Method for preparing retinal precursor cells, photoreceptor precursor cells and cone cells from pluripotent stem cells

Abstract

The present application provides methods of generating retinal precursor cells, photoreceptor precursor cells, and further cone cells from human pluripotent stem cells. The invention also relates to an induction medium used in the method and a combination thereof. The invention also relates to clinical and non-clinical applications of the cells obtained by the invention.

Description

Technical Field

[0001] The present invention relates to a method for producing retinal progenitor cells from human pluripotent stem cells, such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) by directed induction, and further producing photoreceptor precursor cells (PPCs) and cone cells (Cones). The present invention also relates to an induction medium used in the method and a combination thereof. It also relates to clinical and non-clinical applications of cells obtained by the present invention. Background Art

[0002] Cones are essential for human vision of daylight, color and high acuity and are particularly valuable in cell therapy. In fact, macular retinopathy involving cone loss is the leading cause of blindness.

[0003] The classic differentiation method is to first culture iPSCs adherently for 28 days and then switch to suspension culture for 231 days, thereby obtaining human cone cells with lower purity (Gonzalez-Cordero A, et al., Recapitulation of Human Retinal Development from Human Pluripotent Stem Cells Generates Transplantable Populations of Cone Photoreceptors. Stem Cell Reports. 2017 Sep 12; 9 (3): 820-837), and the functionality and maturity of the cells are also lower.

[0004] The method reported later was to first culture iPSCs in suspension to form EBs, culture the cells in suspension for 7 days, then switch to adherent culture for 26 days, and then switch to suspension culture for 168 days or more, wherein cone cells were expanded by adding retinoic acid (RA) and taurine. However, the cone cells obtained in this way were of low purity and needed to be sorted and purified (Cowan CS, et al., Cell Types of the Human Retina and Its Organoids at Single-Cell Resolution. Cell. 2020 Sep 17; 182(6): 1623-1640).

[0005] In order to improve the purity of cone cells, a recent method is to edit human embryonic stem cells to obtain mature cone cells with higher purity (Elisa Cuevas et al., NRL - / - gene edited human embryonic stem cells generate rod-deficient retinal organoids enriched in S-cone-like photoreceptors, Stem Cells, Volume 39, Issue 4, April 2021, Pages 414–428).

[0006] It can be seen that the currently available methods of obtaining cone cells from pluripotent stem cells through induced differentiation culture have problems such as long cycle, low cell purity, and low yield. Obtaining cone cells through gene editing methods will introduce gene mutations into the product cells, thus causing potential risks. In addition, fetal bovine serum (FBS) is introduced in the above differentiation methods, which is not conducive to clinical application.

[0007] Therefore, there is an urgent need in the art for a method for differentiating photoreceptor precursor cells or cone cells that is shorter in time, can obtain higher purity and is suitable for clinical use. Summary of the invention

[0008] The present invention establishes a method for generating RPCs from ESCs or iPSCs through directed differentiation, and further generating PPCs and cone cells. The method is time-saving, does not involve gene editing and fetal bovine serum, and the proportion of cells with desired characteristics in the obtained cell population is high, thereby meeting the above needs and completing the present invention.

[0009] Therefore, in the first aspect, the present invention relates to a method for generating RPCs from pluripotent stem cells, the method comprising forming pluripotent stem cells into embryoid bodies (EBs), and culturing the embryoid bodies in a culture medium containing a specific combination of factors, preferably performing suspension culture and adherent culture in sequence.

[0010] In a second aspect, the present invention relates to RPCs produced by the method of the first aspect. In one embodiment, the majority of the RPCs produced by the method of the first aspect, such as at least 75% or more, preferably at least 80% or more, and more preferably at least 85% or more, are VSX2 + RPCs.

[0011] In a third aspect, the present invention relates to a method for generating PPCs from pluripotent stem cells, the method comprising further culturing the culture containing retinal progenitor cells obtained by the method into photoreceptor progenitor cells.

[0012] In a fourth aspect, the present invention relates to PPCs produced by the method of the third aspect. In one embodiment, the majority of the PPCs produced by the method of the third aspect, such as at least 65% or more, preferably at least 80% or more, are CRX + and RCVRN + PPCs; more preferably, the PPCs are cone-predisposed.

