Application of urine-derived stem cells in replacement of corneal endothelial cells

Abstract

The invention discloses application of urine-derived stem cells in replacement of corneal endothelial cells. The corneal endothelium-like cells obtained after induced differentiation of the urine-derived stem cells can recover corneal transparency, reduce corneal thickness, reconstruct corneal endothelium barrier functions, effectively treat corneal endothelium function decompensation and improve corneal endothelium barrier functions. The method has wide application value and positive social benefits for treatment or recovery of people with impaired vision due to corneal injury.

Description

Technical Field

[0001] The present invention belongs to the field of medicine and relates to the application of urine-derived stem cells in replacing corneal endothelial cells, a method for differentiating urine-derived stem cells into corneal endothelial-like cells, and the application of urine-derived stem cells in alleviating or treating corneal endothelial decompensation. Background Art

[0002] The cornea is a transparent membrane located on the anterior wall of the eyeball. It is composed of five layers, from front to back: epithelial cell layer, Descemet's membrane, stroma, Descemet's membrane, and endothelial cell layer. High corneal transparency and optical properties are essential for normal physiological function, and corneal endothelial cells play a vital role in maintaining corneal transparency and normal physiological function. Corneal endothelial cells are a single layer of hexagonal cells located on the innermost layer of the cornea, forming a physical barrier between the Descemet's membrane and the aqueous humor. Through their ion pump function, they regulate ion concentration and moisture in the cornea, maintaining a semi-dehydrated state and ensuring normal corneal thickness and transparency. Damage to the corneal endothelial cells, dysfunction of their function, or a decrease in the number of normal corneal endothelial cells below a critical value can lead to abnormal corneal thickness, decreased corneal transparency, corneal edema, decreased or lost vision, dry and painful eyes, and even partial or complete corneal blindness.

[0003] Because human corneal endothelial cells lose their ability to proliferate after birth, corneal endothelial cell damage is irreversible. Keratoplasty or endothelial keratoplasty is the only effective clinical treatment for corneal endothelial dysfunction or decompensation. However, there is a significant global shortage of corneal donor material. Consequently, a large number of patients with corneal blindness continue to wait in the dark.

[0004] In recent years, emerging strategies such as tissue-engineered corneal endothelial transplantation, differentiation of pluripotent stem cells, and in vitro expansion technology of human corneal endothelial cells have brought hope for solving the shortage of corneal donors, but all face the fundamental challenge of "lack of seed cells". Currently, the main research strategies for corneal endothelial replacement seed cells include cultured human corneal endothelial cells, adult stem cells such as skin dry progenitor cells, and corneal endothelial-like cells derived from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Although the use of primary cultured human corneal endothelial cells, adult stem cells, or corneal endothelial-like replacement cells derived from pluripotent stem cells can improve corneal endothelial function, the effect is not ideal and there are many problems. For example, corneal endothelial-like cells derived from skin dry progenitor cells have poor purity, are difficult to prepare industrially, and have limited therapeutic effects; hESCs / hiPSCs have the ability of unlimited proliferation and multi-directional differentiation potential, but currently there is no standardized method for the directed differentiation of hESCs / hiPSCs into corneal endothelial cells, and there are problems of unclear long-term effectiveness and tumorigenic safety. Up to now, there has been no ideal corneal endothelial replacement seed cell to achieve long-term corneal transparency and clinical translational application.

[0005] Therefore, the screening of corneal endothelial replacement seed cells has always been a difficult problem that needs to be solved in the field of corneal endothelial treatment. Summary of the Invention

[0006] In order to solve the deficiencies of the existing technology, the objectives of the present invention include but are not limited to providing a corneal endothelial replacement seed cell that is autologous, non-invasive, simple to operate, low-cost, non-invasively obtained, has immune compatibility and ethical advantages, and can replace corneal endothelium to play a corneal transparency repair function; providing a method for differentiating urine-derived stem cells into corneal endothelial-like cells to obtain corneal endothelial replacement cells; providing the application of corneal endothelial-like cells differentiated from urine-derived stem cells as seed cells in the treatment of corneal endothelial injury, corneal endothelial dysfunction, corneal endothelial decompensation, corneal endothelial lesions, etc.; or providing the application of corneal endothelial-like cells differentiated from urine-derived stem cells as seed cells in the preparation for relieving or treating abnormal corneal thickness, decreased corneal transparency, corneal edema, vision loss or loss, dry and painful eyes, etc. A further objective is to improve the function of transplanted cells, optimize the process of preparing cell suspension, and ensure the normal function of transplanted cells.

[0007] In the first aspect of the present invention, a method for inducing the differentiation of urine-derived stem cells into corneal endothelial-like cells is provided, including: culturing urine-derived stem cells in a differentiation medium to promote the expression of ZO-1 (barrier protein), or ZO-1 protein and ATP1A1 protein in urine-derived stem cells.

