Differentiation method of hiPSC-derived corneal endothelial cells

Abstract

A differentiation method of hiPSC-derived corneal endothelial cells comprises the following steps: (1) firstly, inoculating human induced pluripotent stem cells into a 1% Matrigel-coated pore plate, and culturing for 2-4 days; continuously culturing for 5-10 days by adopting a neural crest cell differentiation culture medium to obtain neural crest cells; (2) carrying out differentiation culture on the neural crest cells obtained after culture in the step (1) for 12-20 days by adopting a corneal endothelial cell differentiation culture medium to obtain corneal endothelial cells; the method has the advantages that the obtained corneal endothelial cells are high in purity, sufficient in quantity and safe and reliable in quality.

Description

Technical Field

[0001] The present application relates to the technical field of corneal endothelial cell differentiation, and specifically to a method for differentiating corneal endothelial cells derived from hiPSC (human induced pluripotent stem cells). Background Art

[0002] Corneal blindness refers to a condition in which visual function is significantly reduced or completely lost due to corneal disease or damage. The cornea is a transparent protective layer in front of the eyeball, which is essential for maintaining clear vision. It is not only responsible for blocking dust and microorganisms from entering the eye, but also refracts light and focuses it on the retina to achieve clear vision. The transparency and health of the cornea directly affect the clarity and sharpness of vision. Therefore, any disease or injury that affects the transparency and function of the cornea may lead to corneal blindness. The clinical manifestations of corneal blindness are diverse, mainly depending on the specific cause of corneal damage. Common causes include: congenital abnormalities: such as corneal dystrophy (Fuchs'endothelial dystrophy), keratoconus, etc. Infection: bacterial, viral, fungal and parasitic infections can cause keratitis, which can lead to corneal ulcers and perforations in severe cases. Trauma: direct physical injury, such as scratches, impacts or chemicals splashing into the eyes. Surgical complications: such as corneal endothelial cell damage after cataract surgery. Inflammatory diseases: Chronic inflammatory processes such as dry eyes, blepharitis, etc. Metabolic diseases: Systemic diseases such as diabetes may also affect corneal health.

[0003] Human induced pluripotent stem cells (hiPSC) are stem cells obtained by reprogramming mature human somatic cells. This technology was first realized by Japanese scientist Shinya Yamanaka and his team in 2006, and they won the 2012 Nobel Prize in Physiology or Medicine for this. hiPSC can be cultured on a large scale in vitro and has strong plasticity. It can be induced to differentiate into specific types of cells as needed, including neurons, cardiomyocytes, pancreatic islet cells, immune cells and hepatocytes, which makes them have great potential in regenerative medicine and disease model research.

[0005] Because of the scarcity of corneal donor sources, the differentiation of corneal endothelial cells (CECs) from human induced pluripotent stem cells can provide a cell source for clinical corneal endothelial regeneration. Due to the lack of understanding of the fate regulation mechanism of corneal endothelial cells, most of the early induction systems used conditioned medium for induction, and it was difficult to obtain functional corneal endothelial cells. With the continuous deepening of corneal development research, researchers began to first induce pluripotent stem cells to differentiate into neural crest cells (NCCs). The method of differentiating human pluripotent stem cells into neural crest cells is mainly to use ALK5 / TGF-β type I receptor kinase inhibitor SB431542 combined with bone morphogenetic protein (BMP) antagonist Noggin or Wnt pathway inhibitor SMAD signaling pathway regulators IWP2 and CHIR99021. The obtained neural crest cells are then co-cultured with corneal endothelial cells and lens epithelial cell culture medium to obtain corneal endothelial cells with morphology and expression of specific proteins. However, the corneal endothelial cells obtained using this method are of low purity, and it is difficult to maintain their correct morphology and function in vitro for a long time. In addition, a large amount of recombinant proteins is used, and the resulting safety issues are not conducive to clinical use. Summary of the invention

[0006] In view of the above-mentioned deficiencies in the prior art, the present application provides a method for differentiating corneal endothelial cells derived from hiPSCs with high purity, sufficient quantity, and safe and reliable quality.