[0013] In a fifth aspect, the present invention relates to a method for preparing photoreceptor cells, especially cone cells, comprising further culturing a culture containing retinal progenitor cells obtained by the method of the first aspect into photoreceptor progenitor cells, or comprising further culturing a culture containing photoreceptor progenitor cells obtained by the method of the third aspect into photoreceptor cells.

[0014] In a sixth aspect, the present invention relates to photoreceptor cells, particularly cone cells, produced by the method of the fifth aspect.

[0015] In a seventh aspect, the present invention relates to the use of the RPCs of the second aspect, the PPCs of the fourth aspect, or the photoreceptor cells such as cone cells of the sixth aspect, including clinical and non-clinical uses. For example, the clinical use is the use as a drug. For example, the non-clinical use is the use for scientific research and drug development.

[0016] In an eighth aspect, the present invention relates to one or more culture media for inducing differentiation of pluripotent stem cells into RPCs, PPCs or photoreceptor cells such as cone cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to facilitate a better understanding of the features and advantages of the present invention, the following detailed description of the invention and the accompanying drawings are provided. However, those skilled in the art should understand that these contents are only for the purpose of illustrating the present invention, rather than limiting the present invention. The scope of the present invention shall be subject to the claims.

[0018] Figure 1 The process steps of a representative method of the present invention (Example 1) are shown, including the culture media used and factors added at different stages.

[0019] Figure 2 Bright field images of NR-like structures generated from human pluripotent stem cells (hPSCs) are shown (Example 1, D30).

[0020] Figure 3 Bright field images of RPCs differentiated from hPSCs are shown (Example 1, D45).

[0021] Figure 4The expression levels of markers of cells obtained in Example 1 of the present invention based on RT-qPCR and immunofluorescence staining results are shown. (A) Bar graph of the relative expression levels of PAX6, RAX and VSX2 at day 34 (D34); (B) Bar graph of the relative expression levels of PAX6, RAX and CRX at day 45 (D45); (C) Immunofluorescence staining results of RPCs obtained in Example 1.

[0022] Figure 5 The RT-qPCR-based expression levels of markers of PPCs obtained when different factors (SHH, SAG or DAPT) were used in Example 3 of the present invention are shown. (A) Relative expression level of RAX; (B) Relative expression level of CRX.

[0023] Figure 6 The immunofluorescence staining results of PPCs obtained when different factors (SHH, SAG or DAPT) were used in Example 3 of the present invention are shown.

[0024] Figure 7 The expression levels of markers of cells cultured in Example 5 of the present invention based on RT-qPCR and immunofluorescence staining results are shown. (A) Relative expression levels of RPC markers RAX and VSX2 and PPC marker CRX at different time points (D30, D45, D60); (B) Immunofluorescence staining results of PPCs obtained in Example 5.

[0025] Figure 8 The immunofluorescence staining results of the photoreceptor cell marker (RCVRN) of the cells obtained in Example 7 of the present invention are shown.

[0026] Fig. 9 The results of immunofluorescence staining of the photoreceptor precursor cell marker (CRX) and cone cell marker (ARR3) of the cells obtained in Example 7 of the present invention are shown.

[0027] Fig.10 ARR3 is shown in a zoomed-in view + Cone cell morphology. (A) and (B) are two ARR3 + Cones. DETAILED DESCRIPTION OF THE INVENTION

[0029] Unless otherwise specified in the present invention, the meanings of all technical and scientific terms used herein are to be interpreted according to the common understanding by one of ordinary skill in the art to which the present invention belongs.

[0030] 1. Definition

[0031] "Or" has the same meaning as "and / or" herein unless explicitly stated.

[0032] In the context of the present invention, unless otherwise stated, "comprising", "including" and "containing" should be understood to mean including the listed elements, such as a component, a feature, a step or a group thereof, but not excluding any other elements, such as other components, properties and steps. When used herein, the term "comprising" or any variation thereof can be replaced with "containing", "including" or "having" or synonymous variations. In certain embodiments, "comprising" also includes the situation of "consisting of..."