[0008] Preferably, the differentiation medium contains the TGF-β inhibitor SB431542, the ROCK inhibitor Y27632, and nicotinamide; more preferably, the differentiation medium is prepared by adding the TGF-β inhibitor SB431542, the ROCK inhibitor Y27632, and nicotinamide to the epithelial proliferation medium to promote the monolayer arrangement of cells and form tight junctions; the components of the epithelial proliferation medium consist of 500 ml of basal medium, 10 ml of fetal bovine serum, 5 ml of epithelial cell growth supplement, and 5 ml of penicillin / streptomycin solution.

[0009] Preferably, the culture time is 3 - 10 days.

[0010] Preferably, the urinary stem cells are primary cells, or the first, second, third, fourth, fifth, or sixth generation cells after passage. More preferably, the urinary stem cells are the first, second, third, fourth, or fifth generation cells after passage. Even more preferably, the urinary stem cells are the second, third, or fourth generation cells after passage.

[0011] Preferably, the method for inducing the differentiation of urinary stem cells into corneal endothelial-like cells further includes a pre-step of induction and differentiation: culturing urinary stem cells, including culturing the primary cells of urinary stem cells with a proliferation medium.

[0012] More preferably, the method for inducing the differentiation of urinary stem cells into corneal endothelial-like cells includes: (1) culturing cells to confluence with a proliferation medium; (2) replacing the proliferation medium with a differentiation medium and continuing the culture to obtain corneal endothelial-like cells with monolayer arrangement and tight junction formation.

[0013] Preferably, in step (1), the reagent for coating the porous plate is gelatin, Fibronectin, or Matrigel matrix glue.

[0014] Preferably, in step (2), the differentiation medium contains the TGF-β inhibitor SB431542, the ROCK inhibitor Y27632, and nicotinamide; more preferably, the differentiation medium is prepared by adding the TGF-β inhibitor SB431542, the ROCK inhibitor Y27632, and nicotinamide to the epithelial proliferation medium; more preferably, the differentiation medium is prepared by adding the TGF-β inhibitor SB431542 with a final concentration of 10 μM, the ROCK inhibitor Y27632 with a final concentration of 5 - 10 μM, and nicotinamide with a final concentration of 5 - 10 mM to the epithelial proliferation medium.

[0015] Further, the above method for inducing the differentiation of urine-derived stem cells into corneal endothelial-like cells further includes the previous steps of culturing the primary cells of urine-derived stem cells: isolating, enriching, and washing urine-derived stem cells from urine samples; inoculating the primary cells of urine-derived stem cells, etc.

[0016] In a second aspect of the present invention, there is provided a cell suspension of corneal endothelial-like cells, comprising corneal endothelial-like cells obtained by the above method for inducing the differentiation of urine-derived stem cells into corneal endothelial-like cells.

[0017] Preferably, the cell suspension includes corneal endothelial-like cells and DMEM basal medium. More preferably, the cell suspension further includes one or more specific inhibitors, and the specific inhibitors include Y27632, nicotinamide, and / or TGF-β inhibitor SB431542.

[0018] In a third aspect of the present invention, there is provided the use of corneal endothelial-like cells obtained by the above method for inducing the differentiation of urine-derived stem cells into corneal endothelial-like cells in the preparation of drugs or medical devices for relieving or treating corneal endothelial injury, corneal endothelial lesions, corneal endothelial cell dysfunction, and corneal endothelial cell decompensation.

[0019] In a fourth aspect of the present invention, there is provided the use of corneal endothelial-like cells obtained by the above method for inducing the differentiation of urine-derived stem cells into corneal endothelial-like cells in the preparation of drugs or medical devices for relieving or treating abnormal corneal thickness, decreased corneal transparency, corneal edema, vision decline or loss, and dry and painful eyes.

[0020] In a fifth aspect of the present invention, there is provided the use of urine-derived stem cells in the preparation of drugs or medical devices for relieving or treating corneal endothelial injury, corneal endothelial lesions, corneal endothelial cell dysfunction, and corneal endothelial cell decompensation.

[0021] In a sixth aspect of the present invention, there is provided the use of urine-derived stem cells in the preparation of drugs or medical devices for relieving or treating abnormal corneal thickness, decreased corneal transparency, corneal edema, vision decline or loss, and dry and painful eyes.

[0022] In the fifth and sixth aspects of the present invention, the urine-derived stem cells express ZO-1 protein after induced differentiation, or simultaneously express ZO-1 protein and ATP1A1 protein, and become corneal endothelial-like cells; the cell morphology of the corneal endothelial-like cells is corneal endothelial-like.

[0023] In the fifth and sixth aspects of the present invention, the urine-derived stem cells become corneal endothelial-like cells after induced differentiation by culturing the urine-derived stem cells in a differentiation medium to promote the expression of ZO-1 (barrier protein), or ZO-1 (barrier protein) and ATP1A1 (fluid pump protein) in the urine-derived stem cells.

[0024] In the present invention, the dosage of the corneal endothelial-like cells is not limited and can be selected according to actual situations. For example, it can be 1×10 5 ~1.5×10 6 cells per eye.

