CN110468094B

CN110468094B - Corneal epithelial cells and corneal scaffold, and preparation method and application thereof

Info

Publication number: CN110468094B
Application number: CN201810441589.7A
Authority: CN
Inventors: 徐仁和; 杨娟; 郑德锦; 朴政友
Original assignee: University of Macau
Current assignee: University of Macau
Priority date: 2018-05-10
Filing date: 2018-05-10
Publication date: 2021-04-13
Anticipated expiration: 2038-05-10
Also published as: CN110468094A

Abstract

The invention discloses corneal epithelial cells and a corneal stent as well as a preparation method and application thereof, and relates to the field of corneal disease medicaments. The invention discloses a preparation method of corneal epithelial cells, which is used for inoculating human embryonic stem cells into an E6 culture medium for culture so as to differentiate the human embryonic stem cells into the corneal epithelial cells.

Description

Corneal epithelial cells and corneal scaffold, and preparation method and application thereof

Technical Field

The invention relates to the field of corneal disease drugs, in particular to corneal epithelial cells and a corneal stent, and a preparation method and application thereof.

Background

Corneal disease affects millions of people worldwide. For example, Limbal Stem Cell Deficiency (LSCD) is characterized by the loss of limbal stem cells, which is critical for the regeneration of Corneal Epithelial Cells (CECs). Differentiation of Limbal Stem Cells (LSCs) into CECs by intermediate progenitors^[1]. Thus, loss or deficiency of LSC will lead to corneal keratoma, chronic inflammation and potential vision loss following corneal injury^[2]. Although restoration of the limbal microenvironment may have some effect on the early treatment of LSCD^[3]Corneal and LSC transplants, however, have so far been the most effectiveMethod (2)^[4]. However, transplantation of autologous LSCs may damage the healthy cornea of contralateral donor eyes, and allografting is limited by donor shortages or immune incompatibility^[5,6]. Therefore, human pluripotent stem cells (hPSC), including human embryonic stem cells (hESC), are utilized^[7]And Inducing Pluripotent Stem Cells (iPSCs) [8,9 ]]The interest in the resulting CECs in seeking treatment regimens is rising.

Many methods have been published for differentiation of pluripotent stem cells into CEC. For example, limbal fibroblasts or LSCs^[10-13]The conditioned medium of (4), or PA6 stromal cells as feeder cells^[14]Or on an extracellular matrix-rich surface^[15]And synthetic corneal epithelial arcual membrane^[16]. Recently, differentiation of CEC has been reported using defined media to mimic in vivo signaling (e.g., TGF, WNT and FGF signaling pathways)^[17-20]。

In addition, engineered biomaterials such as modified human amniotic membrane, collagen, fibrin, poly (epsilon-caprolactone), silk fibroin-chitosan and chitosan-gelatin have been investigated as corneal cell carriers^[20-26]. However, they do not generally meet clinical criteria for clarity, mechanical strength, biocompatibility and biosafety. In contrast, Decellularized Cornea (DC) has become a relatively safe and sustainable scaffold for cell delivery because it not only maintains corneal structure, strength and optical properties, but also retains native stromal ultrastructure. The recellularization of rabbit DC corneal cells can establish a rabbit corneal equivalent^[27]. Similar results were obtained with porcine DCs harvested using human corneal cells^[28]. In addition, at 7% O₂Serum-free medium with keratinocytes 1: hESC-derived CEC in 1 Mixed DMEM/F12 seeded on decellularized porcine corneal stroma, followed by air-lift culture to induce epithelial delamination^[29]。

Traditionally, the cornea has been considered one of the unique sites of immunity in vivo. However, immune rejection remains a major cause of corneal allograft failure, including angiogenesis, inflammation and corneal graft failure^[31]. Thus, differences in polymorphic Human Leukocyte Antigen (HLA) molecules between donors and alloreceptors can elicit immune responses following corneal transplantation^[32]. HLA-matched donors are difficult to find and require long-term administration of immunosuppressive agents. To solve these problems, scientists have performed genetic manipulations to generate so-called "universal" hPSCs without HLAI or class II molecules^[33]. Immune cells of recipient against allogeneic cells not having surface expression of HLA molecules are not visible^[34]。

Corneal damage and limbal stem cell deficiency impair vision in millions of people worldwide. Although allogeneic corneal transplantation is one of the major treatment options, the shortage of donated corneas and potential immune rejection remain major obstacles.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a preparation method of corneal epithelial cells.

Another object of the present invention is to provide a corneal epithelial cell.

It is another object of the present invention to provide a method of preparing a corneal scaffold.

Another object of the present invention is to provide a corneal stent.

The invention also aims to provide application of the corneal epithelial cells and the corneal scaffold.

It is another object of the present invention to provide a medicament for treating corneal diseases.

The invention is realized by the following steps:

a method of preparing corneal epithelial cells, comprising: the human embryonic stem cells were inoculated into E6 medium for culture.

In the present study, the present examples used a defined and albumin-free E6 medium to classify human embryonic stem cells (hescs) as Corneal Epithelial Cells (CECs). The gene expression profile of hescs differentiation into CECs mimics the developmental process of CECs. Furthermore, the present examples seeded mouse acellular corneas (DCs) with hESC-derived CECs, which formed a multi-layered epithelium on the surface of the DC, with sustained transparency and restoration of tight junctions.

Further, in some embodiments of the present invention, the human embryonic stem cell is a B2M knockout human embryonic stem cell.

Embodiments of the invention generate low immunogenic CECs by disrupting the non-polymorphic gene β -2-microglobulin (B2M) in hescs using CRISPR/Cas 9. B2M^-/-CECs lose surface expression of HLA class I after injection into the anterior chamber of immunocompetent mice and result in less T cell infiltration than wild-type (WT) CECs.

Thus, it was demonstrated that corneal epithelial cells differentiated from the B2M knock-out human embryonic stem cells have lower immunogenicity, and the corneal epithelial cells can be used for reducing the barrier to immune rejection in allogeneic corneal transplantation.

A corneal epithelial cell prepared by the preparation method.

