Method for preparing photoreceptor precursor cells of human embryonic stem cell-derived retinal organoids and application thereof

Abstract

The present application relates to a preparation method and application of photoreceptor precursor cells of human embryonic stem cell-derived retinal organoids, and belongs to the field of biotechnology and stem cell engineering. The present application constructs a lentiviral vector carrying an exogenous target gene and infects human embryonic stem cells to obtain a stable expression of engineered stem cell clone group by screening; the clone group is subjected to three-dimensional differentiation, and sequentially undergoes the formation of a blastoid, neural epithelial induction and retinal layering stage to obtain a retinal organoid; finally, the co-expression of photoreceptor precursor cell markers and exogenous genes in the organoid is identified. The present application realizes stable integration of genes at the source of stem cells, avoids the problem of low infection efficiency in the later stage, and provides a complete standardized scheme from gene modification, directional differentiation to function verification. The engineered photoreceptor precursor cells obtained by the method have important application value in the construction of retinal disease models, drug screening and cell therapy development.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and stem cell engineering, and relates to a method for preparing and applying photoreceptor precursor cells of retinal organoids derived from human embryonic stem cells. Background Technology

[0002] Retinitis Pigmentosa (RP) is a group of hereditary retinal diseases characterized by progressive death of photoreceptors (PR) and atrophy of retinal pigment epithelium (RPE). In the late stage, patients lose a large number of photoreceptors, and the key outer segment (OS) is difficult to regenerate, resulting in irreversible loss of light signal transduction function [1-3].

[0003] Photoreceptor cell transplantation derived from stem cells is a potential therapeutic strategy. However, traditional photoreceptor cell transplantation relies on the interaction between transplanted cells and photoreceptor progenitor cells (RPEs) and the normal formation of photoreceptor ostomy (OS), which is difficult to achieve in patients with advanced photoreceptor lesions (RP) [3, 4]. To address this issue, researchers have attempted to introduce optogenetic tools into photoreceptor progenitor cells (PPCs) derived from stem cells, enabling cells to respond directly to light stimulation without relying on OS and RPEs. Garita-Hernandez et al. (2019) validated the feasibility of this strategy by introducing optogenetic tools (Jaws) into PPCs derived from induced pluripotent stem cells (iPSCs) in the late differentiation stage [5, 6]. However, PPCs are close to their post-mitotic state when infected in the late differentiation stage, making it difficult to expand and screen them in large quantities, resulting in low positive yields and unstable expression.

[0004] Meanwhile, the technology for differentiating three-dimensional retinal tissues from pluripotent stem cells (PCSs) has become increasingly mature [7-9]. Zhong et al. (2014) established an optimized scheme for differentiating three-dimensional retinal tissues from human induced pluripotent stem cells, and significantly improved the efficiency and reproducibility of differentiation by quantifying the size of embryoid bodies (EBs) (approximately 200 μm) and seeding density (20-30 bodies / cm2)

[10] . Based on this, researchers have been able to obtain retinal-like tissues rich in mature photoreceptors.

[0005] For applications that require the expression of exogenous genes in differentiated cells (such as optogenetic tools, reporter genes, or the delivery of therapeutic proteins), achieving stable integration of exogenous genes at the stem cell stage is a better strategy. Lentivirals can integrate exogenous genes into the host genome and pass them to daughter cells during cell division, thereby achieving continuous expression throughout the differentiation process

[11] .

[0006] In addition, the differentiation process of PPCs can be tracked in real time by combining reporter gene systems (such as CRX-tdTomato reporter cell line)[9], which makes it easier to monitor the emergence of target cells during the differentiation process.

[0007] Currently, there are two main challenges in introducing exogenous genes into photoreceptor precursor cells in retinal organoids: First, gene manipulation (such as viral infection) of already formed organoids or cells in the later stages of differentiation is often inefficient, with low positive cell yields and unstable exogenous gene expression; Second, there is a lack of a complete and standardized technical solution for stable gene integration from the source (stem cell stage) throughout the entire directed differentiation process, ultimately obtaining and validating engineered photoreceptor precursor cells.