[0033] 1.1 Cell types at different stages

[0034] The term "cell population" herein refers to a population comprising a plurality of cells, wherein the plurality of cells may be one type of cell or a plurality of types of cells. In the method of the present invention, cell populations at different culture stages or differentiation stages have different cell compositions. Sometimes a cell population is referred to according to the cell type that is dominant in number, but this does not mean that the cultured cell population at that stage does not contain any other types of cells at all.

[0035] "Cell population enriched in..." in the present invention refers to a situation where a specific cell population contains a higher proportion of a specific type of cells compared to cell populations in other culture stages, especially compared to a starting cell population or an intermediate cell population used to obtain the cell population.

[0036] The term "stem cell" or "pluripotent stem cells (PSCs)" refers to cells in an undifferentiated state, characterized by being able to self-renew under appropriate conditions and to differentiate into various types of differentiated cells under appropriate conditions. Unless otherwise specified, the pluripotent stem cells herein are human pluripotent stem cells (hPSCs).

[0037] The term "embryonic stem cells" or "ESCs" as used herein refers to pluripotent stem cells derived from embryos. The term "induced pluripotent stem cells" or "iPSCs" as used herein refers to pluripotent stem cells produced by reprogramming differentiated somatic cells. The method of the present invention can use ESCs or iPSCs as the starting material for directed differentiation. In a specific embodiment of the present invention, ESCs or iPSCs are mammalian origin, particularly human origin. The cells can be directly obtained from commercial sources or can be prepared, for example, by reprogramming somatic cells into iPSCs. The iPSCs can be obtained by any reprogramming method. In a preferred embodiment, the ESCs and iPSCs are cells that meet clinical applications, such as clinical-grade cells. The ESCs are ESCs isolated from human embryos within 14 days of fertilization without in vivo development.

[0038] The term "embryoid body" or "EBs" as used herein refers to cell aggregates grown from ESCs and iPSCs by three-dimensional culture. EBs can be formed by suspension culture of pluripotent stem cells. However, it is difficult to obtain EBs of uniform size and shape in the traditional way of culturing EBs. In a preferred embodiment, the EBs of the present invention are EBs of relatively uniform size and shape obtained by suspension culture by a specific culture method, for example, each EB is roughly circular. The preferred EB culture method will be described in detail in the culture method section below.

[0039] The term "NR" refers to the neural retina, which is the inner layer of the double-layered optic cup during its development.

[0040] The term "NR-like structure" refers to a structure mainly composed of RPCs and similar to NR during the in vitro differentiation of pluripotent stem cells. In this article, the term "NR-like structure" can be used interchangeably with the term "NR".

[0041] The term "retinal progenitor cells" or "RPCs" is a multipotent cell that is capable of self-renewal and has the ability to develop into all cell types in the retina, including photoreceptor cells such as cones and rods, as well as photoreceptor pigment epithelial cells and Muller glial cells. Retinal progenitor cells express a variety of genes, including early retinal development genes such as VIM (vimentin), NES (nestin), PAX6, SOX2, RAX, OTX2, SIX6 and CHX10 (VSX2). Some of these genes are continuously expressed during the culture of retinal progenitor cells. Retinal progenitor cells can be identified by the transcription factors that are unique to eye cells expressed by them and distinguished from other cells such as pluripotent stem cells. The presence of RPCs can be confirmed by detecting one or more of the above genes, particularly genes that are specifically expressed in RPCs. For example, the formation of RPCs can be confirmed by detecting the expression of PAX6, RAX and / or VSX2. Methods for determining the expression of specific genes are well known in the art.

[0042] The term "RPC aggregates" as used herein refers to spherical aggregates formed by suspension culture of optic nerve fate cells separated from NR-like structures. RPC aggregates are a group consisting essentially of retinal progenitor cells.

[0043] The term "photoreceptor progenitor cells" or "PPCs" refers to genes encoding transcription factors that can be induced to differentiate into photoreceptor cells (including cones and rods) and express transcription factors specific to photoreceptor progenitor cells, such as CRX and RCVRN. The presence of PPCs can be confirmed by detecting one or more of the above genes. In a specific embodiment, a cell population in which the proportion of PPCs expressing CRX and RCVRN is greater than 70% can be obtained by the method of the present invention. "Cone-prone" photoreceptor progenitor cells refer to photoreceptor progenitor cells that are more likely to differentiate into cones than into rods.