A method of making a corneal scaffold, comprising: the corneal epithelial cells described above were seeded onto decellularized corneas.

A method for preparing a corneal scaffold, which comprises the preparation method of the corneal epithelial cells.

A corneal scaffold prepared by the method for preparing the corneal scaffold.

The corneal epithelial cells or the corneal scaffold can be used for preparing medicines for treating or preventing or repairing corneal diseases.

Further, in some embodiments of the invention, the corneal disease is a corneal injury disease or a limbal stem cell deficiency disease.

A medicament for treating corneal diseases, which comprises the corneal epithelial cells or the corneal scaffold.

The invention has the following beneficial effects:

the preparation method of the corneal epithelial cells, provided by the invention, can continuously provide the corneal epithelial cells with rapid proliferation capacity by inoculating the human embryonic stem cells into an E6 culture medium for culture, can be used for preparing corneal scaffolds or medicines for treating corneal diseases.

The corneal epithelial cells provided by the invention have rapid proliferation capacity and lower immunogenicity, and can be used for preparing corneal scaffolds or medicines for treating corneal diseases and reducing the immunological rejection obstacle in allogeneic corneal transplantation.

The method for preparing the corneal scaffold can prepare the corneal scaffold with low immunogenicity, continuous cell transparency, tight connection and the like by applying the corneal epithelial cells to the acellular cornea.

The corneal scaffold provided by the invention has the characteristics of low immunogenicity, continuous cell transparency, tight connection and the like, can be used for preparing a medicine for treating or preventing or repairing corneal diseases, and provides a new treatment means or thought for corneal diseases such as corneal injury diseases or limbal stem cell deficiency diseases.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIGS. 1-A-1-E show the results of hES-CEC identification and differentiation efficiency assays: FIG. 1-A is a flow chart of hESC differentiation into CEC; FIG. 1-B shows cell morphology during differentiation for Day15 and Day 40; FIG. 1-C shows the expression of marker proteins PAX6, TP63, KRT19, KRT15, KRT3 and KRT12 during CEC development, with the length of the scale: 50 μm; FIG. 1-D shows the results of real-time quantitative PCR detection of corneal epithelial development-related gene expression; FIGS. 1-E are flow charts of the results of the expression analysis of NANOG, PAX6, TP63, KRT15 during differentiation;

FIGS. 2-A-2-I show the expression profiles of the relevant genes during differentiation of hESCs into CEC (in the figures: P <0.05, P <0.01, P <0.001, ns: no significance); FIG. 2-A is an expression profile of pluripotency marker genes (POU5F1, NANOG, SOX2, DNMT38, GA8R83, GDF 83, TDGF 83), FIG. 2-B is an expression profile of pro-basal ectodermal marker genes (DLX 83, EYA 83, HES 83, OTX 83, SIX 83), FIG. 2-C is an expression profile of ocular ectodermal marker genes (KLF 83, PAX 83, KLF 83, GJA 83), FIG. 2-D is an expression profile of limbal marker genes (ABCG 83, ID 83, KRT 83, MSX 83, and 83 TP), FIG. 2-E is an embryonic stage marker gene (KRT 83, epithelial marker genes (PAT 83, PAG 83, PAITGA 83, ABITG 83, GAITG 83, ABITG 83, and G83, ABITG 83, and G83, NES, NEUROD, MSL1, PAX6, GFAP, ROR2, NCAM1, SOX2, FABP7), fig. 2-H is the expression profile of crystal precursor cell marker genes (PAX6, SIX3, PITX3, MSX2, FOXE3, PROX1, SOX1, MAF, CRYAA, CRYAB, MAB21L1, MAB21L2), fig. 2-I is the expression profile of retinal precursor cell marker genes (LHX2, PAX6, RAX, SIX3, SIX6, VSX2, HES1, HES5, moth 1, SOX 2);

FIGS. 3-A to 3-D show the results of hES-CEC proliferation assay: FIG. 3-A is a growth curve of Incucyte analysis hES-CEC; FIGS. 3-B1-B3 show the results of cell cycle analysis of hES-CEC after PI staining (H9 hESC in FIG. 3-B1, CEC differentiated from H9hESC strain in FIG. 3-B2, CEC differentiated from CT3hESC strain in FIG. 3-B3); FIG. 3-C shows the results of KRT15 and KI67 immunostaining of hES-CEC; FIG. 3-D is a quantitative analysis of KI67/KRT15 double positive cells;

FIGS. 4-A to 4-D show the results of the recellularization-related assays of mouse decellularized corneas: FIG. 4-A is a flow chart of corneal recellularization; FIG. 4-B shows the results of hematoxylin eosin staining (multiple layers of hES-CEC cell layers formed on the surface of DC, demonstrating successful DC recellularization); FIG. 4-C is a microscopic examination of hES-CEC transparency by observing the letter A of the hES-CEC recellularized DC overlay; FIG. 4-D is the transparency of hES-CEC recellularized DCs at different wavelengths of light;

FIGS. 5-A-5-D show the results of measurements relating to recellularization of the cornea of decellularized mice by hES-CEC and MSC: FIG. 5-A is a comparison of the expression of the Crystallin gene in hES-CEC and MSC; FIG. 5-B is a graph of the detection of expression of PAX6 and KRT3 by recellularized corneal tissue; FIG. 5-C section immunofluorescence assay results for expression of GFP, PAX6, KRT15, KRT12 in ENVYhES-CEC;

FIG. 6-A to FIG. 6-D are B2M^-/-Results of relevant assays for hESC differentiation to CEC: FIG. 6-A is a sequencing test result of knocking out B2M gene in H1hESC by CRIPR/Cas9 and knocking out result; FIG. 6-B is a flow chart showing the analysis of B2M and HLA-A/B/C in wild type and B2M^-/-Results of expression in hescs; FIG. 6-C shows immunofluorescence assays for TP63, PAX6, KRT19, KRT15 at B2M^-/-Expression results in Hes-CEC;

