithelial cells can be induced (
This example used the hiPSC cell line DY0100 to induce retinal pigment epithelial cells.
(1) Directed Differentiation
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.
(3) Preparation of Cell Suspension
Cells were resuspended using DMEM low-glucose basal medium, the number of cells were counted by cell counter for distribution. About 8×105-1×106 cells were dissolved in about 200-300 μl DMEM basal medium with addition of 10 μM Y27632 and 5 mM nicotinamide for transplantation.
(2) Enzymatic digestion of cells, (4) Injection of cell suspension into the anterior chamber and (5) Functional evaluation were the same as Example 1.
Results & Analysis:
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 (
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.
In some embodiments, the hES cell line H1 was used; In some other embodiments, the hiPS cell line DY0100 was used.
(1) Construction of Tryosinase Knockout Cells:
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=(MOI×number 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.
(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.
Results & Analysis:
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 (
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.
(1) Isolation and Culture
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 ¾ 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.
(3) Preparation of Cell Suspension
Cells were resuspended using DMEM low-glucose basal medium, the number of cells was counted by cell counter for distribution. About 5×105-8×106 cells were dissolved in about 200-300 μl DMEM basal medium in addition of 10 μM Y27632 for transplantation.
(2) Enzymatic digestion of cells, (4) Injection of cell suspension into the anterior chamber and (5) Functional evaluation were the same as Example 1.
Results & Analysis:
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 (
(1) Induction of Differentiation: Steps were the Same as Example 1.
(2) Enzymatic Digestion of Cells
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.
(3) Cell Viability and Size Detection
Trypan blue staining was performed and cell viability, size, etc. were counted by cell counter.
Results & Analysis:
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.
In this example, cell suspension was prepared using DMEM high sugar medium (containing 4.5 g/ml glucose).
(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.
(3) Preparation of Cell Suspension
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 5×105-1×106 cells were dissolved in about 200-300 μl DMEM high sugar medium for transplantation.
Results & Analysis:
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 (
The present example prepares cell suspensions with different cell volumes.
(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.
(3) Preparation of Cell Suspensions
Resuspend cells using DMEM low-glucose basal medium, count the number of cells by cell counter for distribution. Dissolve about 1.5×106-1×106, about 1×106-5×105, about 5×105-1×105 cells in about 200-300 μl DMEM basal medium respectively for transplantation.
Results & Analysis:
Postoperative evaluation found that severe exudation of the anterior chamber and persistent cornea edema occurred after transplantation with over 1.2×106 cells; while persistent cornea edema occurred and cornea transparency recovery failed after transplantation with less than 3×105 cells (
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.
This application is a continuation of PCT/CN2022/105465, filed Jul. 13, 2022, which is herein incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CN2022/105465 | Jul 2022 | US |
| Child | 18352690 | US |