[0044] The term "photoreceptor cell" refers to a visual cell capable of phototransduction, which is capable of converting received light signals into signals that can stimulate biological processes. Photoreceptor cells mainly include cones, rods and intrinsically photosensitive retinal ganglion cells.

[0045] The term "cone" or "Cones" refers to a type of photoreceptor cell, which is divided into three types: L, M, and S according to the wavelength of light to which it responds. Cones express ARR3, M / L opsin, and S opsin. The presence of cones can be confirmed by detecting one or more of the above genes. For example, the formation of cones can be confirmed by detecting the expression of ARR3.

[0046] 1.2 Culture medium

[0047] The composition of the culture medium used in each step of the present application is very important for the formation of PPCs and the subsequent successful differentiation of Cones, and particularly important is the composition of factors in the differentiation induction medium used to induce stem cell differentiation.

[0048] The term "culture medium" is a mixture that contains various nutrients required for the growth of a certain type of cell. The culture medium can be prepared by adding "additives" to the "basal culture medium". "Additives" in a broad sense refer to additional components required for cell culture but not included in the basal culture medium, including proteins, lipids, amino acids, vitamins, hormones, cytokines, growth factors, etc. However, in this article, the term "additive" does not include inhibitors such as ROCK inhibitors that are added separately to hPSC culture medium or EB culture medium, nor factors such as various induction factors. "Additives" in the context of the present invention mainly refer to "nutritional additives", particularly those that can be directly purchased commercially and are usually used in conjunction with culture media.

[0049] "Factors" added to the culture medium refer to those agents, such as inhibitors, inducers, etc., that are used in significantly lower amounts (e.g., by weight or volume) than in the basal culture medium and nutrient additives.

[0050] 1.3 Cultivation methods

[0051] The term "suspension culture" in the context of the present invention refers to a culture method in which cells or cell aggregates are allowed to proliferate in a freely suspended state in a liquid culture medium. Suspension culture can be achieved by using a culture container with low adsorption, such as a low adsorption culture plate or an ultra-low adsorption culture plate.

[0052] The term "adherent culture" in the context of the present invention refers to a culture method in which cells are grown on an artificial matrix, wherein the cells are usually grown as a monolayer of cells. Adherent culture usually requires cells to be seeded at a specific density when seeding.

[0053] The term "substrate" or "matrix glue" refers to a substance between the culture container and cells in adherent culture, which helps the cells to attach and grow.

[0054] The term "enzyme treatment" or "digestion" in the context of the present invention refers to a process in which cells (such as aggregates) that are aggregated are broken up by adding enzymes in order to calculate the inoculation density or, in some cases, for better culture results. In some cases, mechanical methods can also be used instead of enzyme treatment to disperse cell aggregates into a looser structure or into single cells.

[0055] The term "culture container" refers to a container for containing culture medium and cells, which may be a culture flask, culture plate, etc. The culture container may be appropriately selected depending on the conditions of a specific culture step.

[0056] 2. Cultivation method of the present invention

[0057] For the purpose of convenient reference only, the method of the present application can be divided into a plurality of steps, each of which includes one or more culture processes and / or treatments. Steps (1) to (5) and optional step (6) of the method are basically divided based on functional changes in the culture medium, changes in the culture mode (adherent or suspension culture) and / or the form of the cell culture obtained at the end of each step.

[0058] 2.1 Step (1): Preparation of human pluripotent stem cells

[0059] Human pluripotent stem cells, such as ESCs or iPSCs, can be obtained through commercial channels, or can be prepared by themselves without violating relevant regulations, such as preparing iPSCs from somatic cells by reprogramming methods. In a preferred embodiment, human pluripotent stem cells are cells suitable for clinical applications, such as clinical-grade human pluripotent stem cells. In this case, it is preferred to use a maintenance medium suitable for clinical applications, such as a GMP-grade or CTS-grade medium.

[0060] Before differentiation to EB, human pluripotent stem cells can be maintained in culture. When the human pluripotent stem cells are from a commercial source, the maintenance culture can be carried out using a culture medium recommended by the supplier. The maintenance culture can also be carried out using a culture medium and a culture method conventionally used in the art for culturing pluripotent stem cells.