[0008] [1]VERBAKEL SK, VAN HUET RAC, BOON CJF, et al. Non-syndromicretinitis pigmentosa [J]. Progress in Retinal and Eye Research, 2018, 66:157-186. [2]DIAS MF, JOO K, KEMP JA, et al. Molecular genetics and emerging therapeutics for retinitis pigmentosa: Basic research and clinical perspectives[J]. Progress in Retinal and Eye Research, 2018, 63: 107-131. [3]JONES BW, PFEIFFER RL, FERRELL WD, et al. Retinal remodeling in human retinitis pigmentosa [J]. Experimental Eye Research, 2016, 150: 149-165. [4]MARC R E, JONES B W. Retinal remodeling in inherited photoreceptordegenerations [J]. Molecular Neurobiology, 2003, 28(2): 139-147. [5]GARITA-HERNANDEZ M, LAMPIC M, CHAFFIOL A, et al. Restoration ofvisual function by transplantation of optogenetically engineeredphotoreceptors [J]. Nature Communications, 2019, 10(1): 4524. [6]CHUONG A S, MIRI M L, BUSSKAMP V, et al. Noninvasive opticalinhibition with a red-shifted microbial rhodopsin [J]. Nature Neuroscience,2014, 17(8): 1123-1129. [7]BOHRER L R, WILEY L A, WRIGHT A T, et al. Production of clinicalgrade patient iPSC-derived 3D retinal organoids containing transplantablephotoreceptor cells [J]. Stem Cell Research&Therapy, 2025, 16(1): 641. [8]MANDAI M, FUJII M, HASHIGUCHI T, et al. iPSC-Derived RetinaTransplants Improve Vision in rd1 End-Stage Retinal-Degeneration Mice [J].Stem Cell Reports, 2017, 8(1): 69-83. [9]PAN D, XIA XX, ZHOU H, et al. COCO enhances the efficiency of photoreceptor precursor differentiation in early human embryonic stem cell-derived retinal organoids [J]. Stem Cell Research&Therapy, 2020, 11(1): 366.

[10] ZHONG X, GUTIERREZ C, XUE T, et al. Generation of three-dimensional retinal tissue with functional photoreceptors from human iPSCs[J]. Nature Communications, 2014, 5: 4047.

[11] LEGER-CHARNAY E, SLEMBROUCK-BREC A, GOUREAU O. EngineeringSpecific Human iPS Reporter Cell Lines to Generate Optogenetically ModifiedPhotoreceptors [J]. Advances in Experimental Medicine and Biology, 2025,1468: 409-414. Summary of the Invention In view of this, the purpose of this invention is to provide an integrated method for achieving stable integration of exogenous genes at the stem cell stage and for producing high-purity, functionally defined engineered photoreceptor precursor cells in batches through an efficient and reproducible three-dimensional differentiation process.