[0007] In order to solve the above technical problems, the technical solution adopted in the present application is: a method for differentiating corneal endothelial cells derived from hiPSCs, the method comprising the following steps:

[0008] (1) First, human induced pluripotent stem cells (hiPSCs) were seeded in a 1% Matrigel (basement membrane matrix)-coated well plate and cultured for 2-4 days; then, neural crest cell (NCC) differentiation medium was used to continue culturing for 5-10 days to obtain neural crest cells;

[0009] (2) The neural crest cells obtained after culturing in step (1) are differentiated and cultured in a corneal endothelial cell differentiation medium for 12-20 days to obtain corneal endothelial cells.

[0010] Furthermore, the human induced pluripotent stem cells (hiPSCs) described in step (1) were seeded in 1% Matrigel (basement membrane matrix) coated in a 6-well plate and cultured for 3 days until the cell confluence reached 30-50%.

[0011] Furthermore, the neural crest cell differentiation medium described in step (1) comprises: 80-90% basal medium DMEM / F12 (Gibco), 10-20% knockout serum replacement (KSR, Gibco), 1-5 mM L-GlutaMAX (Gibco, L-alanyl-L-glutamine solution), 0.1-1 mM non-essential amino acids (MEM nonessential amino acids, NEAA, Gibco), 0.01-0.2 mM β-mercaptoethanol (β-mercaptoethanol, Gibco), 5-30 ng / mL basic fibroblast growth factor (bFGF, R&D Systems), 1-100 μM SB431542 (Sigma) and 0.1-30 μM retinoic acid (RA, Sigma).

[0012] Furthermore, the neural crest cell (NCC) differentiation medium described in step (1) is used to continue culturing for 7 days to obtain neural crest cells.

[0013] Furthermore, after culturing in neural crest cell (NCC) differentiation medium for 7 days in step (1), the cell confluence of the neural crest cells obtained reaches 70-85%.

[0014] Furthermore, in step (1), neural crest cells are obtained by differentiation culture, and the expression positive rate of neural crest cell marker proteins P75 and HNK1 detected by flow cytometry is greater than 90%.

[0015] Furthermore, the corneal endothelial cell (CEC) differentiation medium described in step (2) has the following specific components and contents: 99-99.9% basal medium DMEM / F12 (Gibco), 0.1-1% human serum albumin (HSA, Sigma), 1-50 ng / mL basic fibroblast growth factor (bFGF, R&D Systems), 0.1-60 μg / mL epidermal growth factor (epidermal growth factor, R&D Systems), 0.1-50 μM SB431542 (sigma), 1-300 nM SU5402 (selleck), 1-100 ng / ml DKK-2 (R&D Systems), 1-70 μM CKI-7 (sigma), 1-5 mM L-GlutaMAX (Gibco), 0.1-1 mM non-essential amino acids (NEAA, Gibco), 1-110 μg / mL ascorbic acid (Sigma), 50×B27 (Gibco) and 0.01-0.2 mM β-mercaptoethanol (β-mercaptoethanol, Sigma).

[0016] Furthermore, the culture process of step (2) is completed in a carbon dioxide incubator with a carbon dioxide concentration of 4-6% and a temperature of 37° C. The culture medium is changed once a day during the differentiation process.

[0017] Furthermore, the carbon dioxide concentration in step (2) is 5%.

[0018] Furthermore, in step (2), corneal endothelial cells are differentiated and cultured for 14 days using a corneal endothelial cell differentiation medium to obtain corneal endothelial cells.

[0019] Furthermore, the corneal endothelial cells obtained by differentiation culture in step (2) were detected by flow cytometry for corneal endothelial cell marker proteins CD166, ATP1A1, CD56 and ZO-1, and the positive rates were all greater than 95%.

[0020] Furthermore, the corneal endothelial cells differentiated and cultured in step (2) have a typical hexagonal morphology.