[0061] For example, the medium for maintaining the culture in step (1) of the present method may be selected from the group consisting of: hPSC XF Medium, CTS TM Essential 8 Medium, Basic03 culture medium, StemMACS TM iPS-Brew medium, ACF medium or TeSR-AOF medium.

[0062] In a specific embodiment, the maintenance culture is carried out using hPSC XF medium (Biological Industries, BI) was used.

[0063] In some embodiments, no additional factors are added to the maintenance culture medium in order to maintain the self-renewal and stemness of the pluripotent stem cells.

[0064] The maintenance culture can be carried out in a culture container such as a culture plate, a culture flask or a cell factory. In one embodiment, the maintenance culture is carried out in a 6-well culture plate.

[0065] The seeding density for maintaining the culture can be determined by a person skilled in the art according to conventional methods. For example, it can be about 0.8×10 4 cells / cm 2 Up to about 5×10 5 cells / cm 2 , preferably about 1×10 4 cells / cm 2 To about 1×10 5 cells / cm 2 , more preferably about 1.2×10 4 cells / cm 2 Up to about 5×104 cells / cm 2 , and more preferably about 1.5×10 4 cells / cm 2 to about 1.8×10 4 cells / cm 2 , for example 1.65×10 4 cells / cm 2 .

[0066] In a preferred embodiment, the culture is maintained in an adherent manner. Preferably, the culture vessel is coated with a matrix. The matrix can be any suitable matrix conventionally used in the art. One example of a matrix commonly used in cell culture is laminin. Specific commercially available laminin matrices such as Laminin-521, such as LN521, MX521, and CT521 available from BioLamina.

[0067] Maintenance culture can continue for any length of time, such as 3-5 days, or longer.

[0068] In a preferred embodiment, after the cells are cultured until the confluence reaches about 80% or more, preferably 85% or more or 90% or more, the pluripotent stem cells maintained in culture can be used in subsequent steps.

[0069] The peeling and / or dispersion can be performed by mechanical means or chemical means such as enzyme treatment for subsequent steps, such as resuspension, counting, inoculation, etc. The enzyme treatment can use any suitable digestive enzyme, such as collagenase, recombinant pancreatin. In a preferred embodiment, TrypLE TM Select enzyme (Gibco) to dissociate cells, preferably CTS TM TrypLE TM Select enzyme (Gibco). In order for the enzyme to work better, the culture medium is removed, and the cells are preferably washed before the enzyme treatment. The process of enzymatic digestion can be terminated by adding culture medium. For the convenience of subsequent experiments, the same culture medium as the subsequent culture can be used to terminate enzymatic digestion and resuspend the cells, for example, using a culture medium for inducing human pluripotent stem cells to form embryoid bodies (EBs).

[0070] Preferably, the cells are counted before the next step of culturing. Before the maintenance culture of pluripotent stem cells is used in the subsequent steps of the present invention, it is preferably washed and the cells are counted. In order to count the cells, the cells need to be peeled off and dispersed into single cells. The counting can be performed by methods known in the art.

[0071] 2.2 Step (2): Differentiation of human pluripotent stem cells (hPSCs) to embryoid bodies (EBs)

[0072] In this step, pluripotent stem cells (especially ESCs or iPSCs) are cultured in a medium for embryoid body formation to differentiate into EBs.

[0073] The culture medium used to induce pluripotent stem cells to differentiate into EBs may be the same as or different from the culture medium used to maintain hPSCs in step (1). hPSC XF medium (Biological Industries, BI).

[0074] In a preferred embodiment, a ROCK inhibitor is further added to the culture medium in step (2). ROCK inhibitors are a class of protein kinase inhibitors that inhibit the activity of rho-associated protein kinase (ROCK) belonging to the serine-threonine protein kinase family and prevent cells from apoptosis after digestion or resuscitation. ROCK is involved in regulating the shape and movement of cells by acting on the cytoskeleton, and also regulates the immortalization and differentiation of cells. Exemplary ROCK inhibitors include, but are not limited to, Y-27632. For example, the ROCK inhibitor, such as Y-27632, is supplemented into the culture medium at a concentration of 1-100 μM, preferably about 5-50 μM, and more preferably about 10 μM.

