Method for promoting differentiation of cranial nerve crest cells to corneal endothelial cells

Abstract

The invention discloses a method for promoting differentiation of cranial nerve crest cells into corneal endothelial cells. According to the method, a culture medium with definite chemical components is used for inducing differentiation of the nerve crest cells into the corneal endothelial cells. The method provided by the invention has the advantages of high induced differentiation efficiency, strong stability, clear culture system components, high safety and the like, and is suitable for large-scale induced differentiation culture of corneal endothelial cells; and the prepared corneal endothelial cell is highly consistent with the primary corneal endothelial cell in the aspects of contact inhibition, ion transport, metabolic function and the like, and has huge market value and wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. Specifically, this invention relates to a method for promoting the differentiation of cranial neural crest cells into corneal endothelial cells. Background Technology

[0002] Corneal endothelial cells (CECs) play a crucial role in maintaining normal corneal function. However, human corneal endothelial cells are non-regenerative, and their numbers decrease continuously after birth. When disease damage causes the corneal endothelial cell density to drop to a critical value (400-700 cells / mm²), the cells can no longer maintain the pump and barrier functions of the cornea through proliferation and migration, leading to irreversible corneal endothelial dysfunction, causing corneal edema and opacity, and ultimately irreversible vision loss. Currently, corneal transplantation is considered the only effective treatment for corneal endothelial decompensation; however, the extreme scarcity of donor corneas cannot meet clinical needs. In recent years, anterior chamber injection of corneal endothelial cells has been proposed as a novel therapy, but this therapy is still limited by the lack of donor corneas. Therefore, developing novel corneal endothelial regeneration strategies that can overcome donor limitations has become a critical issue that urgently needs to be addressed in the field of ophthalmic regenerative medicine.

[0003] Neural crest cells (NCCs) are an ideal source of corneal endothelial cells due to their multipotent differentiation potential during embryonic development. Studies have shown that chemically induced neural crest cells derived from fibroblasts can differentiate into corneal endothelial cells through the induction of small molecule compounds. Neural crest cells are precursor cells of corneal endothelial cells. During eye development, when the neural tube closes, cranial neural crest cells migrate to the anterior end of the eyeball and develop into corneal endothelial cells. Current methods for inducing the differentiation of neural crest cells into corneal endothelial cells in vitro typically involve a pluripotent stem cell stage, resulting in low differentiation efficiency, high cell heterogeneity, and the use of numerous culture medium components, raising safety concerns that hinder clinical application. Therefore, providing a novel method with higher differentiation efficiency to promote the differentiation of cranial neural crest cells into corneal endothelial cells is crucial. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, the purpose of this invention is to provide a novel and highly efficient method for promoting the differentiation of cranial neural crest cells into corneal endothelial cells.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] The first aspect of this invention provides a method for promoting the differentiation of neural crest cells into corneal endothelial cells.

[0007] Furthermore, the method includes the following steps: culturing neural crest cells in an induction differentiation medium to obtain corneal endothelial cells;

[0008] The induction differentiation medium comprises the following components: basal medium DMEM / F12, N2 additive, B27 additive, bFGF, EGF, and SB431542.

[0009] Furthermore, the concentrations of each component in the induction differentiation medium are as follows: (0.5-1)× N2 additive, (0.5-1)× B27 additive, (10-25) ng / mL bFGF, (5-15) ng / mL EGF, and (5-10) μM / mL SB431542.

[0010] Furthermore, the concentrations of each component in the induction differentiation medium are as follows: 1× N2 additive, 1× B27 additive, 20 ng / mL bFGF, 10 ng / mL EGF, and 5 μM / mL SB431542.

[0011] Furthermore, the neural crest cells include cranial neural crest cells, vagal neural crest cells, trunk neural crest cells, and sacral neural crest cells.

[0012] Furthermore, the neural crest cells are cranial neural crest cells.

[0013] Furthermore, the method includes the following steps:

[0014] Day 0: Neural crest cells were cultured in an induction differentiation medium.

[0015] Days 7-15: Corneal endothelial cells were obtained.

[0016] Furthermore, the induction differentiation medium should be replaced every 24 hours or 36 hours.

[0017] Furthermore, the induction differentiation medium was changed every 24 hours.

[0018] In some implementations, the neural crest cells are not particularly limited and can be derived from virtually any suitable source, which can be various mammals.