[0009] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a method for preparing photoreceptor precursor cells of retinal organoids derived from human embryonic stem cells, comprising the following steps: S1: H9 CRX-tdTomato reporter cells were cultured in a Matrigel-coated culture vessel and maintained in mTeSR-E8 complete medium. When the confluence reached 80%-90%, the cells were digested with EDTA and passaged into small clumps. S2: Using a lentiviral vector carrying the exogenous target gene hGtACR2, the cells from step S1 were infected with a multiplicity of 30. After 24 hours of infection, the medium was replaced with mTeSR-E8 medium. After amplification, single-cell clones were obtained by dilution and seeding. Stable cell populations that were EYFP positive and co-expressed OCT4, SOX2, and Nanog were selected. S3: Digest the cells selected in step S2 with EDTA, resuspend them in mTeSR-E8 complete medium containing Blebbistatin, and seed them in well plates containing honeycomb culture sheets. Culture overnight to form embryoid bodies. S4: The embryoids cultured in step S3 were transitionally cultured in a mixed medium of mTeSR-E8 complete medium and neural induction medium, and then completely replaced with neural induction medium for further culture. When the embryoid diameter reached about 200 μm, they were seeded into Matrigel-coated culture plates to allow them to adhere and spread. They were first cultured in neural induction medium, and then replaced with retinal differentiation medium for further differentiation culture. The differentiation of photoreceptor precursor cells was monitored using the CRX-tdTomato reporter system. S5: Select cells with vesicle morphology from step S4, transfer them to an ultra-low adsorption culture dish, and suspend them in retinal differentiation medium to obtain the photoreceptor precursor cells; Preferably, the nucleic acid sequence of the hGtACR2 gene is shown in SEQ ID NO:1; Preferably, the concentration of Matrigel is 0.1 mg / mL, dissolved in DMEM / F12 medium; the concentration of Blebbistatin is 10 μM. Preferably, the transition culture is as follows: on day 1, a medium in which mTeSR-E8 complete medium and neural induction medium are mixed at a volume ratio of 3:1 is used; on day 2, a medium in which mTeSR-E8 complete medium and neural induction medium are mixed at a volume ratio of 1:1 is used; and on day 3, the medium is replaced with complete neural induction medium. Preferably, the formulation of the neural induction medium is: 50 mL DMEM / F12 (1:1, 1×), 0.5 mL N-2 Supplement (100×), 0.5 mL NEAA (100×), and 2 μg / mL heparin solution; Preferably, the retinal differentiation culture medium is formulated as follows: 30 mL DMEM / F12 (1:1, 1×), 20 mL DMEM basic (1×), 1 mL B-27 Supplement (50×, vitamin A-free), and 0.5 mL NEAA (100×). The photoreceptor precursor cells prepared by the method described above; This invention provides an engineered photoreceptor precursor cell, wherein the exogenous gene hGtACR2 is stably integrated into the genome of the cell, and the cell co-expresses the photoreceptor precursor cell marker CRX; Furthermore, the use of the aforementioned photoreceptor precursor cells in the preparation of medicaments for treating retinal degenerative diseases; A pharmaceutical composition comprising the engineered photoreceptor precursor cells and a pharmaceutically acceptable carrier.

[0010] The beneficial effects of this invention are as follows: 1. Source integration, stable and efficient: Lentiviral genetic engineering is carried out in the hESC stage, and the exogenous gene is stably inherited to all daughter cells with cell division, ensuring the continuous and stable expression of the target gene throughout the differentiation process, fundamentally avoiding the problems of low infection efficiency and unstable expression in the later stage of differentiation.

[0011] 2. Integrated process and high degree of standardization: This invention provides a complete technical process covering "stem cell genetic engineering → positive clone screening → three-dimensional retinal directed differentiation → target cell identification", providing a standardized and reproducible solution for preparing genetically modified photoreceptor precursor cells.

[0012] 3. Visual tracking and intuitive verification: By using endogenous reporter gene cell lines (such as CRX-tdTomato) and exogenous reporter genes (such as EYFP), the emergence of photoreceptor precursor cells and the expression of exogenous genes can be monitored in real time and non-destructively during differentiation. The results are then confirmed by immunofluorescence co-localization, making the results intuitive and reliable.

[0013] 4. High cell quality and broad application prospects: The engineered photoreceptor precursor cells obtained by this method have high purity and well-defined functions. They can be directly used to construct retinal disease models, screen drug or gene therapy vectors, and provide a high-quality source of "seed cells" for the development of retinal cell transplantation therapy based on engineered stem cells.

[0014] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0015] To make the objectives, technical solutions, and advantages of the present invention clearer, the preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein: Figure 1This is an overall flowchart of the method of the present invention. A represents the preparation of engineered stem cells, B represents the culture of retinal organoids, and C represents the collection and staining identification of optic vesicles.

[0016] Figure 2 To stably express hGtACR2-EYFP, H9 ESCs formed clonal colonies: (ac) Classical clonal colonies formed after limiting dilution for 2 weeks, shown in the following images: bright field image (a), EYFP fluorescence image (b), Merge image (c), (df) After the same clone was cultured for 3 weeks, the images are shown in the following images: bright field image (d), EYFP fluorescence image (e), Merge image (f).