[0025] In the present invention, the corneal endothelial-like cells are administered to the anterior chamber of the patient's eyeball in the form of a cell suspension; the cell suspension includes corneal endothelial-like cells and DMEM basal medium. Preferably, the cell suspension further includes one or more specific inhibitors, and the specific inhibitors include Y27632, nicotinamide, and / or TGF-β inhibitor SB431542.

[0026] The urinary stem cells and the corneal endothelial-like cells differentiated from the urinary stem cells provided by the present invention can be provided in any convenient dosage form. Preferred dosage forms include injection solutions, cell sheets, patches, or kits. No matter in what dosage form it is provided, it is administered to the anterior chamber of the patient's eyeball.

[0027] The beneficial technical effects of the present invention: Through a non-invasive autologous cell source and an efficient induction and differentiation system, the present invention solves the core problems such as donor dependence, immune rejection, insufficient cell function, and high cost, providing a safer, more economical, and scalable new technical path for the treatment of corneal endothelial decompensation. Specifically, it includes:

[0028] 1) Non-invasive acquisition: Directly isolate urinary stem cells (USCs) from the patient's own urine, without the need for donor corneas, avoiding donor shortages and ethical controversies.

[0029] 2) Strong proliferation ability: USCs can be stably amplified to more than the 5th passage. Combined with the induction and differentiation system, it breaks through the proliferation bottleneck of primary human corneal endothelial cells.

[0030] 3) Autologous cell source: USCs are derived from the patient themselves, and the differentiated corneal endothelial-like replacement cells have low immunogenicity, significantly reducing the risk of rejection.

[0031] 4) No exogenous gene integration: Through small molecule-induced differentiation (non-viral vector), it avoids the safety hazards brought by gene editing or viral transfection.

[0032] 5) Low cost and high accessibility: Urine samples are easily obtained, and the separation and amplification processes of USCs are standardized, reducing production costs.

[0033] 6) Wide applicability: USCs can be isolated from the urine of patients of any age, gender, or health status, especially suitable for patients with scarce donors or immune sensitivity (such as children, the elderly, or immunodeficient patients). In addition, the viability and function of patient-derived urinary cells can be improved or enhanced through in vitro engineering of the patient-derived urinary cells. Brief Description of the Drawings

[0034] Figure 1 Morphology of urine-derived cells isolated from urine stored at 4°C for different times.

[0035] Figure 2 ZO1 and ATP1A1 staining identification results of corneal endothelial-like replacement cells differentiated from urine-derived stem cells.

[0036] Figure 3 Observation results of corneal transparency after transplantation of corneal endothelial-like replacement cells differentiated from urine-derived stem cells from a 38-year-old adult male volunteer.

[0037] Figure 4 Detection results of corneal thickness after transplantation of corneal endothelial-like replacement cells differentiated from urine-derived stem cells from a 38-year-old adult male volunteer.

[0038] Figure 5 Detection results of corneal transparency after transplantation of corneal endothelial-like replacement cells differentiated from urine-derived stem cells from a 9-year-old male child. Detailed Description of the Invention

[0039] In recent years, a cell population isolated from urine has the biological characteristics and differentiation potential of stem cells, which is called urine-derived stem cells (USC). Regarding the source of USC, there is currently no conclusive evidence, but its proliferation ability and multi-directional differentiation ability have been confirmed. The main advantages of USCs include convenient material collection, non-invasive operation, low cost, no tumorigenicity and immunogenicity, etc.

[0040] The present invention provides the application of urine-derived stem cells in replacing corneal endothelial cells. Urine-derived stem cells are obtained by collecting cells from human urine and culturing and expanding them in vitro. Corneal endothelial-like replacement cells are induced by a combination of small molecules and a differentiation medium, and are identified to express the corneal endothelial marker barrier function gene ZO1 and / or the fluid pump function gene ATP1A1. An animal model of corneal endothelial dysfunction is constructed using New Zealand rabbits, and the corneal endothelial-like replacement cells derived from USC are transplanted by intracameral injection, which proves that the cells can reconstruct corneal endothelial function, restore corneal transparency and normal corneal thickness in the long term, and achieve the restoration of corneal visual function.

[0041] In the present invention, corneal endothelial-like cells and corneal endothelial-like replacement cells have the same meaning, referring to cells that are not derived from corneal endothelial cells, express the corneal endothelial cell marker barrier function gene ZO1, and have obtained some or all of the functions of corneal endothelial cells; they can be used as seed cells to replace corneal endothelial cells.

[0042] The present invention is further illustrated by the following examples, which are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. Unless otherwise specified, the technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention pertains. For those conditions not specified in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained commercially.

[0043] At each stage of the present invention (for example, the primary cell stage of culturing urinary stem cells, the stage of inducing differentiation of urinary stem cells), regardless of the medium used, the present invention places no restrictions on the specific operations of cell culture (such as changing the medium, cell passage, etc.) and culture conditions (such as temperature, humidity, culture time, etc.). Those skilled in the art can complete them according to actual needs according to conventional operations. For example, cells are cultured at 37 °C and 5% carbon dioxide.