FIGS. 7-A and 7-B show wild type and B2M^-/-hES-CEC was assayed for expression in these cells by flow analysis of B2M and HLA-A/B/C following stimulation with IFN-. gamma.TNF-. alpha.and LPS, respectively: FIG. 7-A, top row, shows the results of flow assay of B2M expression in wild type hES-CEC after stimulation with IFN-. gamma.TNF-. alpha.and LPS, respectively; lower behavior B2M^-/-hES-CEC was flow-analyzed for B2M expression in these cells after stimulation with IFN-. gamma.TNF-. alpha.and LPS, respectively; FIG. 7-B, top row, shows the results of flow assay of HLA-A/B/C expression in wild type hES-CEC after stimulation with IFN-. gamma.TNF-. alpha.and LPS, respectively; lower behavior B2M^-/-After being stimulated by IFN-gamma, TNF-alpha and LPS respectively, hES-CEC analyzes the expression detection result of HLA-A/B/C in the cells by flow analysis;

FIG. 8 shows B2M^-/-CEC and wild type CEC injection into mouse limbal region of the eye after anterior chamber of eye CD3⁺And (5) detecting the cell number.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

hESC induced differentiation CEC in defined and albumin-free E6 medium

H9 hescs routinely cultured in E8 differentiated CECs in E6 according to the flow chart (fig. 1A). By day15 differentiation, cells developed mostly into epithelial morphology, some of which had a typical CEC polygonal morphology (fig. 1B). This example passaged cells on day40 and immunostained cells within one week after passage. Most cells were positive for CEC progenitor markers TP63 and PAX6, another CEC progenitor keratin-19 (KRT19) and the basal CEC marker KRT 15. After 20 days of culture in E6 medium, many cells were also positive for the two terminally differentiated CEC markers KRT3 and KRT12 (fig. 1C).

Gene expression was analyzed by RT-qPCR, and the expression lineage specific marker genes were studied every two weeks of collected differentiated hescs. Expression of the pluripotency marker OCT4 was down-regulated during differentiation, whereas expression of the CEC markers PAX6, TP63, KRT15, KRT12 and KRT3 and CRYAA, CRYAB and ALDH1a1 described above was up-regulated and time-dependent (fig. 1D).

Flow cytometric analysis showed that 81% and 92.7% of the cells were PAX6, respectively, by differentiation at day15⁺And TP63⁺Indicating efficient differentiation of hescs into early epithelial CECs (fig. 1E). At day40, 65.2% of the cells became KRT15⁺And up to 96% of the cells become NANOG-. Interestingly, selective digestion of epithelial cells with EDTA/DPBS resulted in KRT15⁺The proportion of cells increased up to 90% (FIG. 1E). These data indicate that this example almost uniformly obtained KRT15 by simple passage of CEC in EDTA/DPBS⁺A cell.

Example 2

Gene expression profiling of hESC differentiation into CEC

As expected, the expression of pluripotency genes decreased significantly at week 2 of differentiation and remained low. This example detected increased expression of surface ectodermal markers at

weeks

2,4, and LSCs and embryonic CEC markers at weeks 6 and 8, respectively. At the same time, expression of neural and retinal markers increased at week 2, peaked at week 4 and then gradually declined, while expression of lens progenitor markers continued to increase during all 8 weeks of differentiation. However, expression of adult CEC markers remains low in differentiated cells. These data indicate that expression of eye developmental genes, particularly genes associated with epidermal ectoderm and embryonic CECs, is induced in E6 differentiated cells, and that the multistep differentiation process somehow recapitulates the embryonic process of CEC development (fig. 2A-2I).

Example 3

Proliferative capacity of hESC-derived CEC

LSCs differentiate into CEC progenitors, migrate to the basal layer of the cornea, and finally differentiate into CECs during corneal regeneration and homeostasis^[42]. One of the characteristics of CEC progenitors is their ability to proliferate rapidly. Thus, this example analyzes whether hESC-derived CECs are proliferative. First, this example monitors the proliferative capacity of CECs differentiated from H9 and CT3hESC, respectively. Although neither CEC line proliferated as rapidly as the parental hescs, CECs proliferated at a relatively high rate throughout the 3-day observation period (fig. 3A). Second, cell cycle assays indicated that about 40% CEC were in S and G2/M phases (FIGS. 3-B1-3-B3). Third, approximately half of CECs from H9 or CT3 have KRT15⁺The cells are Ki67⁺(FIGS. 3C and 3D). All these data indicate the proliferative nature of the cells.

Example 4

Recellularization of acellular mouse cornea using hESC-derived CECs

To test whether newly generated CEC was normal, this example decided to plant cells on mouse DCs, rather than artificial matrices used by others^[21-25]. This example first isolated the cornea from a mouse and decellularized it with 0.5M NaOH to produce DCs as corneal scaffolds. This example then compares GFP⁺CECs were seeded onto the surface of DCs for regeneration (fig. 4A), which formed multiple layers of epithelium in DCs (fig. 4-B). Mice DC inoculated with CEC remained clear, with the letter a below being clearly visible, similar to the letter a below DC that had been controlled for non-inoculated cells. However, the letter a appears blurry under DCs of Mesenchymal Stem Cells (MSCs) differentiated from the same hESC (fig. 4-C).

Consistently, transmission of light at various wavelengths of 400 to 800nm for CEC-seeded DCs was similar to that through cell-free DCs, but 2-3 times higher than MSC-seeded DCs (fig. 4-D). The RT-qPCR results also showed that expression of the two clear related genes CRYAA and ALDH1a1 was much higher in CEC-inoculated DCs than control MSCs (fig. 5A). Furthermore, CECs inoculated on DCs remained positive for KRT12 and the tight junction marker ZO-1 (FIG. 4-C) as well as PAX6 and KRT3 (FIG. 5-B) by immunostaining analysis. Tightly connected surround CEC as a barrier to isolate the cornea from the external environment^[43]. Vertical sections of DC re-epithelialized hESC-CEC also showed that the top CEC layer in DC remained positive for PAX6, KRT15, KRT12 and VINCULIN (fig. 5-C). Taken together, these data indicate that CEC is characterized by corneal cell transparency and tight junctions after DC inoculation, and retains CEC marker proteins.