[0075] In the culture of step (2), for reference, when using a low-adsorption 96-well plate for culture, approximately 1×10 3 cells / well to 3×10 4 The cells are seeded at a density of approximately 1 × 10 cells / well, preferably approximately 1 × 10 4 cells / well to 2×10 4 cells / well, more preferably about 1×10 4 cells / well.

[0076] In a preferred embodiment, the reagents and culture media used in step (2), such as the culture medium and ROCK inhibitor for differentiating pluripotent stem cells into EBs, are reagents and culture media suitable for clinical applications, such as GMP-grade or CTS-grade reagents and culture media.

[0077] The culture in step (2) can be carried out in a culture container such as a culture plate or a culture bottle. In one embodiment, the maintenance culture is carried out in a 96-well culture plate. In a preferred embodiment, a low-adsorption or ultra-low-adsorption culture plate is used.

[0078] The culture temperature of step (2) is preferably 37° C. Preferably, the culture is carried out under 5% CO2. The culture can last for 12 to 72 hours, preferably 2 days. Usually, the formation of embryoid bodies can be observed on the first day of inoculation. The embryoid bodies formed are similar in size, spherical, and have a smooth surface.

[0079] The present invention may also use other methods for forming embryoid body aggregates from pluripotent stem cells.

[0080] 2.3 Step (3): Directed differentiation of embryoid bodies (EBs) into retinal progenitor cells (RPCs)

[0081] Step (3) is a key step in this application and is crucial to the determination of cell fate. The inventors of the present invention innovatively designed a culture method that combines suspension culture and adherent culture, and used a specific combination of factors to enable embryoid bodies to differentiate into retinal progenitor cells.

[0082] Step (3) includes at least two stages: a suspension culture stage of embryoid bodies, such as step (3a), and an adherent culture stage, such as steps (3b) and (3c).

[0083] 2.3.1 Step (3a): EB suspension culture

[0084] The medium used for the suspension culture of EBs in step (3a) contains factors that help induce neural differentiation of neuroectoderm.

[0085] In some embodiments, the factors in step (3a) comprise one or more Wnt signaling pathway inhibitors, and one or more TGF-β superfamily signaling pathway inhibitors; or consist of one or more Wnt signaling pathway inhibitors, and one or more TGF-β superfamily signaling pathway inhibitors.

[0086] The Wnt signaling pathway inhibitor may be selected from one or more of the following groups, preferably one or two: XAV939, DDK1, IwR1e and CKI-7.

[0087] The TGF-β superfamily is divided into the TGF-β subfamily and the BMP subfamily. For the convenience of description below, the TGF-β subfamily signaling pathway inhibitor is referred to as a "TGF-β signaling pathway inhibitor", and the BMP subfamily signaling pathway inhibitor is referred to as a "BMP signaling pathway inhibitor". The TGF-β signaling pathway inhibitor can be selected from one or more of the following groups: SB431542, A83-01 and Repsox. The BMP signaling pathway inhibitor refers to an inhibitor of the bone morphogenetic protein signaling pathway. Different BMP signaling pathway inhibitors can play a role by inhibiting different targets in the signaling pathway, such as a BMP inhibitor that inhibits BMP or an ALK inhibitor that inhibits ALK. The BMP signaling pathway inhibitor used in step (3a) can be selected from one or more of the following groups, preferably one or two: Noggin, DMH-1, Dorsomorphin, COCO and LDN-193189.

[0088] In a preferred embodiment, the one or more TGF-β superfamily signaling pathway inhibitors in step (3a) comprise at least one TGF-β signaling pathway inhibitor, and at least one BMP signaling pathway inhibitor.

[0089] In a preferred embodiment, the culture medium in step (3a) comprises or consists of one or more of Noggin, SB431542, XAV939, DMH-1, DDK1, CKI-7, IwR1e and Dorsomorphin.