[0044] In the embodiments of the present invention, the sources of some materials are as follows:

[0045] Human urine: Urine specimens of healthy adult male and male child volunteers were collected with the approval of the Ethics Review Committee of the Affiliated Eye Institute of Shandong First Medical University. All volunteers signed informed consent forms. Under sterile conditions, the urine source of the patients was collected.

[0046] New Zealand white rabbits were purchased from Jinan Xilingjiao Breeding Center.

[0047] The present invention places no restrictions on the collection of urine samples, the preservation of urine samples, the isolation, purification, and washing of urinary stem cells, the inoculation, culture, and passage of primary urinary stem cells, and the identification methods of urinary stem cells. Those skilled in the art can choose to implement according to the existing technology. For example, urinary stem cells are isolated and obtained by basic culture of urinary-derived stem cells; primary culture is achieved by centrifuging to collect urine cells; refer to relevant literature (Zhou T et al. Generation of human induced pluripotent stem cells from urine samples. Nat Protoc. 2012;7(12):2080 - 2089. Wang Fuping, et al. Isolation and identification of human urinary stem cells, Biotechnology Bulletin, 2018, 34(8):190 - 198), etc.

[0048] Some preferred experimental methods and reagent preparation methods of the present invention are as follows:

[0049] Preferably, when collecting urine samples, 30 - 500 mL of midstream urine is collected in a sterile container under a sterile environment.

[0050] Preferably, the urine samples are stored at 4 °C.

[0051] Preferably, the storage time of the urine sample does not exceed 2 days; more preferably, the storage time of the urine sample does not exceed 1 day; even more preferably, the storage time of the urine sample does not exceed 15 hours, such as 1 - 10 hours, to ensure cell viability.

[0052] A method for separating, enriching and washing urine-derived stem cells from a urine sample, comprising: centrifuging the urine sample (preferably at 400×g for 10 minutes), discarding the supernatant and retaining the precipitate; adding a washing solution to the precipitate and centrifuging (preferably at 200×g for 10 minutes) to remove impurities; repeating the washing of the precipitate (for example, 1 - 3 times).

[0053] Preferably, in the method for separating, enriching and washing urine-derived stem cells from a urine sample, the washing solution is a PBS washing solution containing penicillin, streptomycin and amphotericin B. More preferably, the washing solution is a PBS washing solution containing 100 U / mL penicillin, 100 μg / mL streptomycin and 0.5 μg / mL amphotericin B.

[0054] A method for inoculating primary cells with urine-derived stem cells, comprising: resuspending the precipitate in a primary basal medium and then inoculating it into an experimental vessel, such as a well of a 12-well cell culture plate. Preferably, the experimental vessel is coated, for example, the reagent for coating the well plate is gelatin, Fibronectin or Matrigel. More preferably, 2 - 10 μM Y27632 is added to the reagent to promote cell proliferation and adhesion efficiency.

[0055] Preferably, the primary basal medium is prepared by mixing DMEM high-glucose medium (Gibco, Thermo Fisher Scientific Inc., catalog number 11965092) and Ham’s F12 nutrient mixture (Gibco, Thermo Fisher Scientific Inc., catalog number 31765035) in a 1:1 ratio to form DMEM / F12 medium; adding FBS, penicillin, streptomycin, epithelial cell growth supplement (ScienCell, catalog number: 4152) and amphotericin B. Preferably, the concentrations of FBS, penicillin, streptomycin, epithelial cell growth supplement and amphotericin B in the primary basal medium are 10% FBS, 100 U / ml penicillin, 0.1 mg / ml streptomycin, 5 - 50 ng / ml epithelial cell growth supplement and 0.25 μg / ml amphotericin B.

[0056] A method for culturing primary cells of urine-derived stem cells, comprising: culturing the primary cells of urine-derived stem cells with a proliferation medium.

[0057] Preferably, the method for culturing primary cells of urinary stem cells includes: culturing primary cells of urinary stem cells with a primary basal medium; after culturing for a period of time, switching to a proliferation medium for continuous culture.

[0058] In some embodiments of the present invention, the method for culturing primary cells of urinary stem cells is to add a primary basal medium (select the addition amount as needed, such as 1 milliliter) to an experimental vessel (such as a well of a 12-well plate) inoculated with primary cells of urinary stem cells at different times after inoculation (such as 24, 48, and 72 hours after inoculation); after culturing for a period of time (such as 96, 120 hours), aspirate most of the medium in the experimental vessel, and then add a proliferation medium (such as only leaving about 1 milliliter of the medium, and then adding 1 milliliter of the proliferation medium); aspirate the medium in the experimental vessel every day, and then add the proliferation medium for continuous culture (such as replacing half of the medium in the experimental vessel every day, keeping the other half unchanged, and then adding the proliferation medium, and culturing for 1 - 2 weeks).