[0021] Furthermore, the corneal endothelial cells differentiated and cultured in step (2) can be continuously passaged for 8 times.

[0022] Furthermore, the corneal endothelial cells differentiated and cultured in step (2) have a cell population doubling time between 39 and 53 hours.

[0023] Furthermore, the corneal endothelial cells differentiated and cultured in step (2) were subjected to qPCR detection of corneal endothelial cell marker genes by extracting mRNA from the 1st, 2nd, 4th, 6th and 8th generations of corneal endothelial cells. Compared with primary human CEC (phCEC), the genes AQP1 and ZO-1 encoding the water channel protein (AQP1) and the tight junction protein (ZO-1) had higher expression levels; the gene expression levels of the 2nd, 4th and 6th generations gradually increased, and the gene expression level of the 8th generation was lower than that of the 6th generation. The gene ATP1A1 encoding the sodium-potassium ATPase (ATP1A1) in the differentiated corneal endothelial cells was lower than that of the primary human CEC.

[0024] Furthermore, the telomerase activity of the corneal endothelial cells differentiated and cultured in step (2) was negative.

[0025] Furthermore, the chromosome karyotype of the corneal endothelial cells differentiated and cultured in step (2) is mainly 2n=46,XX, and the chromosome structure has no deletion, duplication, or displacement.

[0026] Advantages and beneficial effects of this application:

[0027] 1. The method of the present application has sufficient cell sources: human induced pluripotent stem cells (hiPSCs) have the ability to proliferate and differentiate into any cell type in the body indefinitely, which means that the desired corneal endothelial cells can be expanded and differentiated in large quantities starting from a small number of primitive cells; the corneal endothelial cells obtained by the method of the present application can be propagated for up to 8 generations in vitro, and the cell mass can be expanded by more than 10 times in each generation, thereby effectively solving the problems of limited number of human corneal donors and limited in vitro expansion capacity of primary human corneal endothelial cells.

[0028] 2. The method of the present application has a precise cell differentiation effect. By using small molecule compounds (such as SU5402, CKI-7, DKK-2, etc.) to precisely control the differentiation conditions, it ensures that the resulting cells are highly functional and mature; this includes maintaining the hexagonal morphology and pump function of the cells, which are necessary for the normal function of corneal endothelial cells; and small molecule compounds are superior to recombinant proteins in terms of both safety and cost.

[0029] 3. The method of the present application can realize a standardized and controllable differentiation process, which makes the differentiation process more standardized and controllable, ensures the quality and consistency of cells, and can be mass-produced, thus solving the problem of the drugability of differentiated corneal endothelial cells and laying the foundation for clinical application.

[0030] 4. The method of the present application can induce the differentiation of neural crest cells with a purity of more than 90% from human induced pluripotent stem cells, and the neural crest cells can further differentiate into corneal endothelial cells with a purity of more than 95%, thus solving the problem of low in vitro differentiation purity.

[0031] 5. By extracting mRNA from the 1st, 2nd, 4th, 6th and 8th generations of corneal endothelial cells differentiated by the present application and performing qPCR detection of CEC marker genes, compared with primary human CECs, the genes AQP1 and ZO-1 encoding water channel proteins and tight junction proteins had higher expression levels, the gene expression levels of the 2nd, 4th and 6th generations gradually increased, and the gene expression level of the 8th generation decreased compared with the 6th generation. The gene ATP1A1 encoding sodium-potassium ATPase in the differentiated CECs was decreased compared with the primary human CECs; the telomerase activity of the differentiated corneal endothelial cells was negative; the chromosome karyotype was mainly 2n=46, and the chromosome structure had no deletion, duplication or displacement. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagram of corneal endothelial cell differentiation from human induced pluripotent stem cells.

[0033] Figure 2 Morphological diagram of corneal endothelial cells at different stages of differentiation from human induced pluripotent stem cells.

[0034] Figure 3 hiPSC-CEC can be serially passaged eight times.

[0035] Figure 4 The population doubling time of hiPSC-CECs at each generation.