Example 5

Low immunogenicity B2M^-/-hESC induced differentiation CEC

The eye is generally considered to be an immunologically privileged organ^[44]Thus resulting in a high success rate of corneal transplantation^[45]. However, immune rejection remains a serious threat, particularly in high-risk recipients with ocular inflammation and abnormal angiogenesis^[46]. In addition, HLAI-like matching significantly increases survival of allogeneic corneal grafts^[32,47]. To minimize potential immune rejection due to HLA mismatch, this example generated a universal hESC line by knocking out B2M. This example knocks out B2M in both alleles in H1hESC using CRISPR/Cas9 technology and confirmed the B2M mutation by sanger sequencing of the target allele (fig. 6-a). Flow cytometric analysis confirmed that B2M and HLA-A/B/C were derived from B2M^-/-Cell surface deletion of hescs, wild-type (WT) hescs expressing B2M and HLA-a/B/C (fig. 6-B).The B2M knockout did not affect hESC pluripotency or ability to differentiate into CECs (fig. 6-C).

Example 6

B2M^-/-In vitro and in vivo assay for hESC-induced CECs

To confirm B2M^-/-Low immunogenicity of CEC, this example WT and B2M treated with 100ng/ml recombinant human interferon-gamma (IFN γ), 100ng/ml tumor necrosis factor- (TNF)^-/-CEC, or 100ng/ml Lipopolysaccharide (LPS), respectively, were cultured for 48 hours. After stimulation, the cells were analyzed by flow cytometry for their effect on the expression of B2M and HLA-A/B/C on the cell surface. WT CEC expressed B2M and HLA-A/B/C even in the absence of inflammatory factors, and the level of expression increased slightly upon stimulation, as evidenced by an increase in fluorescence intensity (FIGS. 7-A and 7-B).

Previous studies have shown that microinjection of syngeneic islets into the anterior chamber of a mouse eye does not cause T cell infiltration, however, the injection of allogeneic islets results in alloroejection^[49]. To test B2M^-/-Whether CEC is less immunogenic than WT control in vivo, this example injected cells into the anterior chamber of murine eyes, inducing T lymphocytes (CD 3)⁺) Penetrate into the eye. 4 days after injection, this example observed a B2M acceptance compared to WT CEC injected eyes^-/-CD3 of the limbic region of the mouse eye injected with CEC⁺The number of cells was significantly reduced (fig. 8). Taken together, these data show that B2M^-/-CECs are less immunogenic both in vitro and in vivo.

It was previously reported that inhibition of WNT and TGF signaling and activation of FGF signaling promote CEC derivatization of human ipscs in human serum albumin defined media^[17]. In this study, this example has shown that basal medium E6 is sufficient to induce differentiation of hescs into CECs. While the exact mechanism by which E6 differentiates hescs into CECs is currently unclear, the time course gene expression data for differentiated cells suggest that down-regulation of WNT/b-catenin signaling between

weeks

2 and 4 of differentiation may be important for differentiation of CECs. Indeed, previous studies have shown that the inhibition of WNT/-catenin and TGF signaling is the development of ocular surface ectoderm, CEC specification and corneal epitheliumIs necessary for formation of^[39,40,50]. Given the interaction between IGF-1 and WNT signaling, the presence of insulin in E6 may also act to modulate WNT/β -catenin signaling through GST3, as shown by head formation and eye structure development in hESC^[41]。

The method of the embodiment of the invention realizes that KRT15 with the generation rate of 90 percent is generated by a two-step method⁺A cell. Expression of the terminally differentiated CEC markers KRT3 and KRT12 by day40 of differentiation after expression of TP63 (92%) and PAX6 (81%) before day15 of differentiation^[17]. Cell proliferation, cell cycle assays and immunostaining of Ki67 showed highly proliferative properties of the cells, indicating that CECs differentiated from hescs in E6 are likely to be more like CEC progenitors.

DCs inoculated with hESC-derived CECs may help solve the problem of corneal donor deficiency. Although many alternative approaches have been developed to date, such as corneal prostheses, xenografts, tissue engineered constructs, DCs and DCs harvested with human corneal cells. However, corneal prostheses require rigorous maintenance of the device, often resulting in wound leakage, tissue melting and glaucoma^[51-54]. The xenograft has higher rejection rate^[55]And cross-species transmission of animal viruses [56,57]. Tissue engineering constructs lack the properties of corneal structure and various corneal cell types. The DC does not cause any immune or inflammatory reaction in vivo^[27,58]. Porcine DCs transplanted onto damaged rabbit corneas can be reimplanted by host CEC^[59]. However, in patients with complete LSCD, DCs cannot be properly epithelialized. Therefore, xenogeneic DCs seeded with hESC-derived CEC cells may be a viable solution.

Here, the present examples have demonstrated that CEC efficiently epithelializes mouse DCs, and demonstrated expression of CEC markers and formation of tight junction barriers. In addition, transparency, one of the most important features of the cornea, was clearly demonstrated on extracellular DCs. DCs from larger animals, such as pigs, then expanded with hESC-derived CECs, may ultimately be suitable for transplantation in LSCD patients. Furthermore, the albumin-free nature of E6 completely eliminates any possible remaining concerns of xenogenic or allogeneic proteins and lipids [35,60]

Another major problem with cell transplantation is immune rejection. Although the eye is considered to be a relatively immune specific organ, the success rate of allogeneic corneal transplantation exceeds 90% in low risk patients, but immune rejection in high risk patients remains a major cause of graft failure. HLA antigens present on allogeneic corneas can elicit an immune response in the recipient's eye, leading to epithelial, chronic stromal, hyperacute and endothelial rejection^[6,61]. This study was first conducted by examining B2M^-/-hescs differentiate into CECs to address this problem. As expected, B2M even after stimulation with the inflammatory factors IFN, TNF and LPS^-/-hescs and CECs derived therefrom lack molecules of the class HLAI on their cell surface. Nevertheless, in the presence or absence of immunostimulatory molecules, at B2M^-/-Expression of neither HLAI nor class II molecules in hESC-derived CECs indicates that the cells have lower immune rejection potential than WT controls. In fact, B2M was injected by anterior chamber injection^-/-CEC, a much reduced lymphocyte infiltration in the limbal region of the mouse compared to WT CEC-injected mice, indicating B2M^-/-Advantages of CEC.