[0059] Preferably, the proliferation medium is obtained by mixing an epithelial proliferation medium and a basal proliferation medium; more preferably, it is obtained by mixing in a ratio of 1:1.

[0060] Preferably, the epithelial proliferation medium is composed of 500 milliliters of basal medium, 10 milliliters of fetal bovine serum, 5 milliliters of epithelial cell growth supplement, and 5 milliliters of penicillin / streptomycin solution (ScienCell Research Laboratories, Epithelial Cell Medium (EpiCM), catalog number: 4101).

[0061] Preferably, the basal proliferation medium includes fetal bovine serum, GlutaMAX additive, non-essential amino acids, penicillin / streptomycin, basic fibroblast growth factor 2 (bFGF), platelet-derived growth factor, and epidermal growth factor (EGF).

[0062] Preferably, the proliferation medium is obtained by mixing an epithelial proliferation medium and a basal proliferation medium, for example, in a ratio of 1:1, 1:2, 2:1, 1.5:2, 2:1.5, 1:3.

[0063] More preferably, the epithelial proliferation medium is composed of 500 milliliters of basal medium, 10 milliliters of fetal bovine serum, 5 milliliters of epithelial cell growth supplement, and 5 milliliters of penicillin / streptomycin solution (ScienCell Research Laboratories, Epithelial Cell Medium (EpiCM), catalog number: 4101).

[0064] More preferably, the basal proliferation medium comprises fetal bovine serum, GlutaMAX additive, non-essential amino acids, penicillin / streptomycin, fibroblast growth factor 2 (bFGF), platelet-derived growth factor (preferably PDGF-AB), and epidermal growth factor (EGF).

[0065] In some other embodiments, the basal proliferation medium comprises 20 - 150 mL of fetal bovine serum, 2 - 15 mL of GlutaMAX additive (Gibco, Thermo Fisher Scientific Inc., catalog number 35050061), 2 - 15 mL of non-essential amino acids (Non-Essential Amino Acids, NEAA) (Wuhan Shang'en Biotechnology Co., Ltd., catalog number: SNSP-005), 2 - 15 mL of penicillin / streptomycin (final concentration 1%) (Gibco, Thermo Fisher Scientific Inc., catalog number 15140122), 50 - 200 μL of 5 ng / mL fibroblast growth factor 2 (bFGF), 100 - 500 μL of 5 ng / mL platelet-derived growth factor (preferably PDGF-AB), and 10 - 100 μL of 5 ng / mL epidermal growth factor (EGF).

[0066] In some other embodiments, the basal proliferation medium comprises 50 mL of fetal bovine serum, 5 mL of GlutaMAX additive, 5 mL of non-essential amino acids, 5 mL of penicillin / streptomycin (final concentration 1%), 100 μL of 5 ng / mL fibroblast growth factor 2 (bFGF), 250 μL of 5 ng / mL platelet-derived growth factor (preferably PDGF-AB), and 50 μL of 5 ng / mL epidermal growth factor (EGF).

[0067] In some embodiments, the basal proliferation medium consists of fetal bovine serum, GlutaMAX additive, non-essential amino acids, penicillin / streptomycin, fibroblast growth factor 2 (bFGF), platelet-derived growth factor (preferably PDGF-AB), epidermal growth factor (EGF), and high-glucose DMEM medium.

[0068] In this embodiment, the basal proliferation medium is prepared by mixing 50 mL of fetal bovine serum with 5 mL of GlutaMAX additive (Gibco, Thermo Fisher Scientific Inc., catalog number 35050061), 5 mL of non-essential amino acids (Non-Essential Amino Acids, NEAA) (Wuhan Shangen Biotechnology Co., Ltd., catalog number: SNSP-005), 5 mL of penicillin / streptomycin (final concentration 1%) (Gibco, Thermo Fisher Scientific Inc., catalog number 15140122), 100 μL of 5 ng / mL fibroblast growth factor 2 (bFGF), 250 μL of 5 ng / mL platelet-derived growth factor (preferably PDGF-AB), and 50 μL of 5 ng / mL epidermal growth factor (EGF); after filtration sterilization, it is made up to 500 mL with high-glucose DMEM medium to obtain the basal proliferation medium.

[0069] The present invention provides a method for inducing differentiation of urinary stem cells into corneal endothelial-like cells, comprising:

[0070] Culturing urinary stem cells in a differentiation medium to promote the expression of ZO-1 (barrier protein) in the urinary stem cells.

[0071] Preferably, ZO-1 (barrier protein) and ATP1A1 (fluid pump protein) are expressed in the urinary stem cells. More preferably, ZO-1 (barrier protein) and ATP1A1 (fluid pump protein) are expressed in the urinary stem cells, and the cells exhibit a regular polygonal-like structure.

[0072] Preferably, the differentiation medium is prepared by adding TGF-β inhibitor SB431542, ROCK inhibitor Y27632, and nicotinamide to the epithelial proliferation medium (ScienCell Research Laboratories, Epithelial Cell Medium (EpiCM), catalog number: 4101) to promote monolayer arrangement of cells and formation of tight junctions.