[0036] Figure 5 The expression of hiPSC-CEC marker genes.

[0037] Figure 6 Shows the expression of hiPSC-NCC marker proteins.

[0038] Figure 7 The expression of hiPSC-CEC marker proteins.

[0039] Figure 8 The results of hiPSC-CEC telomerase activity detection (red is the TERT curve, blue is the internal reference curve).

[0040] Fig. 9 The karyotype of hiPSC-CEC 46 chromosomes is G-banded (2n=46, XX).

[0041] Fig.10 Expression of hiPSC-NCC marker proteins in Example 2.

[0042] Fig.11 Expression of hiPSC-CEC marker proteins in Example 2.

[0043] Fig.12Expression of hiPSC-NCC marker proteins in Example 3.

[0044] Fig.13 Expression of hiPSC-CEC marker proteins in Example 3. DETAILED DESCRIPTION

[0045] The following will combine the embodiments and drawings to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the embodiments described are only preferred embodiments, not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present application;

[0046] Example 1

[0047] (1) hiPSCs (human induced pluripotent stem cells) were seeded in a 6-well plate coated with 1% Matrigel (basement membrane matrix). After culturing for 3 days, the confluence reached about 40%, and then the medium was changed to neural crest cell (NCC) differentiation medium and continued to culture to obtain neural crest cells;

[0048] The neural crest cell culture medium included: 80% DMEM / F12 (Gibco), 20% knockout serum replacement (KSR, Gibco), 2mM L-GlutaMAX (Gibco), 0.1mM non-essential amino acids (NEAA, Gibco), 0.1mM β-mercaptoethanol (Gibco), 8ng / mL basic fibroblast growth factor (bFGF, R&D Systems), 10μM SB431542 (Sigma) and 1μM retinoic acid (RA, Sigma); after 7 days of culture, neural crest cells (hiPSC-NCC) were obtained, the cell confluence reached about 80%, and the expression positive rate of NCC marker proteins P75 and HNK1 detected by flow cytometry was greater than 90% (see the attached for specific test results). Figure 6 As shown), it was confirmed that the obtained cells were neural crest cells (hiPSC-NCC);

[0049] (2) At this time, the medium is replaced with corneal endothelial cell (CEC) differentiation medium to culture and differentiate neural crest cells to obtain corneal endothelial cells;

[0050] Corneal endothelial cell differentiation medium includes: 99.9% DMEM / F12 (Gibco), 0.1% human serum albumin (HSA, Sigma), 10ng / mL basic fibroblast growth factor (bFGF, R&D Systems), 2μg / mL b-epidermal growth factor (EGF, R&D Systems), 1μM SB431542 (sigma), 100nM SU5402 (selleck), 10ng / ml DKK-2 (R&D Systems), 5μM CKI-7 (sigma), 2mM L-GlutaMAX (Gibco), 0.1mM NEAA (Gibco), 50μg / mL ascorbic acid (AA, Sigma), 50×B27 (Gibco) and 0.01mM β-mercaptoethanol (Sigma); after 14 days of differentiation culture, corneal endothelial cells were obtained;

[0051] Attached Figure 1 Schematic diagram of the differentiation of corneal endothelial cells from induced pluripotent stem cells. The differentiation culture process allows NCC to differentiate into CEC; Figure 7 As shown, by using flow cytometry to detect CEC marker proteins CD166, ATP1A1, CD56 and ZO-1, the positive rates were all greater than 95%, which fully demonstrated that the method of the present application indeed obtained corneal endothelial cells; all culture processes were completed in a carbon dioxide incubator with a carbon dioxide concentration of 5% and a temperature of 37°C, and the culture medium was changed once a day during the differentiation process.

[0052] As attached Figure 2 The following are the typical cell morphologies at each stage of hiPSC-induced differentiation into CECs. Figure 2 It can be seen that the differentiated CEC morphology is a typical hexagonal shape; as shown in the attached Figure 3 As shown in Figure 2, CECs differentiated by this method can be passaged 8 times in a row; Figure 4 As shown, the cell population doubling time of CECs differentiated by this method is between 39 and 53 hours.