In summary, the present example has established a simple and effective protocol to induce differentiation of hESCs into CECs, vaccinating DCs to epithelialize them, and demonstrated the expression from B2M^-/-The immune response of hESC differentiated CEC was reduced. Some important issues remain to be solved. First, testing CEC cell expansion is critical to the efficacy of corneal epithelial damage and LSCD animal models. Second, the data of the present examples show that hESC-derived CECs are more mature CECs (KRT 3)⁺/KRT12⁺) Contains more progenitor cells (KRT 15)⁺) CEC. Therefore, it is important to further determine the nature of the cells prior to transplantation. Establishing hESC lines with reporter genes may help isolate pure CEC populations for therapeutic applications, as well as eliminate any residual hESC to avoid teratoma formation. Finally, B2M^-/-While CECs reduce T-cell and B-cell mediated immune rejection, natural killer cell-mediated cytotoxicity may be encountered, particularly in xenogeneic animal models. At this pointIn this case, transgenic expression of HLA-E fused to B2M could solve the reported problems^[34]. Solving these remaining problems will help B2M^-/-Use of hESC-derived CECs for direct transplantation, either directly or via DC, to patients with corneal epithelial damage or LSCD.

Examples 1-6 relate to the following relevant detection methods:

hescs cultured in E6 differentiated towards CECs.

hESCs were digested into small clumps with EDTA/DPBS and inoculated into new six-well plates at a density of about 10% to 15%, and the medium was changed to E6 medium (insulin, selenium, transferrin, L-ascorbic acid, NaHCO) the next day after inoculation₃DMEM/F12). Each well contained 2-ml of E6, and the solution was changed daily. The expression of the related marker protein is detected after the cells are differentiated for 40 days and passaged.

Microarray Gene expression analysis

mRNA (Life technologies) was extracted from H9 undifferentiated and differentiated to various stages (2,4,6,8 weeks) using Purlink RNA kit for cDNA library synthesis and processing, and mRNA expression was further detected using the HumanHT-12v4 expression chip. Analysis used the Beads Studio software (Illumina), Cluster, TreeView and Excel software. First, embodiments of the present invention use a quantile normalization method to normalize each data point relative to the sampled data in the other arrays. The normalized data set was filtered for CECs to remove data points with a detected P value of <0.95 in all samples. The data sets were further normalized for gene analysis by calculating fold changes in the intensity of each probe, compared to the probe intensity of each gene in undifferentiated H9 cells. The microarray dataset has stored a national universal library of gene expression. The center for biotech information in the united states (GSE 107287). 3. Flow cytometry analysis

Adherent cultured cells were broken down into single cells with 1 × TrypLE and treated with 4% PFA for 30 min. At room temperature. The fixed cells were incubated in 0.1% Triton X-100 for 10 min, blocked in 5% BSA for 1h, and incubated with antibodies NANOG (cell Signaling), PAX6(Invitrogen), TP63(Boster) or KRT15(Santa Cruz), HLA-A/B/C (ebioscience), B2M (Sigma) at a dilution ratio of 1: 100. The cells are then washed in cold PBS for 15 minutes and incubated with secondary antibodies, such as goat anti-mouse, goat anti-rabbit or donkey anti-goat igg (invitrogen), for 30 minutes, and control cells are incubated with secondary antibodies only. Cells were washed 3 to 4 times with cold PBS and analyzed on an Accuri C6 flow cytometer.

4. Immunofluorescence assay

Cells were fixed with 4% PFA for 20 min. And permeabilized with 0.1% Triton X-100 for 10 min. Subsequently, the cells were contacted with primary anti-PAX 6(Thermo Fisher) TP63, Ki67(Boster), KRT19, KRT15, KRT12 or KRT3(Santa Cruz). Nuclei were counterstained with 4, 6-diamidino-2-phenylindole (DAPI). Fluorescence images were taken using a carlszeiss Axio Observer. Examination of expression markers for CECs on re-cellularized DCs, treated in the same manner as the above eyeballs and incubated with antibodies against PAX6, KRT12, KRT15 and vinculin (abcam), followed by counterstaining with DAPI of secondary antibodies against the isotype of the primary antibody. Carr photographed a bright scene and a fluorescence image zeiss Axio observer. All primary and secondary antibodies above were used at dilutions of 1: 200 and 1:1,000.

5.IncuCyte^TMLive cell imaging

In IncuCyte^TMCell proliferation was measured on a live cell imaging system (Essenbioscience). Cells were seeded at 105 cells per well in Matrigel coated 6-well plates and images were taken at 9 locations every 2 hours over 4 days. Proliferation cell confluence reached around 16% as determined by calculating the total area occupied by the cells (% confluence).

Decellularization and recellularization of DCs

The corneas of BALB/c mice were separated, treated with 0.5M sodium hydroxide for 10 minutes, washed thoroughly in three corneas/well of a 96-well plate, and then subjected to ultraviolet irradiation for 30 minutes to obtain DCs. Next, three DCs were recellularized in one well (96-well plate) and seeded at 2.5X 10⁵CECs derived from GFP + Envy hESC or MSCs derived from CT2 and Envy hESC differentiation.

Reference documents:

[1]S.C.Tseng,Concept and application of limbal stem cells,Eye 3(Pt 2)(1989)141-57.

[2]M.H.Strungaru,D.Mah,C.C.Chan,Focal limbal stem cell deficiency in Turner syndrome:report of two patients and review of the literature,Cornea 33(2)(2014)207-9.

[3]B.Y.Kim,K.M.Riaz,P.Bakhtiari,C.C.Chan,J.D.Welder,E.J.Holland,S.Basti,A.R.Djalilian,Medically reversible limbal stem cell disease:clinical features and management strategies,Ophthalmology 121(10)(2014)2053-8.