[0073] Preferably, the concentration of SB431542 in the differentiation medium is 5 - 20 μM, the concentration of Y27632 is 5 - 15 μM, and the concentration of nicotinamide is 5 - 15 mM. More preferably, the concentration of SB431542 is 10 - 20 μM, the concentration of Y27632 is 5 - 10 μM, and the concentration of nicotinamide is 5 - 10 mM. Even more preferably, the concentration of SB431542 is 10 μM, the concentration of Y27632 is 5 μM, and the concentration of nicotinamide is 5 mM.

[0074] Preferably, the culture time is 3 - 10 days.

[0075] Preferably, the urine-derived stem cells are primary cells, or the first, second, third, fourth, fifth, or sixth generation cells after passage. More preferably, the urine-derived stem cells are the first, second, third, fourth, or fifth generation cells after passage. Even more preferably, the urine-derived stem cells are the second, third, or fourth generation cells after passage.

[0076] The method for inducing the differentiation of urine-derived stem cells into corneal endothelial-like cells further includes a pretreatment step for induction and differentiation: culturing urine-derived stem cells, including culturing the primary cells of urine-derived stem cells with a proliferation medium. Further, the method for inducing the differentiation of urine-derived stem cells into corneal endothelial-like cells also includes a pre-step for culturing the primary cells of urine-derived stem cells: separating, enriching, and washing urine-derived stem cells from a urine sample; inoculating the primary cells of urine-derived stem cells, etc. See the content of the preservation of the urine sample, the separation, purification, and washing of urine-derived stem cells, and the inoculation, culture, and passage of primary urine-derived stem cells described above for details.

[0077] A method for inducing the differentiation of urine-derived stem cells into corneal endothelial-like cells specifically includes:

[0078] (1) Culturing urine-derived stem cells with a proliferation medium until confluence;

[0079] (2) Replacing the proliferation medium with a differentiation medium for culture and continuing the culture (for example, 5 - 7 days) to obtain corneal endothelial-like cells formed by monolayer arrangement and tight connection of cells.

[0080] Preferably, in step (1), the urine-derived stem cells are the second, third, or fourth generation urine-derived stem cells obtained after passage. The urine-derived stem cells are cultured until more than 70% (for example, 80%, 90%, 100%) confluence.

[0081] Preferably, in step (1), the reagent for coating the multi-well plate is gelatin, Fibronectin, or Matrigel.

[0082] Preferably, in step (2), the differentiation medium is prepared by adding the TGF-β inhibitor SB431542, the ROCK inhibitor Y2763 and nicotinamide to the epithelial proliferation medium (ScienCell Research Laboratories, Epithelial Cell Medium (EpiCM), catalog number: 4101) to promote monolayer arrangement of cells and form tight connections.

[0083] Preferably, the concentration of SB431542 in the differentiation medium is 5-20 μM, the concentration of Y27632 is 5-15 μM, and the concentration of niacinamide is 5-15 mM. More preferably, the concentration of SB431542 is 10-20 μM, the concentration of Y27632 is 5-10 μM, and the concentration of niacinamide is 5-10 mM. More preferably, the concentration of SB431542 is 10 μM, the concentration of Y27632 is 10 μM, and the concentration of niacinamide is 10 mM. Even more preferably, the concentration of SB431542 is 10 μM, the concentration of Y27632 is 5 μM, and the concentration of niacinamide is 5 mM.

[0084] Cell identification: Immunofluorescence detection is performed on the cells obtained after induction of differentiation. If the corneal endothelial marker barrier function gene ZO1 is expressed, it is considered that the corneal endothelial-like cells derived from urine-derived stem cells have been successfully induced to differentiate and have acquired the function of corneal endothelial cells. In some embodiments of the present invention, the cells ZO-1 (barrier protein) obtained after induction of differentiation are expressed continuously and linearly, and the cells present a regular polygonal structure, confirming that corneal endothelial-like cells are obtained. In some embodiments of the present invention, the cells ZO-1 (barrier protein) obtained after induction of differentiation are expressed continuously and linearly, confirming that corneal endothelial-like cells are obtained. In other embodiments of the present invention, the cells ZO-1 (barrier protein) obtained after induction of differentiation are expressed continuously and linearly, and the liquid pump function protein ATP1A1 is positively expressed, and the cells present a regular polygonal structure, confirming that corneal endothelial-like replacement cells are obtained.

[0085] The corneal endothelial cell suspension is prepared by placing the corneal endothelial cell suspension in a DMEM low-glucose medium. Preferably, the ratio of corneal endothelial cell suspension to DMEM low-glucose medium is 1×10 5 ~1.5×10 6 Quantity: 200-300 μl, for example 3×10 5 5×10 5 1×10 6 Each 200 to 300 microliters. Specifically including:

[0086] 1) Enzymatic treatment: corneal endothelial-like cells cultured in culture medium were treated with cell digestion enzymes, and the enzymatic reaction was terminated with complete culture medium;

[0087] 2) Single cell collection: The corneal endothelial-like cells after enzymatic hydrolysis were collected into a centrifuge tube, and after centrifugation, the supernatant was discarded and the cell pellet was retained;

[0088] 3) Preparation of cell suspension: resuspend corneal endothelial cells in DMEM low-glucose basal medium, dissolve 1×10 5 ~1.5×10 6 cells to obtain a cell suspension.