[0053] As attached Figure 5 As shown, by extracting mRNA from the 1st, 2nd, 4th, 6th and 8th generations of CECs obtained through differentiation culture, and performing qPCR detection of CEC marker genes, compared with primary human CECs (phCECs), the genes AQP1 and ZO-1 encoding the water channel protein (AQP1) and the tight junction protein (ZO-1) had higher expression levels, and the gene expression levels in the 2nd, 4th and 6th generations gradually increased, and the gene expression level in the 8th generation was lower than that in the 6th generation; the gene ATP1A1 encoding the sodium-potassium ATPase (ATP1A1) in the differentiated CECs was lower than that in primary human CECs.

[0054] As attached Figure 8 As shown in Figure 2, the telomerase activity of CECs (i.e., hiPSC-CECs) obtained through differentiation culture was negative; Fig. 9 As shown, the karyotype of hiPSC-CEC is mainly 2n=46, XX, female; the chromosome structure has no deletion, duplication, or translocation.

[0055] Example 2

[0056] (1) hiPSCs (human induced pluripotent stem cells) were seeded in a 6-well plate coated with 1% Matrigel (basement membrane matrix). After culturing for 2 days, the confluence reached about 30%, and the medium was changed to neural crest cell (NCC) differentiation medium to obtain neural crest cells;

[0057] The neural crest cell culture medium included: 80% DMEM / F12 (Gibco), 20% knockout serum replacement (KSR, Gibco), 1mM L-GlutaMAX (Gibco), 0.1mM non-essential amino acids (NEAA, Gibco), 0.01mM β-mercaptoethanol (Gibco), 5ng / mL basic fibroblast growth factor (bFGF, R&D Systems), 1μM SB431542 (Sigma) and 0.1μM retinoic acid (RA, Sigma); after 5 days of culture, the cell confluence reached about 70%, and the expression positive rate of NCC marker proteins P75 and HNK1 detected by flow cytometry was greater than 90% ( Fig.10 );

[0058] (2) At this time, the medium is replaced with corneal endothelial cell (CEC) differentiation medium to differentiate neural crest cells and obtain corneal endothelial cells;

[0059] Corneal endothelial cell differentiation medium included: 99% DMEM / F12 (Gibco), 1% human serum albumin (HSA, Sigma), 1 ng / mL basic fibroblast growth factor (bFGF, R&D Systems), 0.1 μg / mL epidermal growth factor (EGF, R&D Systems), 0.1 μM SB431542 (sigma), 1 nM SU5402 (selleck), 1 ng / ml DKK-2 (R&D Systems), 1 μM CKI-7 (sigma), 1 mM L-GlutaMAX (Gibco), 0.1 mM NEAA (Gibco), 1 μg / mL ascorbic acid (AA, Sigma), 50×B27 (Gibco) and 0.01 mM β-mercaptoethanol (Sigma); after 12 days of differentiation culture, the differentiation culture process allowed NCC to differentiate into CEC; as shown in the attached Fig.11 As shown, by using flow cytometry to detect CEC marker proteins CD166, ATP1A1, CD56 and ZO-1, the positive rates were all greater than 95%, indicating that the scheme of the present application can indeed obtain corneal endothelial cells; step (2) all culture processes were completed in a carbon dioxide incubator with a carbon dioxide concentration of 5% and a temperature of 37°C, and the culture medium was changed once a day during the differentiation process.