[0017] Figure 3 Immunofluorescence staining results of OCT4, a marker of clonal pluripotency for H9 ESCs stably expressing hGtACR2: (a) DAPI-labeled cell nuclei; (b) OCT4-positive (red) staining; (c) EYFP fluorescence image; (d) Merge image; (e) bright-field image.

[0018] Figure 4 Immunofluorescence staining results for SOX2, a marker of clonal pluripotency of H9 ESCs stably expressing hGtACR2: (a) DAPI-labeled cell nuclei; (b) EYFP fluorescence image; (c) SOX2 positive staining (red); (d) Merge image; (e) bright field image.

[0019] Figure 5 Nanog immunofluorescence staining results for H9 ESCs clonal pluripotency markers stably expressing hGtACR2: (a) DAPI-labeled cell nuclei; (b) EYFP fluorescence image; (c) Nanog-positive (red); (d) Merge image; (e) bright-field image.

[0020] Figure 6 Suspension culture of embryoid bodies (EBs) and hGtACR2 expression: (a) Bright field image; (b) EYFP fluorescence image; (c) Merge image.

[0021] Figure 7 OCT4 staining results for EB sections on day 3: (a) DAPI-labeled cell nuclei; (b) EYFP fluorescence image; (c) OCT4-positive (red); (d) Merge image; (e) bright-field image.

[0022] Figure 8 Confocal microscopy image of retinal organoids (ROs) on day 39 of differentiation.

[0023] Figure 9Fluorescence images of retinal organoids on day 43 of differentiation: (a) EYFP fluorescence image; (b) tdTomato fluorescence positive (red); (c) Merge image; (d) bright field image.

[0024] Figure 10 Immunofluorescence colocalization results of retinal organoid sections on day 79 of differentiation: (a) DAPI-labeled cell nuclei; (b) EYFP fluorescence image; (c) tdTomato fluorescence image; (d) Merge image; (e) bright-field image. Detailed Implementation

[0025] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments are only schematic representations of the basic concept of the present invention. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0026] The accompanying drawings are for illustrative purposes only and are schematic diagrams, not actual pictures. They should not be construed as limiting the invention. To better illustrate the embodiments of the invention, some parts in the drawings may be omitted, enlarged, or reduced, and do not represent the actual product dimensions. It is understandable to those skilled in the art that some well-known structures and their descriptions may be omitted in the drawings.

[0027] In the accompanying drawings of the embodiments of the present invention, the same or similar reference numerals correspond to the same or similar components. In the description of the present invention, it should be understood that if terms such as "upper," "lower," "left," "right," "front," and "rear" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, they are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, the terms used to describe positional relationships in the drawings are only for illustrative purposes and should not be construed as limiting the present invention. For those skilled in the art, the specific meaning of the above terms can be understood according to the specific circumstances.

[0028] This invention uses the optogenetic protein hGtACR2-EYFP as the exogenous target gene and the H9 CRX-tdTomato reporter cell line as the starting cell.

[0029] Example 1 Preparation and identification of engineered H9 CRX-tdTomato reporter cell line photoreceptor precursor cells stably expressing hGtACR2-EYFP 1. Culture and maintenance of H9 CRX-tdTomato reporter cells: H9 CRX-tdTomato reporter cells were routinely cultured in Matrigel (0.1 mg / mL, in DMEM / F12) coated six-well plates, maintained in mTeSR-E8 complete medium, with 2 mL of fresh medium replaced daily. When cell confluence reached 80%-90%, passage was performed: the medium was aspirated, the cells were washed 1-2 times with 1 mL PBS, 1 mL of 0.5 mM EDTA (in PBS) was added, and the cells were incubated at 37°C for 5-7 min; the EDTA was aspirated, and the cells were gently dispersed into small cell clumps (3-5 cells) by pipetting into new Matrigel-coated wells at a ratio of 1:3-1:5; if obvious differentiation zones (>5%) appeared, they were mechanically scraped off with a sterile needle or pipette tip before changing the medium or passage.