[0089] Preferably, the DMEM basal medium is a low-glucose medium; preferably, one or more specific small molecule compounds are added to the DMEM basal medium, and the specific small molecule compounds include Y27632 and / or nicotinamide.

[0090] Example 1: Isolation and culture of USC

[0091] Let the donor drink a large amount of water about 1 hour before collection. Discard the first segment of urine, and collect 100 - 500 ml of midstream urine into a sterile container. Keep the samples refrigerated at 4°C for half an hour, 24 hours, and 48 hours respectively. Transfer the urine to a sterile 50 ml centrifuge tube, centrifuge at 400 g for 10 minutes at room temperature, aspirate the supernatant, and leave only 1 ml. Gently resuspend the precipitate in the remaining urine. Add 10 ml of washing buffer, centrifuge at 200 g for 10 minutes at room temperature, discard the supernatant, and leave about 0.2 ml and the precipitate. Add 1 ml of primary medium to resuspend the cell precipitate, and transfer it to a single well of a coated 12-well plate for culture. Figure 1 The cell morphologies after culturing for 11 days with the urine processed half an hour after collection, the cell morphologies after isolation and culture for 6 days with the urine stored for 1 day, and the cell morphologies after isolation and culture for 7 days with the urine stored for 2 days are respectively shown. These results suggest that the isolated urine can be stored at 4°C for at least 2 days without affecting the isolation and culture of urinary source cells.

[0092] Example 2: Differentiation and identification of corneal endothelial-like replacement cells derived from USC

[0093] To induce the acquisition of corneal endothelial-like replacement cells, the urinary source cells derived from adult volunteers and adolescent volunteers were inoculated into a coated six-well plate and cultured using a differentiation medium. 10 μM SB431542, 5 μM Y27632, and 5 mM nicotinamide were added to induce differentiation for 7 days to obtain regular and dense monolayer cells. Immunofluorescence staining was used to detect the expression of corneal endothelial marker functional genes, and corneal endothelial-like cells derived from urinary source stem cells expressing the barrier function gene ZO-1, or corneal endothelial-like cells derived from urinary source stem cells expressing both the barrier function gene ZO-1 and the fluid pump function gene ATP1A1 were obtained. As Figure 2 It is shown that the differentiated cells are positive for the barrier function gene ZO-1 and the fluid pump function gene ATP1A1.

[0094] As long as the barrier function gene ZO-1 is expressed, it can be considered to have the function of corneal endothelial cells.

[0095] Example 3: Verification of the corneal function repair by differentiating corneal endothelial-like replacement cells from urinary source cells of adult volunteers

[0096] Urine-derived cells were collected from adult male volunteers aged 38, and corneal endothelial-like replacement cells expressing the barrier function gene ZO1 were induced. At the same time, a rabbit model of corneal endothelial decompensation was constructed using a conventional method, which is applicable to simulate related diseases such as corneal endothelial injury, corneal endothelial lesions, corneal endothelial cell dysfunction, and corneal endothelial cell decompensation, as well as their clinical symptoms, including abnormal corneal thickness, decreased corneal transparency, corneal edema, vision decline or loss, dry eyes, eye pain, etc. The treatment methods and their therapeutic effects used in this animal model can be used to evaluate the therapeutic effects of the same or similar treatment methods when applied to human patients suffering from the above diseases or having the above symptoms.

[0097] The operations are as follows:

[0098] (1) The corneal endothelial cells within a range of Φ7.0 mm in the center of the right cornea of experimental New Zealand white rabbits were gently scraped off using a 20G soft silicone needle to avoid damaging the Descemet's membrane, and a rabbit model of corneal endothelial cell decompensation was established.

[0099] (2) The corneal endothelial-like replacement cells derived from 38-year-old volunteers were resuspended in a mixture of low-glucose DMEM medium and 10 μM Y-27632, and were administered into the eyes of rabbits through intracameral injection (preferably, 1×10 5 ~1×10 6 cells were injected into each eye). The experimental rabbits were kept in the right lateral position for 3 hours using a syringe (2 mL) to ensure that the cells adhered to the surface of the Descemet's membrane.

[0100] Transplantation function detection and efficacy evaluation:

[0101] (1) Corneal transparency detection: At 1 day, 1 week, 2 weeks, 4 weeks, and 8 weeks after transplantation, a slit lamp microscope was used to observe the degree of corneal opacity. As Figure 3 shown in the gross images of 2 animals transplanted with urine-derived cells from 38-year-old volunteers, the cornea regained corneal transparency within 1 week and maintained corneal transparency for at least 8 weeks.