[0060] Example 3

[0061] (1) hiPSCs (human induced pluripotent stem cells) were seeded in a 6-well plate coated with 1% Matrigel (basement membrane matrix). After 4 days of culture, the confluence reached about 50%, and the medium was changed to neural crest cell (NCC) differentiation medium to obtain neural crest cells;

[0062] The neural crest cell culture medium included 90% DMEM / F12 (Gibco), 10% knockout serum replacement (KSR, Gibco), 5mM L-GlutaMAX (Gibco), 1mM non-essential amino acids (NEAA, Gibco), 0.2mM β-mercaptoethanol (Gibco), 30ng / mL basic fibroblast growth factor (bFGF, R&D Systems), 100μM SB431542 (Sigma) and 30μM retinoic acid (RA, Sigma); after 10 days of culture, the cell confluence reached about 85%, and the expression of NCC (hiPSC-NCC) marker proteins P75 and HNK1 was greater than 90% by flow cytometry (see the test results). Fig.12 As shown, it was confirmed that this step can obtain neural crest cells);

[0063] (2) After obtaining neural crest cells, the medium is replaced with corneal endothelial cell (CEC) differentiation medium and differentiation culture is performed to obtain corneal endothelial cells;

[0064] Corneal endothelial cell differentiation medium included: 99.9% DMEM / F12 (Gibco), 0.1% human serum albumin (HSA, Sigma), 50 ng / mL basic fibroblast growth factor (bFGF, R&D Systems), 60 μg / mL epidermal growth factor (EGF, R&D Systems), 50 μM SB431542 (sigma), 300 nM SU5402 (selleck), 100 ng / ml DKK-2 (R&D Systems), 70 μM CKI-7 (sigma), 5 mM L-GlutaMAX (Gibco), 1 mM NEAA (Gibco), 110 μg / mL ascorbic acid (AA, Sigma), 50×B27 (Gibco) and 0.2 mM β-mercaptoethanol (Sigma); after 20 days of differentiation culture, the differentiation culture process makes NCC differentiate into CEC; by using flow cytometry to detect CEC marker proteins CD166, ATP1A1, CD56 and ZO-1, the positive rate is greater than 95% (see the attached Fig.13 All the culture processes in this step were completed in a carbon dioxide incubator with a carbon dioxide concentration of 5% and a temperature of 37°C. The culture medium was changed once a day during the differentiation process.

[0065] The above embodiments and test results show that the corneal endothelial cells obtained by the differentiation and culture method of the present application have a purity of more than 95% and can be continuously passaged for 8 times. By extracting mRNA from the 1st, 2nd, 4th, 6th and 8th generations of neural crest cells for qPCR detection of CEC marker genes, compared with human primary CECs, the genes AQP1 and ZO-1 encoding water channel proteins and tight junction proteins have higher expression levels, the gene expression levels of the 2nd, 4th and 6th generations gradually increase, and the gene expression level of the 8th generation is lower than that of the 6th generation. The gene ATP1A1 encoding sodium-potassium ATPase in the differentiated CECs is lower than that of the human primary CECs; the telomerase activity of the differentiated corneal endothelial cells is negative; the chromosome karyotype is mainly 2n=46, and the chromosome structure has no deletion, duplication or displacement.

Claims

1. A method for differentiating hiPSC-derived corneal endothelial cells, characterized in that: The steps of the method include: (1) First, human induced pluripotent stem cells are seeded in a 1% Matrigel-coated well plate and cultured for 2-4 days; then, neural crest cell differentiation medium is used to continue culturing for 5-10 days to obtain neural crest cells; (2) The neural crest cells obtained after culturing in step (1) are differentiated and cultured in a corneal endothelial cell differentiation medium for 12-20 days to obtain corneal endothelial cells.

2. The hiPSC-derived corneal endothelial cell differentiation method according to claim 1, characterized in that: The human induced pluripotent stem cells described in step (1) were seeded in 6-well plates coated with 1% Matrigel and cultured for 3 days until the cell confluence reached 30-50%.

3. The hiPSC-derived corneal endothelial cell differentiation method according to claim 1, characterized in that: The neural crest cell differentiation medium described in step (1) has the following specific components and contents: 80-90% DMEM / F12, 10-20% knockout serum replacement, 1-5mM L-GlutaMAX, 0.1-1mM nonessential aminoacids, 0.01-0.2mM β-mercaptoethanol, 5-30ng / mL basic fibroblast growth factor, 1-100μM SB431542 and 0.1-30μM retinoic acid.