[0030] 2. Lentiviral infection and screening of stable positive cell populations: The human codon-optimized hGtACR2-EYFP fragment was inserted into the lentiviral backbone vector pcRLenti-EF1α-MCS-WPRE via EcoRI and BamHI restriction sites to obtain the recombinant lentiviral vector pcRLenti-EF1α-hGtACR2-EYFP-WPRE. The viral titer was 3.29 × 10⁻⁶. 8 TU / mL.

[0031] H9 CRX-tdTomato reporter cells were infected with lentivirus 24 h after passage, with a multiplicity of infection (MOI) of 30. After adding the virus solution, the cells were incubated for 24 h, followed by a complete replacement with fresh E8 medium. Cells were expanded to passage density after infection, filtered through a 40 μm screen to obtain a single-cell suspension, serially diluted, and seeded at a density of 3-5 cells per well into Matrigel-coated 96-well plates. The medium was not changed for 2-3 days after seeding, then half-volume medium was changed daily thereafter. After clear colony formation, colonies were manually scraped and transferred to 48-well plates for amplification, and then transferred to 6-well plates for further expansion.

[0032] like Figure 2 As shown, clones with strong EYFP fluorescence were observed after 2 weeks of culture. By 3 weeks, the clones had further enlarged, and the fluorescence was uniform and stable, indicating that the exogenous gene had been stably integrated. Pluripotency identification of this cell population yielded the following results: Figures 3-5As shown, EYFP-positive cells co-expressed OCT4, SOX2, and Nanog, demonstrating that the engineering modification did not affect the undifferentiated state of stem cells.

[0033] 3. Three-dimensional differentiation-induced photoreceptor precursor cells: Day 0 of differentiation (D0): Cells were digested with 0.5 mM EDTA, resuspended in mTeSR-E8 complete medium containing 10 μM Blebbistatin, and seeded at 2 × 10⁵ cells / well into 24-well plates with honeycomb culture plates. The cells were incubated overnight at 37°C with 5% CO₂.

[0034] D1: Transfer the EBs in the honeycomb culture plate to a 100 mm low-adsorption culture dish and replace it with 12 mL of transition solution made of E8 complete medium and neural induction medium (NIM) in a 3:1 ratio.

[0035] D2: Replace with a transition solution of E8 complete medium and NIM mixed in a 1:1 ratio.

[0036] Day 3: Replace completely with NIM basal medium. NIM formulation: 50 mL DMEM / F12 (1:1, 1×), 0.5 mL N-2 Supplement (100×), 0.5 mL NEAA (100×), 50 μL heparin (2 mg / mL in PBS, final concentration 2 μg / mL). The resulting embryoids were uniform in size and exhibited bright and uniform EYFP fluorescence under a fluorescence microscope. Figure 6 Section staining confirmed that the internal cells still expressed OCT4 ( Figure 7 ).

[0037] D5-D7: When the diameter of EBs grows to about 200 μm, they are uniformly seeded into Matrigel-coated six-well plates at a density of 20-30 cells / cm2 to allow them to adhere and spread.

[0038] D8-D9: After all EBs are fully attached and developed, add 2 mL of fresh NIM to each well.

[0039] D12: Use NIM for half-volume fluid replacement.

[0040] D16: Completely replace with retinal differentiation medium (RDM), and thereafter change half the medium every 2-3 days. RDM formula: 30 mL DMEM / F12 (1:1, 1×), 20 mL DMEM basic (1×), 1 mL B-27 Supplement (50×, vitamin A-free), 0.5 mL NEAA (100×).

[0041] D28-D35: Monitoring of tdTomato+ fluorescence signal using the CRX-tdTomato reporter system indicated the initiation of CRX expression and the beginning of differentiation of photoreceptor precursor cells.

[0042] After day 30: Under a microscope, all structures exhibiting optic vesicle morphology were manually picked up using a sterile syringe needle and transferred to a new 100 mm ultra-low adsorption culture dish. 10 mL of RDM was added for suspension culture, and the medium was changed by half every 2-3 days thereafter. By day 39, the retinal organoids had formed a clearly defined neuroepithelial layered structure. Figure 8 At D43, distinct organoid morphology was visible under bright field, tdTomato fluorescence was further enhanced, and EYFP green fluorescence was also visible. Figure 9 ).