[0019] In some implementations, the mammals include rodents, carnivores, chiropterans, hedgehogs, and insectivores.

[0020] In some embodiments, the rodents include those belonging to the families Cricetidae, Cricetidae, Muridae, Falconidae, Spineridae, Moleidae, Echidnae, and Rockrats.

[0021] In a specific embodiment of the present invention, the neural crest cells are derived from Wnt1-Cre / ROSA26 cells during the embryonic period or 14 days after birth. tdTomato It was extracted from double transgenic mice.

[0022] In some implementations, the presence or absence of neural crest cells in the extracted cell population can be determined by observing neural crest cell marker genes. Detecting marker gene expression refers to measuring the relative levels of the gene's RNA transcript or its expression product. Methods for detecting gene expression / transcription, i.e., gene expression / transcription profiling, include multinucleotide-based hybridization analysis, multinucleotide-based sequencing, immunohistochemistry, and proteomics-based methods. These methods generally detect the expression / transcription products (e.g., mRNA) of the gene of interest. In some implementations, PCR-based methods such as reverse transcription PCR (RT-PCR) and array-based methods such as microarrays can be used.

[0023] In some embodiments, the neural crest cell marker genes include P75, HNK1, AP2α, and Sox10.

[0024] In some implementations, the success of inducing neural crest cells to differentiate into corneal endothelial cells can be determined by observing changes in cell morphology at different stages and detecting the expression of corneal endothelial cell marker genes.

[0025] In some implementations, corneal endothelial cells exhibit a regular, flattened hexagonal geometry and highly express ZO-1, AQP1, Slc4a11, N-cadherin, Abcg2, and ATPA1.

[0026] The corneal endothelial cells prepared using the method described in this invention exhibit highly consistent contact inhibition, ion transport, and metabolic functions with primary CECs. The completeness of these functional characteristics provides a solid experimental basis for the clinical application of iCECs, which can be directly applied to various medical and scientific research purposes.

[0027] A second aspect of the present invention provides a culture medium for promoting the differentiation of neural crest cells into corneal endothelial cells.

[0028] Furthermore, the culture medium comprises the following components: basal medium DMEM / F12, N2 additive, B27 additive, bFGF, EGF, and SB431542.

[0029] Furthermore, the concentrations of each component in the culture medium are as follows: (0.5-1)× N2 additive, (0.5-1)× B27 additive, (10-25) ng / mL bFGF, (5-15) ng / mL EGF, and (5-10) μM / mL SB431542.

[0030] Furthermore, the concentrations of each component in the culture medium are as follows: 1× N2 additive, 1× B27 additive, 20 ng / mL bFGF, 10 ng / mL EGF, and 5 μM / mL SB431542.

[0031] In some embodiments, the present invention does not impose any particular restrictions on the specific content of each component in the culture medium. As long as the culture medium composed of each component in a specific content can perform or substantially perform the expected effect (e.g., can effectively or substantially effectively induce neural crest cells to differentiate into corneal endothelial cells), it is within the protection scope of the present invention.

[0032] A third aspect of the present invention provides a population of functional corneal endothelial cells derived from neural crest cells.

[0033] Furthermore, the cell population is obtained by inducing differentiation using the method described in the first aspect of the present invention.

[0034] Furthermore, the cell population highly expresses corneal endothelial characteristic genes ZO-1, AQP1, Slc4a11, N-cadherin, Abcg2, and ATPA1.

[0035] A fourth aspect of the present invention provides a pharmaceutical composition for the prevention and / or treatment of corneal endothelial decompensation.

[0036] Furthermore, the pharmaceutical composition comprises the cell population described in the third aspect of the present invention.

[0037] Furthermore, the pharmaceutical composition also comprises a pharmaceutically acceptable carrier and / or excipients.

[0038] In some embodiments, the carrier and / or excipients include pharmaceutically acceptable carriers, diluents, fillers, binders and other excipients, depending on the route of administration and the designed dosage form.

[0039] In some embodiments, the pharmaceutical composition is any pharmaceutically acceptable dosage form, including at least one of tablets, capsules, injections, granules, suspensions, and solutions.

[0040] In some embodiments, the appropriate dosage of the pharmaceutical composition can be prescribed in various ways depending on factors such as formulation method, route of administration, patient's age, weight, sex, condition, diet, administration time, route of administration, excretion rate, and responsiveness. Skilled physicians can usually easily determine the prescription and the dosage that is effective for the desired treatment.