4. The method for differentiating hiPSC-derived corneal endothelial cells according to claim 3, characterized in that: The neural crest cell differentiation medium described in step (1) is used to continue culturing for 7 days to obtain neural crest cells.

5. The hiPSC-derived corneal endothelial cell differentiation method according to claim 3, characterized in that: After culturing in a neural crest cell differentiation medium for 7 days in step (1), the cell confluence reaches 70-85%.

6. The hiPSC-derived corneal endothelial cell differentiation method according to claim 3, characterized in that: The expression rate of marker proteins P75 and HNK1 of the neural crest cells obtained by differentiation culture in step (1) was detected by flow cytometry and was greater than 90%.

7. The hiPSC-derived corneal endothelial cell differentiation method according to claim 1, characterized in that: The corneal endothelial cell differentiation medium described in step (2) comprises the following specific components and contents: 99-99.9% DMEM / F12, 0.1-1% human serum albumin, 1-50 ng / mL basic fibroblast growth factor, 0.1-60 μg / mL epidermal growth factor, 0.1-50 μM SB431542, 1-300 nM SU5402, 1-100 ng / ml DKK-2, 1-70 μM CKI-7, 1-5 mM L-GlutaMAX, 0.1-1 mM NEAA, 1-110 μg / mL ascorbic acid, 50×B27 and 0.01-0.2 mM β-mercaptoethanol.

8. The hiPSC-derived corneal endothelial cell differentiation method according to claim 7, characterized in that: The culture process of step (2) is completed in a carbon dioxide incubator with a carbon dioxide concentration of 4-6% and a temperature of 37° C. The culture medium is changed once a day during the differentiation process.

9. The hiPSC-derived corneal endothelial cell differentiation method according to claim 7, characterized in that: Step (2) uses corneal endothelial cell differentiation medium to perform differentiation culture for 14 days to obtain corneal endothelial cells.

10. The method for differentiating hiPSC-derived corneal endothelial cells according to claim 7, characterized in that: Step (2) The corneal endothelial cells obtained by differentiation culture were detected by flow cytometry for the corneal endothelial cell marker proteins CD166, ATP1A1, CD56 and ZO-1, and the positive rates were all greater than 95%.

11. The method for differentiating hiPSC-derived corneal endothelial cells according to claim 7, characterized in that: The corneal endothelial cells differentiated and cultured in step (2) have a typical hexagonal morphology.

12. The hiPSC-derived corneal endothelial cell differentiation method according to claim 7, characterized in that: The corneal endothelial cells differentiated and cultured in step (2) can be continuously passaged for 8 times.

13. The hiPSC-derived corneal endothelial cell differentiation method according to claim 7, characterized in that: The corneal endothelial cells differentiated and cultured in step (2) have a cell population doubling time between 39 and 53 hours.

14. The method for differentiating hiPSC-derived corneal endothelial cells according to claim 7, characterized in that: The corneal endothelial cells cultured in step (2) are differentiated, and the mRNA of the 1st, 2nd, 4th, 6th and 8th generations of corneal endothelial cells are extracted for qPCR detection of corneal endothelial cell marker genes. Compared with primary human corneal endothelial cells, the genes AQP1 and ZO-1 encoding water channel proteins and tight junction proteins have higher expression levels; the gene expression levels of the 2nd, 4th and 6th generations gradually increase, and the gene expression level of the 8th generation is lower than that of the 6th generation. The gene ATP1A1 encoding sodium-potassium ATPase in the differentiated corneal endothelial cells is lower than that of primary human corneal endothelial cells.

15. The method for differentiating hiPSC-derived corneal endothelial cells according to claim 7, characterized in that: The telomerase activity of the corneal endothelial cells differentiated and cultured in step (2) was negative.

16. The hiPSC-derived corneal endothelial cell differentiation method according to claim 7, characterized in that: The chromosome karyotype of the corneal endothelial cells differentiated and cultured in step (2) is mainly 2n=46, and the chromosome structure has no deletion, duplication or displacement.

Images