[0043] 4. Immunofluorescence identification: Organoids from D60 to D120 were collected and frozen sections (10 μm thick). Immunofluorescence staining was performed on the sections, and the nuclei were counterstained with DAPI (2 μg / mL). Images were then observed and acquired under a fluorescence microscope. Figure 10 As shown, in organoid sections on day 79 of differentiation, there is a clear overlap between EYFP-labeled exogenous proteins (green) and tdTomato-labeled photoreceptor precursor cells (red), indicating that the exogenous gene is successfully and stably expressed in CRX-positive photoreceptor precursor cells.

[0044] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing photoreceptor precursor cells of retinal organoids derived from human embryonic stem cells, characterized in that, Includes the following steps: S1: H9 CRX-tdTomato reporter cells were cultured in a Matrigel-coated culture vessel and maintained in mTeSR-E8 complete medium. When the confluence reached 80%-90%, the cells were digested with EDTA and passaged into small clumps. S2: Using a lentiviral vector carrying the exogenous target gene hGtACR2, the cells from step S1 were infected with a multiplicity of 30. After 24 hours of infection, the medium was replaced with mTeSR-E8 medium. After amplification, single-cell clones were obtained by dilution and seeding. Stable cell populations that were EYFP positive and co-expressed OCT4, SOX2, and Nanog were selected. S3: Digest the cells selected in step S2 with EDTA, resuspend them in mTeSR-E8 complete medium containing Blebbistatin, and seed them in well plates containing honeycomb culture sheets. Culture overnight to form embryoid bodies. S4: The embryoids cultured in step S3 were transitionally cultured in a mixed medium of mTeSR-E8 complete medium and neural induction medium, and then completely replaced with neural induction medium for further culture. When the embryoid diameter reached about 200 μm, they were seeded into Matrigel-coated culture plates to allow them to adhere and spread. They were first cultured in neural induction medium, and then replaced with retinal differentiation medium for further differentiation culture. The differentiation of photoreceptor precursor cells was monitored using the CRX-tdTomato reporter system. S5: Select cells with vesicle morphology from step S4, transfer them to an ultra-low adsorption culture dish, and suspend them in retinal differentiation medium to obtain the photoreceptor precursor cells.

2. The preparation method according to claim 1, characterized in that, The nucleic acid sequence of the hGtACR2 gene is shown in SEQ ID NO:

1.

3. The preparation method according to claim 1, characterized in that, The concentration of Matrigel was 0.1 mg / mL, dissolved in DMEM / F12 medium; the concentration of Blebbistatin was 10 μM.

4. The preparation method according to claim 1, characterized in that, The transition culture was as follows: on day 1, a medium containing a 3:1 volume ratio of mTeSR-E8 complete medium and neural induction medium was used; on day 2, a medium containing a 1:1 volume ratio was used; and on day 3, the medium was replaced with complete neural induction medium.

5. The preparation method according to claim 1, characterized in that, The formulation of the neural induction medium is as follows: 50 mL DMEM / F12 (1:1, 1×), 0.5 mL N-2 Supplement (100×), 0.5 mL NEAA (100×), and 2 μg / mL heparin solution.

6. The preparation method according to claim 1, characterized in that, The formulation of the retinal differentiation culture medium is as follows: 30 mL DMEM / F12 (1:1, 1×), 20 mL DMEM basic (1×), 1 mL B-27 Supplement (50×, without vitamin A), and 0.5 mL NEAA (100×).

7. Photoreceptor precursor cells prepared by the preparation method according to any one of claims 1-6.

8. An engineered photoreceptor precursor cell, characterized in that, The cells have a stable integration of the exogenous gene hGtACR2 into their genome, and the cells co-express the photoreceptor precursor cell marker CRX.

9. Use of the photoreceptor precursor cell of claim 7 or the engineered photoreceptor precursor cell of claim 8 in the preparation of a medicament for treating retinal degenerative diseases.

10. A pharmaceutical composition, characterized in that, It comprises the photoreceptor precursor cells of claim 7 or the engineered photoreceptor precursor cells of claim 8, and a pharmaceutically acceptable carrier.

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