[0041] In some embodiments, the actual dose of the active ingredient (the cell population described in the third aspect of the invention) in the pharmaceutical composition should be determined based on a variety of relevant factors, including the severity of the disease to be treated, the route of administration, the patient's age, sex, and weight.

[0042] In some implementations, corneal endothelial cells are administered to the subject via one or more routes selected from systemic, local, intravenous, subcutaneous, intra-articular, intramuscular, intrathecal, and intraperitoneal routes.

[0043] In some embodiments, the object includes one or more animals, including, for example, cattle, horses, sheep, primates, birds, and rodents. In some embodiments, the object can be a mammal, such as a human or a non-human mammal. In other embodiments, the object can be a mouse, rat, hamster, weasel, gerbil, rabbit, monkey, chimpanzee, horse, pony, donkey, sheep, pig, chicken, goat, cat, or dog. In a preferred embodiment, the object is a human.

[0044] The term "prevention and / or treatment" as used herein refers to the prevention, reversal, mitigation, or inhibition of the progression of the disorder or condition to which the term applies, or one or more symptoms of such disorder or condition. Treatment of a disease or condition includes improving at least one symptom of a particular disease or condition, even if the underlying pathophysiology is not affected. For example, treating a subject with a corneal endothelial decompensation-related disease, such as corneal edema, decreased corneal transparency, or decreased vision, by administering a medication, even though the medication does not treat the cause of the condition. For instance, "prevention and / or treatment of corneal endothelial decompensation" as used herein includes one or more of the following:

[0045] (1) Preventing corneal endothelial decompensation;

[0046] (2) Inhibit the development of corneal endothelial decompensation;

[0047] (3) Cures corneal endothelial decompensation;

[0048] (4) Relieves related symptoms in patients with corneal endothelial decompensation;

[0049] (5) Reduce the severity of corneal endothelial decompensation;

[0050] (6) Prevent recurrence of corneal endothelial decompensation.

[0051] The fifth aspect of this invention provides the use of the culture medium described in the second aspect of this invention in inducing neural crest cells to differentiate into corneal endothelial cells.

[0052] The sixth aspect of this invention provides the application of the cell population described in the third aspect of this invention in the construction of cell models of corneal endothelial decompensation, drug screening, or in vitro experimental studies.

[0053] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0054] The terms "preferred," "more preferably," etc., used in this invention refer to embodiments of the invention that provide certain beneficial effects under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are unavailable, nor is it intended to exclude other embodiments from the scope of this invention.

[0055] In the description of this invention, the term "and / or" includes all and any combination of one or more of the associated listed items.

[0056] In the description of this invention, the terms "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0057] Advantages and beneficial effects of the present invention:

[0058] This invention provides a scheme for inducing neural crest cells to differentiate into corneal endothelial cells with a clearly defined chemical composition. It has the advantages of high differentiation efficiency, strong stability, clear culture system composition, and high safety, and is suitable for large-scale corneal endothelial cell induction and differentiation culture. Moreover, the prepared corneal endothelial cells are highly consistent with primary CECs in terms of contact inhibition, ion transport and metabolic functions, and have great market value and broad application prospects. Attached Figure Description

[0059] Figure 1 The image shows the results of mouse cranial neural crest cell isolation and culture; among them, Figure 1 A shows mouse cranial neural crest cells isolated with red fluorescence, scale bar 400 μm; Figure 1 B represents the expansion culture of mouse cranial neural crest cells, with a scale bar of 400 μm. Figure 1 C represents the results of cell flow cytometry screening; Figure 1 D is Wnt1-Cre / ROSA26 tdTomato Schematic diagram of the construction model of double transgenic mice;

[0060] Figure 2 The image shows the results of inducing cranial neural crest cells to differentiate into corneal endothelial cells; among them, Figure 2 A is a schematic diagram of the induction of cranial neural crest cells into corneal endothelial cells; Figure 2 B represents the morphological changes of tdTomato-positive cranial neural crest cells at different days during the induction of differentiation into corneal endothelial cells. The scale bar is 400 μm.