[0090] In a preferred embodiment, the culture medium in step (3a) comprises one or more of Dorsomorphin, SB431542 and IwR1e, more preferably all of them. In a specific embodiment, the combination of factors in the culture medium in step (3a) is Dorsomorphin, SB431542 and IwR1e. In another specific embodiment, the combination of factors in the culture medium in step (3a) is Dorsomorphin, SB431542, XAV939 and IwR1e. In another specific embodiment, the combination of factors in the culture medium in step (3a) is Dorsomorphin, SB431542, Noggin, IwR1e and XAV939. In these embodiments, the concentration of Dorsomorphin can be 0.5-2 μM, preferably 0.75-1.5 μM, and most preferably 1 μM; the concentration of SB431542 can be 2-15 μM, preferably 2-10 μM, more preferably 5-10 μM, and most preferably 5 μM; the concentration of IwR1e can be 1-9 μM, preferably 2-5 μM, and most preferably 3 μM; the concentration of Noggin can be 10-500 ng / mL, preferably 20-300 ng / mL, and most preferably 50 ng / mL; the concentration of XAV939 can be 0.5-2 μM, preferably 0.75-1.5 μM, and most preferably 1 μM.

[0091] As the basic culture medium in step (3a), a culture medium suitable for neural cell culture can be used, for example, the culture medium can be selected from: DMEM / F-12 culture medium, DMEM culture medium, Neurobasal TM In one embodiment, the basal medium is DMEM / F-12 medium. In one embodiment, the basal medium is DMEM / F-12 medium and Neurobasal TM A mixture of culture media, for example, a mixture obtained by mixing two culture media at a volume ratio of about 1:1.

[0092] Preferably, nutritional supplements are added to the basal medium. Exemplary nutritional supplements include N-2, B27, non-essential amino acids (NEAA), and L-glutamine. These additives can be obtained commercially. In a specific embodiment, the medium in step (3a) is supplemented with N-2 additive, NEAA and / or L-glutamine (e.g., GlutaMAX TM -I additives). The added concentration of the nutritional additives can be determined according to the guidance concentration in the commercial instructions.

[0093] In a specific embodiment, the EBM medium in Example 1, 3 or 5 is used in step (3a).

[0094] In one embodiment, the suspension of EBs is cultured in a culture container such as a culture plate or a culture flask, preferably a low-adhesion culture plate, more preferably a low-adhesion 6-well culture plate.

[0095] Typically, the culturing in step (3a) is performed at 37° C. The EBs are cultured for about 3 to 10 days, preferably about 5 days, and then they are advanced to the next step.

[0096] Preferably, the EB culture medium is replaced with fresh culture medium every day. At the end of step (3a), the EBs have a spherical shape with a smooth surface and an enlarged volume.

[0097] 2.3.2 Step (3b) - Step (3c): EB adherence culture

[0098] Step (3b) of the method is to allow EBs grown to a desired stage to grow in an adherent manner to induce the formation of NR-like structures.

[0099] After the previous step, the EBs are transferred to a culture vessel coated with a matrix for adherent culture. The matrix can be any suitable matrix conventionally used in the art, such as laminin. Specific commercially available laminin matrices are, for example, Laminin-521, such as LN521, MX521 or CT521 available from BioLamina.

[0100] In the context of the present invention, the culture medium used in step (3b)-step (3c) is referred to as NRM culture medium. Step (3b)-step (3c) can use different culture media or the same culture medium. When different culture media are used, they are respectively referred to as NRM1 and NRM2.

[0101] 2.3.2.1 Culture medium for step (3b)

[0102] During the first few days of adherent culture, for example, the first 5-25 days, such as the first about 2 weeks, a medium having the same composition as that of the previous step (3a) is used, ie, step (3b).

[0103] 2.3.2.2 Culture medium for step (3c)

[0104] After step (3b), the culture medium is replaced with a culture medium having a different combination of induction factors, or the culture medium is not replaced and the culture is continued for about 5-40 days, such as 5-15 days, such as about 9 days.

[0105] When the culture medium is replaced, the culture medium in step (3c) is changed to NRM2 culture medium. NRM2 culture medium preferably uses factors that promote retinal formation. In this case, the factors in the culture medium in step (3c) include:

[0106] (i) one or more of pyruvic acid, retinoic acid, and taurine, preferably at least two, such as a combination of retinoic acid and taurine, or a combination of pyruvic acid, retinoic acid, and taurine;

[0107] (ii) optionally one or more Notch signaling pathway inhibitors, and

[0108] (iii) optionally one or more Hedgehog / Smoothened signaling pathway agonists.