[4]S.Kolli,S.Ahmad,M.Lako,F.Figueiredo,Successful clinical implementation of corneal epithelial stem cell therapy for treatment of unilateral limbal stem cell deficiency,Stem cells 28(3)(2010)597-610.

[5]H.S.Dua,A.Azuara-Blanco,Autologous limbal transplantation in patients with unilateral corneal stem cell deficiency,The British journal of ophthalmology84(3)(2000)273-8.

[6]Y.Qazi,P.Hamrah,Corneal Allograft Rejection:Immunopathogenesis to Therapeutics,J Clin Cell Immunol 2013(Suppl 9)(2013).

[7]J.A.Thomson,J.Itskovitz-Eldor,S.S.Shapiro,M.A.Waknitz,J.J.Swiergiel,V.S.Marshall,J.M.Jones,Embryonic stem cell lines derived from human blastocysts,Science 282(5391)(1998)1145-7.

[8]K.Takahashi,K.Tanabe,M.Ohnuki,M.Narita,T.Ichisaka,K.Tomoda,S.Yamanaka,Induction of pluripotent stem cells from adult human fibroblasts by defined factors,Cell 131(5)(2007)861-72.

[9]J.Yu,M.A.Vodyanik,K.Smuga-Otto,J.Antosiewicz-Bourget,J.L.Frane,S.Tian,J.Nie,G.A.Jonsdottir,V.Ruotti,R.Stewart,Slukvin,II,J.A.Thomson,Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells,Science(2007).

[10]S.Ahmad,R.Stewart,S.Yung,S.Kolli,L.Armstrong,M.Stojkovic,F.Figueiredo,M.Lako,Differentiation of human embryonic stem cells into corneal epithelial-like cells by in vitro replication of the corneal epithelial stem cell niche,Stem cells 25(5)(2007)1145-55.

[11]J.Zhu,K.Zhang,Y.Sun,X.Gao,Y.C.Li,Z.J.Chen,X.Y.Wu,Reconstruction of Functional Ocular Surface by Acellular Porcine Cornea Matrix Scaffold and Limbal Stem Cells Derived from Human Embryonic Stem Cells,Tissue Eng Pt A 19(21-22)(2013)2412-2425.

[12]J.Brzeszczynska,K.Samuel,S.Greenhough,K.Ramaesh,B.Dhillon,D.C.Hay,J.A.Ross,Differentiation and molecular profiling of human embryonic stem cell-derived corneal epithelial cells,Int J Mol Med 33(6)(2014)1597-1606.

[13]R.Shalom-Feuerstein,L.Serror,S.De La Forest Divonne,I.Petit,E.Aberdam,L.Camargo,O.Damour,C.Vigouroux,A.Solomon,C.Gaggioli,J.Itskovitz-Eldor,S.Ahmad,D.Aberdam,Pluripotent stem cell model reveals essential roles for miR-450b-5p and miR-184 in embryonic corneal lineage specification,Stem cells 30(5)(2012)898-909.

[14]R.Hayashi,Y.Ishikawa,M.Ito,T.Kageyama,K.Takashiba,T.Fujioka,M.Tsujikawa,H.Miyoshi,M.Yamato,Y.Nakamura,K.Nishida,Generation of corneal epithelial cells from induced pluripotent stem cells derived from human dermal fibroblast and corneal limbal epithelium,PloS one 7(9)(2012)e45435.

[15]K.J.Hewitt,Y.Shamis,M.W.Carlson,E.Aberdam,D.Aberdam,J.A.Garlick,Three-Dimensional Epithelial Tissues Generated from Human Embryonic Stem Cells,Tissue Eng Pt A 15(11)(2009)3417-3426.

[16]C.Hanson,T.Hardarson,C.Ellerstrom,M.Nordberg,G.Caisander,M.Rao,J.Hyllner,U.Stenevi,Transplantation of human embryonic stem cells onto a partially wounded human cornea in vitro,Acta ophthalmologica 91(2)(2013)127-30.

[17]A.Mikhailova,T.Ilmarinen,H.Uusitalo,H.Skottman,Small-molecule induction promotes corneal epithelial cell differentiation from human induced pluripotent stem cells,Stem cell reports 2(2)(2014)219-31.

[18]Z.Y.Wang,Q.J.Zhou,H.Y.Duan,Y.Wang,M.C.Dong,W.Y.Shi,Immunological Properties of Corneal Epithelial-Like Cells Derived from Human Embryonic Stem Cells,Plos One 11(3)(2016).

[19]R.Hayashi,Y.Ishikawa,Y.Sasamoto,R.Katori,N.Nomura,T.Ichikawa,S.Araki,T.Soma,S.Kawasaki,K.Sekiguchi,A.J.Quantock,M.Tsujikawa,K.Nishida,Co-ordinated ocular development from human iPS cells and recovery of corneal function,Nature 531(7594)(2016)376-80.

[20]P.J.Susaimanickam,S.Maddileti,V.K.Pulimamidi,S.R.Boyinpally,R.R.Naik,M.N.Naik,G.B.Reddy,V.S.Sangwan,I.Mariappan,Generating minicorneal organoids from human induced pluripotent stem cells,Development 144(13)(2017)

2338-2351.

[21]J.Y.Lai,P.R.Wang,L.J.Luo,S.T.Chen,Stabilization of collagen nanofibers with L-lysine improves the ability of carbodiimide cross-linked amniotic membranes to preserve limbal epithelial progenitor cells,Int J Nanomedicine 9(2014)5117-30.

[22]E.J.Orwin,A.Hubel,In vitro culture characteristics of corneal epithelial,endothelial,and keratocyte cells in a native collagen matrix,Tissue Eng 6(4)(2000)307-19.

[23]M.Talbot,P.Carrier,C.J.Giasson,A.Deschambeault,S.L.Guerin,F.A.Auger,R.Bazin,L.Germain,Autologous transplantation of rabbit limbal epithelia cultured on fibrin gels for ocular surface reconstruction,Molecular vision 12(2006)65-75.