[0061] Figure 3 The image shows the results of the biological characteristic verification of iCECs; among them, Figure 3 Figure A shows the results of qRT-PCR detection of the expression of neural crest cell marker genes P75, HNK1, AP2α, Sox10, and TTF. The gene expression levels (log2) have been normalized. Figure 3 Image B shows the immunofluorescence staining results of iCECs, with green fluorescent labeling of ZO-1 and Na. + / K + -ATPase, AQP1, Laminin, blue fluorescently labeled DAPI, scale bar 50 μm; Figure 3 C is the result of qRT-PCR detection of the expression of corneal endothelial cell marker genes ZO-1, Slc4a11, N-cadherin, Abcg2, mCECs and iCECs. The gene expression level (log2) was normalized.

[0062] Figure 4 Figure showing the results of flow cytometry detection of AQP1 and ATPA1 expression in iCECs;

[0063] Figure 5 The results are shown in the figure to verify that iCECs originated from the differentiation of NCCs; among them, Figure 5 A shows the results of NCCs immunofluorescence staining. Green fluorescence represents P75, HNK1, Sox10, and AP2α, while blue fluorescence represents DAPI. The scale bar is 50 μm. Figure 5B represents tdTomato-positive cranial neural crest cells (NCCs) induced to differentiate into corneal endothelial cells (iCECs), scale bar 400 μm; Figure 5 C shows the immunofluorescence staining results of iCECs, with green fluorescence representing ZO-1 and blue fluorescence representing DAPI. The scale bar is 50 μm.

[0064] Figure 6 The graph shows the results of iCECs' uptake function of low-density lipids (LDL); red fluorescence represents Dil-Ac-LDL, green fluorescence represents FITC-EUA, and the scale bar is 100 μm.

[0065] Figure 7 This is a graph showing the results of transcriptome sequencing analysis; in which, Figure 7 A is a heatmap of differentially expressed genes in samples at different time points. The numbers below the heatmap represent independent biological replicates, and red and blue represent upregulated and downregulated genes, respectively. Figure 7 B is a heatmap showing the differentially expressed genes among samples at a specified time point. The numbers below the heatmap represent independent biological replicates. The labels on the right side of the heatmap represent the hallmark genes of neural crest cells and corneal endothelial cells. Red and blue represent upregulated and downregulated genes, respectively.

[0066] Figure 8 This image shows the isolation and induction culture of cranial neural crest cells after birth; among them, Figure 8 A represents primary cranial neural crest cells extracted from mice 14 days after birth; Figure 8 B represents the morphological changes of cranial neural crest cells at different days during the induction of corneal endothelial cell differentiation, with a scale bar of 400 μm. Detailed Implementation

[0067] The technical solutions in the embodiments of this application will be clearly and completely described below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are merely preferred embodiments, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0068] Unless otherwise specified in the following examples, the conditions should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0069] The experimental materials used in the following examples were obtained from the following sources:

[0070] Experimental animals: Wnt1-cre(Tg(Wnt1-cre)11Rth mice and ROSA26 mice tdTomato(Gt(ROSA)26Sortm14(CAG) tdTomato The Hze mice were purchased from The Jackson Laboratory, and all animal experiments were conducted in accordance with the guidelines of the Animal Ethics Committee of Wenzhou Medical University. All animals were housed in the Animal Center of Wenzhou Medical University under constant temperature and humidity control (21±2°C), with a 12-hour day-night alternating light cycle and free access to food.

[0071] Antibodies: P75 (Rabbit, Abcam, ab52987); HNK1 (Rabbit, Abcam, ab221756); Sox10 (Rabbit, Abcam, ab155279); AP2α (Rabbit, Abcam, ab108311); ZO-1 (Rabbit, Abcam, ab216880); Na + / K + -ATPase (Mouse, Thermo, MA3-912); Laminin (Rabbit, Abcam, ab11575); AQP1 (Rabbit, Abcam, ab168387).

[0072] Small molecule compound: SB431542 (Sigma, S4317).