[0102] (2) Corneal thickness measurement: At 8 weeks after transplantation, an optical coherence scanner was used to detect the central corneal thickness. As Figure 4 shown, compared with the edematous control corneas, the corneas transplanted with cells could maintain normal corneal thickness.

[0103] The results showed that the corneal endothelial-like replacement cells differentiated from urine-derived cells from adult males could eliminate corneal edema, restore normal corneal thickness, and maintain corneal transparency for a long time, achieving normal corneal visual function after transplantation.

[0104] Example 4: Verification of the repair of corneal function by corneal endothelial-like replacement cells differentiated from urine-derived cells of adolescent volunteers

[0105] Urine-derived cells were collected from 9-year-old male children, and corneal endothelial-like replacement cells that simultaneously expressed the barrier function gene ZO-1 and the fluid pump function gene ATP1A1 were induced. Meanwhile, a rabbit model of corneal endothelial decompensation was constructed using a conventional method. The corneal endothelial-like replacement cells derived from 9-year-old volunteers were resuspended and administered into the eyes of rabbits by intracameral injection. At 1 day, 1 week, 2 weeks, 4 weeks, and 8 weeks after transplantation, a slit lamp microscope was used to observe the degree of corneal opacity. As Figure 5 shown, the cornea became transparent within 1 week after transplantation of urine-derived cells from 9-year-old volunteers and remained transparent for at least 8 weeks.

[0106] The results suggest that corneal endothelial-like replacement cells differentiated from urine-derived cells of adolescent males can eliminate corneal edema, maintain corneal transparency in the long term, and achieve normal corneal visual function after transplantation.

[0107] In summary, using corneal endothelial-like cells differentiated from urine-derived cells to replace corneal endothelial cells can treat related diseases such as corneal endothelial injury, corneal endothelial lesions, corneal endothelial cell dysfunction, and corneal endothelial cell decompensation, as well as their clinical symptoms, including abnormal corneal thickness, decreased corneal transparency, corneal edema, decreased or loss of vision, dry eyes, eye pain, etc.

[0108] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand. Based on all the teachings that have been disclosed, various modifications and substitutions can be made to those details and dosages, and these changes are all within the protection scope of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. Use of urinary stem cells in the preparation of a drug or medical device for relieving or treating corneal endothelial injury, corneal endothelial disease, corneal endothelial cell dysfunction, or corneal endothelial cell decompensation.

2. Use of urinary stem cells in the preparation of a drug or medical device for relieving or treating abnormal corneal thickness, decreased corneal transparency, corneal edema, vision decline or loss, or dry and painful eyes.

3. The application according to claim 1 or 2, characterized in that, Urinary stem cells express ZO-1 protein, or simultaneously express ZO-1 protein and ATP1A1 protein.

4. The application according to claim 1 or 2, characterized in that, Cultivate urinary stem cells in a differentiation medium to promote the expression of ZO-1 protein in urinary stem cells, or the simultaneous expression of ZO-1 protein and ATP1A1 protein; the differentiation medium contains TGF-β inhibitor SB431542, ROCK inhibitor Y27632, and nicotinamide.

5. A method for inducing the differentiation of urinary stem cells into corneal endothelial-like cells, comprising: Promote the expression of ZO-1 protein in urinary stem cells to obtain corneal endothelial-like cells; Or, promote the expression of ZO-1 protein and ATP1A1 protein in urinary stem cells to obtain corneal endothelial-like cells.

6. The method according to claim 5, characterized in that, Cultivate urinary stem cells in a differentiation medium to promote the expression of ZO-1 protein, or ZO-1 protein and ATP1A1 protein, in urinary stem cells to obtain corneal endothelial-like cells; the differentiation medium is obtained by adding TGF-β inhibitor SB431542, ROCK inhibitor Y27632, and nicotinamide to an epithelial proliferation medium to promote monolayer cell arrangement and form tight junctions.

7. The method according to claim 5, characterized in that, It also includes a previous step: culturing primary cells of urinary stem cells with a proliferation medium; The proliferation medium is obtained by mixing an epithelial proliferation medium and a basal proliferation medium; wherein, the basal proliferation medium includes fetal bovine serum, GlutaMAX additive, non-essential amino acids, penicillin / streptomycin, fibroblast growth factor 2, platelet-derived growth factor, and epidermal growth factor.

8. Use of corneal endothelial-like cells obtained by the method of inducing differentiation of urinary stem cells into corneal endothelial-like cells according to any one of claims 5-7 in the preparation of a drug or medical device for relieving or treating corneal endothelial injury, corneal endothelial disease, corneal endothelial cell dysfunction, or corneal endothelial cell decompensation.

9. The application according to claim 8, characterized in that, The corneal endothelial-like cells are administered to the anterior chamber of the patient's eye in the form of a cell suspension.

10. A suspension of corneal endothelial-like cells, characterized in that, It contains corneal endothelial-like cells obtained by the method of inducing differentiation of urinary stem cells into corneal endothelial-like cells according to any one of claims 5-7.

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