[24]S.Sharma,D.Gupta,S.Mohanty,M.Jassal,A.K.Agrawal,R.Tandon,Surface-modified electrospun poly(epsilon-caprolactone)scaffold with improved optical transparency and bioactivity for damaged ocular surface reconstruction,Investigative ophthalmology&visual science 55(2)(2014)899-907.

[25]L.Guan,H.Ge,X.Tang,S.Su,P.Tian,N.Xiao,H.Zhang,L.Zhang,P.Liu,Use of a silk fibroin-chitosan scaffold to construct a tissue-engineered corneal stroma,Cells Tissues Organs 198(3)(2013)190-7.

[26]A.de la Mata,T.Nieto-Miguel,M.Lopez-Paniagua,S.Galindo,M.R.Aguilar,L.Garcia-Fernandez,S.Gonzalo,B.Vazquez,J.S.Roman,R.M.Corrales,M.Calonge,Chitosan-gelatin biopolymers as carrier substrata for limbal epithelial stem cells,J Mater Sci Mater Med 24(12)(2013)2819-29.

[27]Y.G.Xu,Y.S.Xu,C.Huang,Y.Feng,Y.Li,W.Wang,Development of a rabbit corneal equivalent using an acellular corneal matrix of a porcine substrate,Molecular vision 14(2008)2180-9.

[28]E.Yoeruek,T.Bayyoud,C.Maurus,J.Hofmann,M.S.Spitzer,K.U.Bartz-Schmidt,P.Szurman,Decellularization of porcine corneas and repopulation with human corneal cells for tissue-engineered xenografts,Acta ophthalmologica90(2)(2012)e125-31.

[29]C.Zhang,L.Du,K.Pang,X.Wu,Differentiation of human embryonic stem cells into corneal epithelial progenitor cells under defined conditions,PLoS One12(8)(2017)e0183303.

[30]Z.Zhang,G.Niu,J.S.Choi,M.Giegengack,A.Atala,S.Soker,Bioengineered multilayered human corneas from discarded human corneal tissue,Biomed Mater 10(3)(2015)035012.

[31]H.Chen,W.Wang,H.Xie,X.Xu,J.Wu,Z.Jiang,M.Zhang,L.Zhou,S.Zheng,A pathogenic role of IL-17 at the early stage of corneal allograft rejection,Transplant immunology 21(3)(2009)155-61.

[32]T.H.van Essen,D.L.Roelen,K.A.Williams,M.J.Jager,Matching for Human Leukocyte Antigens(HLA)in corneal transplantation-to do or not to do,Progress in retinal and eye research 46(2015)84-110.

[33]D.Zheng,X.Wang,R.H.Xu,Concise Review:One Stone for Multiple Birds:Generating Universally Compatible Human Embryonic Stem Cells,Stem Cells34(9)(2016)2269-75.

[34]G.G.Gornalusse,R.K.Hirata,S.E.Funk,L.Riolobos,V.S.Lopes,G.Manske,D.Prunkard,A.G.Colunga,L.A.Hanafi,D.O.Clegg,C.Turtle,D.W.Russell,HLA-E-expressing pluripotent stem cells escape allogeneic responses and lysis by NK cells,Nature biotechnology 35(8)(2017)765-772.

[35]G.Chen,D.R.Gulbranson,Z.Hou,J.M.Bolin,V.Ruotti,M.D.Probasco,K.Smuga-Otto,S.E.Howden,N.R.Diol,N.E.Propson,R.Wagner,G.O.Lee,J.Antosiewicz-Bourget,J.M.Teng,J.A.Thomson,Chemically defined conditions for human iPSC derivation and culture,Nat Methods 8(5)(2011)424-9.

[36]P.J.Gage,M.Qian,D.Wu,K.I.Rosenberg,The canonical Wnt signaling antagonist DKK2 is an essential effector of PITX2 function during normal eye development,Dev Biol 317(1)(2008)310-24.

[37]S.Fuhrmann,Wnt signaling in eye organogenesis,Organogenesis 4(2)(2008)60-7.

[38]R.M.Arkell,P.P.Tam,Initiating head development in mouse embryos:integrating signalling and transcriptional activity,Open Biol 2(3)(2012)120030.

[39]S.Dupont,L.Zacchigna,M.Cordenonsi,S.Soligo,M.Adorno,M.Rugge,S.Piccolo,Germ-layer specification and control of cell growth by Ectodermin,a Smad4 ubiquitin ligase,Cell 121(1)(2005)87-99.

[40]L.Richard-Parpaillon,C.Heligon,F.Chesnel,D.Boujard,A.Philpott,The IGF pathway regulates head formation by inhibiting Wnt signaling in Xenopus,Dev Biol 244(2)(2002)407-17.

[41]C.B.Mellough,J.Collin,M.Khazim,K.White,E.Sernagor,D.H.Steel,M.Lako,IGF-1 Signaling Plays an Important Role in the Formation of Three-Dimensional Laminated Neural Retina and Other Ocular Structures From Human Embryonic Stem Cells,Stem cells 33(8)(2015)2416-30.

[42]G.Cotsarelis,S.Z.Cheng,G.Dong,T.T.Sun,R.M.Lavker,Existence of slow-cycling limbal epithelial basal cells that can be preferentially stimulated to proliferate:implications on epithelial stem cells,Cell 57(2)(1989)201-9.

[43]S.P.Sugrue,J.D.Zieske,ZO1 in corneal epithelium:association to the zonula occludens and adherens junctions,Experimental eye research 64(1)(1997)11-20.

[44]P.B.Medawar,Immunity to homologous grafted skin；the fate of skin homografts transplanted to the brain,to subcutaneous tissue,and to the anterior chamber of the eye,British journal of experimental pathology 29(1)(1948)58-69.

[45]K.A.Williams,S.M.Muehlberg,R.F.Lewis,D.J.Coster,How successful is corneal transplantationA report from the Australian Corneal Graft Register,Eye 9(Pt 2)(1995)219-27.