[0073] Cell Culture Reagents: DMEM / F12 Medium: Gibco, USA; Trypsin-EDTA: Gibco, USA; N2: Gibco, USA; B27: Gibco, USA; Basic Fibroblast Growth Factor (bFGF): PeproTech, USA; Epidermal Growth Factor (EGF): PeproTech, USA; Fetal Bovine Serum (FBS): Gibco, USA; Non-essential Amino Acids (NEAAs): Sigma, USA; GlutaMAX: Gibco, USA; Penicillin-Streptomycin-Secondary Antibody: Gibco, USA; Trizol Reagent: Invitrogen, USA; Dimethyl Sulfoxide (DMSO): Sigma, USA; Fetal Bovine Serum: Gibco, USA; Bovine Serum Albumin (BSA): Sigma, USA; RNeasy Plus mini Kit: Qiagen, Germany; iScript cDNA Synthesis Kit: Bio-Rad, USA; KSR: Gibco, USA; Triton X-100: Merck KGaA, Germany; Paraformaldehyde (PFA): Sigma, USA; Triton X-100: Merck KGaA, Germany; Trizol reagent: Invitrogen, USA; DAPI: Beyotime, China; Antifluorescence quenching mounting medium: Thermo Scientific, USA; iScript cDNA synthesis kit: Bio-Rad, USA; FAST SYBR Green Master Mix kit: ABI, USA.

[0074] Example 1: Isolation of mouse cranial neural crest cells

[0075] I. Experimental Methods

[0076] 1. Cell isolation

[0077] Cranial neural crest cell isolation: After ensuring that the mice used were healthy and uncontaminated, and that the operating environment met aseptic requirements, Wnt1-Cre / ROSA26 cells were isolated under a microscope. tdTomato Mice were euthanized by cervical dislocation after disinfection. The head and torso were separated using sterile scissors and forceps. The separated skin was cut into small pieces (approximately 1 mm³) and placed in sterile culture dishes. Then, 0.25% trypsin solution was added to completely submerge the tissue pieces, and the dishes were incubated at 37°C for 15 minutes. During this time, the culture dishes were gently shaken every 5 minutes to ensure uniform digestion. The culture dishes were kept tightly sealed to prevent contamination.

[0078] Termination of digestion: When the clumps were observed to have digested into single cells under a microscope, Dulbecco modified Eagle medium (DMEM) / F12 was added for washing twice, and the supernatant was discarded after each wash to completely remove trypsin.

[0079] 2. Cell Culture

[0080] (1) Prepare the culture medium: Prepare the culture medium under sterile conditions. The culture medium consists of DMEM / F12 supplemented with 1×N2, 1×B27, bFGF (20 ng / mL) and EGF (10 ng / mL) to ensure that the culture medium is completely dissolved and uniform.

[0081] (2) Cell inoculation: After gently mixing the prepared cell suspension by pipetting, inoculate it into a culture dish. Add an appropriate amount of the prepared culture medium to ensure that the cells are completely submerged in the medium and evenly distributed. Finally, place the culture dish in an incubator at 37°C and 5% CO2 for incubation.

[0082] (3) Cell culture: During the culture process, the culture medium should be changed regularly (it is recommended to change it every 2-3 days) to maintain the cell growth environment. Each time the culture medium is changed, the old culture medium should be gently aspirated to avoid physical damage, and fresh NCCs culture medium should be added. The amount of culture medium changed each time should be consistent to maintain a stable culture environment.

[0083] (4) Cell observation: During the culture process, observe the morphology and growth status of the cells regularly using an inverted microscope. Record the cell adhesion, morphological changes, and growth density. If abnormal cell morphology or slow growth is found, adjust the culture conditions or address the problem promptly.

[0084] II. Experimental Results

[0085] In this study, to ensure that the cell population used in the experiments consisted of cranial neural crest cells (NCCs), we used Wnt1-Cre transgenic mice. Wnt1-Cre, as a widely validated lineage-tracing marker system for neural crest cell (NCC) development, has had its reliability and specificity well-established in marking neural crest cells. By using Wnt1-Cre mice and ROSA26... tdTomato Through hybridization with mice, we successfully constructed Wnt1-Cre / ROSA26. tdTomato Double transgenic mouse model ( Figure 1 (D), thereby ensuring specific and stable expression of tdTomato protein in NCCs. This system drives the expression of Cre recombinase through the Wnt1 promoter, which in turn activates the expression of tdTomato fluorescent protein at the ROSA26 site, thus achieving specific labeling of neural crest cells and their derivatives.

[0086] NCCs isolated from double transgenic mice at 13.5 days of embryonic development exhibited significant red fluorescence under a fluorescence microscope, indicating that the isolated cell population could successfully express tdTomato fluorescent protein. Figure 1 A). Next, the cells were expanded and cultured in vitro until the confluence reached over 80% ( Figure 1 B), the cell population was sorted by flow cytometry, and a tdTomato-positive cell population was successfully screened, with a positive rate of 68.2% ( Figure 1 C). The sorted cell population has high purity, providing a reliable cellular basis for subsequent induction differentiation experiments.

[0087] Example 2: Induction of cranial neural crest cells (NCCs) into corneal endothelial cells (iCECs)

[0088] I. Experimental Methods

[0089] Differentiation induction medium components: The medium was prepared under aseptic conditions. It consisted of DMEM / F12 supplemented with 1×N2, 1×B27, bFGF (20 ng / mL), EGF (10 ng / mL) and SB431542 (5 μM / mL) to ensure complete dissolution and homogeneity.

[0090] First, cranial neural crest cells (NCCs) were seeded in well plates coated with 1% Matrigel (basement membrane matrix) and cultured for 1-3 days. The culture medium consisted of DMEM / F12 supplemented with 1×N2, 1×B27, bFGF (20 ng / mL), and EGF (10 ng / mL). Once the cell confluence reached 60%, induction differentiation medium was added and the cells were cultured for another 9-15 days, with the medium changed every other day.

[0091] II. Experimental Results

[0092] Experimental results showed that after induction treatment using the culture medium components of this application, by day 9, the cell population formed a monolayer clonal-like structure through tight junctions. The cell morphology exhibited homogenization characteristics, displaying a regular, flattened hexagonal geometric structure, consistent with the typical morphological characteristics of primary corneal endothelial cells. This morphological remodeling indicates that cranial neural crest cells successfully transdifferentiated into corneal endothelial cells. Furthermore, by day 12, the area of ​​the clusters increased, and by day 15, they merged into even larger cell populations. These phenomena demonstrate that the cells possess strong proliferative capacity. Figure 2 B). In view of this, this study formally names these corneal endothelial-like cells induced from cranial neural crest cells as induced corneal endothelial cells (iCECs).

[0093] Example 3 Characterization and gene expression profiling of iCECs

[0094] To verify whether iCECs possess the characteristics of corneal endothelial cells, this study first used quantitative reverse transcription polymerase chain reaction (qRT-PCR) technology to analyze Wnt1-positive neural crest cells (Wnt1... + -NCC) and Wnt1-negative tail tip fibroblasts (Wnt1 - Comparative analysis of Wnt1 gene expression (-TTF). Results showed that Wnt1 + -NCC significantly expressed neural crest cell marker genes (P75, HNK1, AP2α, Sox10), and the expression levels of these genes were significantly higher than those of the control group Wnt1. - -TTF ( Figure 3 A). This result indicates that the initial cell population used in the induced differentiation experiment was neural crest cells, providing a reliable cellular basis for subsequent directed differentiation research.

[0095] To further verify whether iCECs obtained through induced NCCs-directed differentiation possess corneal endothelial functional characteristics, this study used immunofluorescence assays to detect the expression of key functional proteins in iCECs. The results showed that iCECs specifically expressed corneal endothelial cell marker proteins, including laminin, tight junction protein ZO-1, aquaporin 1 (AQP1), and the ion transport pump Na⁺ / K⁺-ATPase (Na⁺ / K⁺-ATPase). Figure 3 B).

[0096] To systematically verify the lineage specificity of iCECs, this study used qRT-PCR to compare and analyze the gene expression of primary mouse corneal endothelial cells (mCECs) and iCECs. The results showed that iCECs significantly overexpressed corneal endothelial characteristic genes (ZO-1, Slc4a11, N-cadherin, Abcg2), and their mRNA levels were higher than those of mCECs. Figure 3 C). This gene expression pattern is highly consistent with that of primary corneal endothelial cells, indicating that iCECs not only exhibit corneal endothelial phenotypes at the morphological and functional levels, but their transcriptomic characteristics are also significantly similar to those of primary cells, further demonstrating the effectiveness of this induction protocol.

[0097] Furthermore, flow cytometry analysis of iCECs showed that the positive expression rates of CEC marker proteins AQP1 and ATPA1 were both greater than 95%. Figure 4This further demonstrates the effectiveness of the induction scheme.

[0098] In summary, cranial neural crest cells can be successfully differentiated into iCECs with corneal endothelial cell characteristics under the induction of the method described in this application.