[46]M.G.Maguire,W.J.Stark,J.D.Gottsch,R.D.Stulting,A.Sugar,N.E.Fink,A.Schwartz,Risk factors for corneal graft failure and rejection in the collaborative corneal transplantation studies.Collaborative Corneal Transplantation Studies Research Group,Ophthalmology 101(9)(1994)1536-47.

[47]T.Reinhard,D.Bohringer,J.Enczmann,G.Kogler,S.Mayweg,P.Wernet,R.Sundmacher,HLA class I and II matching improves prognosis in penetrating normal-risk keratoplasty,Developments in ophthalmology 36(2003)42-9.

[48]M.Iwata,A.Kiritoshi,M.I.Roat,A.Yagihashi,R.A.Thoft,Regulation of HLA class II antigen expression on cultured corneal epithelium by interferon-gamma,Investigative ophthalmology&visual science 33(9)(1992)2714-21.

[49]M.H.Abdulreda,G.Faleo,R.D.Molano,M.Lopez-Cabezas,J.Molina,Y.Tan,O.A.Echeverria,E.Zahr-Akrawi,R.Rodriguez-Diaz,P.K.Edlund,I.Leibiger,A.L.Bayer,V.Perez,C.Ricordi,A.Caicedo,A.Pileggi,P.O.Berggren,High-resolution,noninvasive longitudinal live imaging of immune responses,Proceedings of the National Academy of Sciences of the United States of America108(31)(2011)12863-8.

[50]M.Mukhopadhyay,M.Gorivodsky,S.Shtrom,A.Grinberg,C.Niehrs,M.I.Morasso,H.Westphal,Dkk2 plays an essential role in the corneal fate of the ocular surface epithelium,Development 133(11)(2006)2149-54.

[51]C.Hicks,G.Crawford,T.Chirila,S.Wiffen,S.Vijayasekaran,X.Lou,J.Fitton,M.Maley,A.Clayton,P.Dalton,S.Platten,B.Ziegelaar,Y.Hong,A.Russo,I.Constable,Development and clinical assessment of an artificial cornea,Progress in retinal and eye research 19(2)(2000)149-70.

[52]P.A.Netland,H.Terada,C.H.Dohlman,Glaucoma associated with keratoprosthesis,Ophthalmology 105(4)(1998)751-7.

[53]H.F.Chew,B.D.Ayres,K.M.Hammersmith,C.J.Rapuano,P.R.Laibson,J.S.Myers,Y.P.Jin,E.J.Cohen,Boston keratoprosthesis outcomes and complications,Cornea 28(9)(2009)989-96.

[54]B.L.Zerbe,M.W.Belin,J.B.Ciolino,G.Boston Type 1 Keratoprosthesis Study,Results from the multicenter Boston Type 1 Keratoprosthesis Study,Ophthalmology 113(10)(2006)1779 e1-7.

[55]D.F.Larkin,K.A.Williams,The host response in experimental corneal xenotransplantation,Eye 9(Pt 2)(1995)254-60.

[56]C.Patience,Y.Takeuchi,R.A.Weiss,Infection of human cells by an endogenous retrovirus of pigs,Nat Med 3(3)(1997)282-6.

[57]D.Niu,H.J.Wei,L.Lin,H.George,T.Wang,I.H.Lee,H.Y.Zhao,Y.Wang,Y.Kan,E.Shrock,E.Lesha,G.Wang,Y.Luo,Y.Qing,D.Jiao,H.Zhao,X.Zhou,S.Wang,H.Wei,M.Guell,G.M.Church,L.Yang,Inactivation of porcine endogenous retrovirus in pigs using CRISPR-Cas9,Science 357(6357)(2017)1303-1307.

[58]Y.Hashimoto,S.Funamoto,S.Sasaki,T.Honda,S.Hattori,K.Nam,T.Kimura,M.Mochizuki,T.Fujisato,H.Kobayashi,A.Kishida,Preparation and characterization of decellularized cornea using high-hydrostatic pressurization for corneal tissue engineering,Biomaterials 31(14)(2010)3941-8.

[59]E.Yoeruek,T.Bayyoud,C.Maurus,J.Hofmann,M.S.Spitzer,K.U.Bartz-Schmidt,P.Szurman,Reconstruction of corneal stroma with decellularized porcine xenografts in a rabbit model,Acta ophthalmologica 90(3)(2012)e206-10.

[60]Y.Lin,G.Chen,Embryoid body formation from human pluripotent stem cells in chemically defined E8 media,StemBook,Cambridge(MA),2008.

[61]A.Panda,M.Vanathi,A.Kumar,Y.Dash,S.Priya,Corneal graft rejection,Survey of ophthalmology 52(4)(2007)375-96.

[62]G.Lin,K.Martins-Taylor,R.H.Xu,Human embryonic stem cell derivation,maintenance,and differentiation to trophoblast,Methods in molecular biology 636(2010)1-24.

[63]M.Costa,M.Dottori,E.Ng,S.M.Hawes,K.Sourris,P.Jamshidi,M.F.Pera,A.G.Elefanty,E.G.Stanley,The hESC line Envy expresses high levels of GFP in all differentiated progeny,Nat Methods 2(4)(2005)259-60.

[64]X.Wang,A.S.Lazorchak,L.Song,E.Li,Z.Zhang,B.Jiang,R.H.Xu,Immune Modulatory Mesenchymal Stem Cells Derived from Human Embryonic Stem Cells Through a Trophoblast-like Stage,Stem Cells(2015).

[65]F.A.Ran,P.D.Hsu,J.Wright,V.Agarwala,D.A.Scott,F.Zhang,Genome engineering using the CRISPR-Cas9system,Nature protocols 8(11)(2013)2281-2308.

the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing corneal epithelial cells, comprising: the human embryonic stem cells were inoculated into the E6 medium and cultured to differentiate the human embryonic stem cells into corneal epithelial cells.

2. The method of claim 1, wherein the human embryonic stem cell is a B2M knock-out human embryonic stem cell.

3. A method for producing a corneal scaffold, which comprises the method for producing corneal epithelial cells according to claim 1 or 2.