[0099] Example 4: Verifying the differentiation of NCCs into iCECs through lineage tracing

[0100] First, using Wnt1-Cre / ROSA26 -tdTomato After successfully isolating primary cranial neural crest cells from a mouse model, their characteristics were verified by immunofluorescence staining. The results showed that the isolated cell population simultaneously expressed tdTomato protein and characteristic markers of neural crest cells (P75, HNK1, Sox10, AP2α). Figure 5 A) indicates that the initial cell population used in the experiment was Wnt1-positive cranial neural crest cells, ensuring the specificity of the cell source for subsequent induced differentiation.

[0101] Based on the previously established induction protocol, tdTomato-positive neural crest cells were directionally induced to differentiate. After induction, the differentiation products consistently and stably expressed the tdTomato red fluorescent signal (…). Figure 5 (B) This lineage tracing result directly confirms that the induced iCECs originate from the directed differentiation of Wnt1-positive neural crest cells, ruling out the possibility of non-specific cell contamination.

[0102] To further verify the cellular characteristics of iCECs, induced tdTomato cells were used. + After iCECs were expanded and cultured to a monolayer fusion state, their functional protein expression was detected by immunofluorescence staining. The results showed that iCECs specifically expressed the corneal endothelial cell marker protein ZO-1 (tight junction protein), and its expression exhibited a typical cell membrane localization pattern. Figure 5 C). The sustained expression of the tdTomato lineage marker indicates that the induced cells acquired the functional characteristics of corneal endothelial cells while maintaining lineage tracing.

[0103] In summary, by combining tdTomato lineage tracing technology with functional phenotypic analysis, we have confirmed from the two dimensions of cell origin and terminal differentiation that the induction protocol of this application can specifically drive Wnt1⁺ cranial neural crest cells to differentiate into functional corneal endothelial cells.

[0104] Example 5: iCECs have normal physiological functions in vitro.

[0105] Corneal endothelial cells (iCECs) participate in lipid metabolism and cell membrane repair by phagocytizing acetylated low-density lipoprotein (Ac-LDL), a function crucial for maintaining corneal dehydration and transparency. FITC-EUA is a labeling technique that uses the green fluorescent probe FITC to specifically bind to fucose residues on the surface of endothelial cells. To assess whether iCECs possess this key physiological function, we performed a phagocytosis assay using acetylated low-density lipoprotein (Dil-Ac-LDL) labeled with Dil for functional validation. The results showed that iCECs could efficiently take up Dil-Ac-LDL, with significant intracellular fluorescence signal accumulation (…). Figure 6 ).

[0106] This result demonstrates that iCECs possess a significant and specific uptake capacity for Dil-Ac-LDL, confirming not only their intact endocytic function but also suggesting that their metabolic pathway is highly consistent with that of primary corneal endothelial cells. This further supports the claim that iCECs possess the normal physiological functions of corneal endothelial cells in vitro.

[0107] Example 6: Transcriptome sequencing to assess the expression profile of iCECs

[0108] To systematically evaluate the characteristics of iCECs, this study used RNA-seq analysis to analyze their transcriptome-level changes during transdifferentiation. Whole-gene expression analysis showed that the gene expression patterns of iCECs differed significantly from those of neural crest cells. Figure 7 A).

[0109] To further verify the transdifferentiation process of cranial neural crest cells into corneal endothelial cells, the focus was on the expression changes of marker genes in corneal endothelial cells and neural crest cells. RNA-seq analysis showed that the expression of corneal endothelial marker genes (such as Htr1d, Mcam, Prom1) was significantly upregulated in iCECs, while the expression of neural crest cell marker genes (such as Snai1, Sox10, Twist1) was significantly downregulated. Figure 7 B).

[0110] It is noteworthy that transcriptome sequencing analysis of primary corneal endothelial cells (pCECs) after passage revealed significant changes in gene expression patterns due to endothelial-mesenchymal transition (EndMT). Figure 7 A, 7B).

[0111] In summary, RNA-seq analysis results indicate that iCECs possess typical corneal endothelial cell characteristics. Furthermore, these data further support the induction protocol of this invention's ability to effectively promote the directed differentiation of cranial neural crest cells into corneal endothelial cells.

[0112] Example 7: Isolation and Induction Culture of Cranial Neural Crest Cells After Birth

[0113] This study aimed to investigate whether cranial nerve cells in mice still possess the potential to differentiate into corneal endothelial cells after birth. We collected Wnt1-Cre / ROSA26 cells from mice 14 days after birth. tdTomato Primary cranial neural crest cells were isolated from double transgenic mice. After culture, a large number of primary cranial neural crest cells were successfully obtained by day 8. Figure 8 A).

[0114] When cell confluence reached over 80%, the cells were induced using a previously established induction protocol. As the culture progressed, the tdTomato fluorescence expression level gradually decreased. By day 7 of induced differentiation, cells began to aggregate and form a monolayer clonal cluster; by day 14, the induced cells exhibited the typical morphological characteristics of corneal endothelial cells, namely a flattened hexagonal structure. Figure 8 B). The above experimental results indicate that SB431542 can effectively induce cranial neural crest cells in newborn mice to differentiate into corneal endothelial-like cells.

[0115] In summary, the induction protocol developed in this study successfully induced the differentiation of cranial neural crest cells into corneal endothelial cells (iCECs) during the embryonic period and after birth, providing a new cell source for the in vitro expansion and regeneration of corneal endothelial cells. This protocol avoids the potential risks of traditional viral vectors or gene editing, and the prepared iCECs exhibit high consistency with primary CECs in terms of contact inhibition, ion transport, and metabolic functions. These well-developed functional characteristics provide solid experimental evidence for the clinical application of iCECs.

[0116] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A method of promoting differentiation of neural crest cells into corneal endothelial cells, characterized by, The method comprises the following steps: culturing neural crest cells in an induction differentiation medium to obtain corneal endothelial cells. The induction differentiation medium comprises the following components: a basic medium DMEM / F12, an N2 supplement, a B27 supplement, bFGF, EGF, and SB431542.

2. The method of claim 1, wherein, The concentrations of the components in the induction differentiation medium are as follows: (0.5-1) × N2 supplement, (0.5-1) × B27 supplement, (10-25) ng / mL bFGF, (5-15) ng / mL EGF, and (5-10) μM / mL SB431542. Preferably, the concentrations of the components in the induction differentiation medium are as follows: 1 × N2 supplement, 1 × B27 supplement, 20 ng / mL bFGF, 10 ng / mL EGF, and 5 μM / mL SB431542.

3. The method of claim 1, wherein, The neural crest cells comprise cranial neural crest cells, vagal neural crest cells, trunk neural crest cells, and sacral neural crest cells. Preferably, the neural crest cells are cranial neural crest cells.

4. The method of claim 1, wherein, The method comprises the following steps: On Day 0, the neural crest cells are cultured in the induction differentiation medium; On Day 7-15, the corneal endothelial cells are obtained. Preferably, the induction differentiation medium is replaced every 24 h or 36 h. Preferably, the induction differentiation medium is replaced every 24 h.

5. A medium for promoting differentiation of neural crest cells into corneal endothelial cells, characterized by, The medium comprises the following components: a basic medium DMEM / F12, an N2 supplement, a B27 supplement, bFGF, EGF, and SB431542.

6. The medium of claim 5, wherein, The concentrations of the components in the medium are as follows: (0.5-1) × N2 supplement, (0.5-1) × B27 supplement, (10-25) ng / mL bFGF, (5-15) ng / mL EGF, and (5-10) μM / mL SB431542. More preferably, the concentrations of the components in the medium are as follows: 1 × N2 supplement, 1 × B27 supplement, 20 ng / mL bFGF, 10 ng / mL EGF, and 5 μM / mL SB431542.

7. A population of functional corneal endothelial cells of neural crest cell origin, characterized in that, The cell population is obtained by inducing differentiation using the method of any one of claims 1-4. Preferably, the cell population highly expresses the corneal endothelial characteristic genes ZO-1, AQP1, Slc4a11, N-cadherin, Abcg2, and ATPA1.

8. A pharmaceutical composition for preventing and / or treating corneal endothelial decompensation, characterized by, The pharmaceutical composition comprises the cell population of claim 7. Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

9. Use of the medium of any one of claims 5-6 in inducing differentiation of neural crest cells into corneal endothelial cells.

10. Use of the cell population of claim 7 in constructing a cell model of corneal endothelial decompensation, drug screening, or in vitro experimental research.

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