[0109] In the culture medium of step (3c), the concentration of pyruvate may be 0.5-2 mM, preferably 0.75-1.5 mM, most preferably 1 mM; the concentration of retinoic acid may be 0.05-2 μM, preferably 0.1-1 μM, most preferably 0.1 μM; and / or the concentration of taurine may be 50-200 μM, preferably 75-150 μM, most preferably 100 μM.

[0110] The Notch signaling pathway inhibitor is preferably DAPT. The concentration of DAPT can be 5-20 μM, preferably 7.5-15 μM, and most preferably 10 μM.

[0111] The Hedgehog / Smoothened signaling pathway agonist may be selected from SAG or Sonic Hedgehog protein (SHH). The SHH may be a recombinant Sonic Hedgehog protein. The concentration of the SAG may be 50-500 nM, preferably 100-300 nM, and most preferably 100 nM. The concentration of the SHH may be 10-500 ng / mL, preferably 20-300 ng / mL, and most preferably 50 ng / mL.

[0112] In a preferred embodiment, step (3c) only requires adding one of a Notch signaling pathway inhibitor and a Hedgehog / Smoothened signaling pathway agonist, preferably a Hedgehog / Smoothened signaling pathway agonist such as SAG or SHH.

[0113] In a specific embodiment, the factors in the culture medium of step (3c) are pyruvic acid, retinoic acid and taurine. In some specific embodiments, the factors in the culture medium of step (3c) are pyruvic acid, retinoic acid and taurine, and one of SAG, SHH and DAPT.

[0114] As the basic culture medium in step (3c), a culture medium suitable for neural cell culture can be used, for example, the culture medium can be selected from: DMEM / F-12 culture medium, DMEM culture medium, Neurobasal TMIn a specific embodiment, the basal medium is a mixture of DMEM / F-12 medium and DMEM medium, for example, a mixture obtained by mixing the two culture media at a volume ratio of about 1:1. In a specific embodiment, the basal medium is a mixture of DMEM / F-12 medium and Neurobasal TM A mixture of culture media, for example, a mixture obtained by mixing two culture media at a volume ratio of about 1:1.

[0115] Preferably, nutritional supplements are added to the basal medium. Exemplary nutritional supplements include N-2, B27, and L-glutamine. These additives can be obtained commercially. In a specific embodiment, the medium in step (3c) is supplemented with N-2 additive, B-27 additive, and L-glutamine (e.g., GlutaMAX TM -I additives). The added concentration of the nutritional additives can be determined according to the guidance concentration in the commercial instructions.

[0116] In another embodiment, step (3c) uses the same culture medium and factors as step (3a) and step (3b). For example, the NRM culture medium in steps (3b) and (3c) and the EBM culture medium in step (3a) have exactly the same composition. Such a technical solution can make the entire method simpler and more convenient to operate.

[0117] In a specific embodiment, the NRM2 medium in Examples 1 and 3, or the NRM medium in Example 5 is used in step (3c).

[0118] At the end of step (3c), EBs differentiated to form NR-like structures composed of RPCs.

[0119] 2.4 Step (4): Formation of RPC aggregates from NR-like structures

[0120] This step is intended to allow the NR-like structures generated in step (3) to form RPC aggregates through suspension culture. The culture medium used in step (4) is referred to as "RPCM".

[0121] As the basic culture medium in step (4), a culture medium suitable for neural cell culture can be used, for example, the culture medium can be selected from: DMEM / F-12 culture medium, DMEM culture medium, Neurobasal TM In a specific embodiment, the basal medium is Neurobasal TM Culture medium.

[0122] Preferably, a nutritional supplement is added to the basal medium. Exemplary nutritional supplements include N-2, B27, and L-glutamine. These additives can be obtained commercially. In a specific embodiment, the medium in step (4) is supplemented with B-27 additives and L-glutamine, such as GlutaMAX TM -I Additives. The added concentration of the nutritional additives can be determined according to the guidance concentration in the commercial instructions.

[0123] In some embodiments, the RPCM medium used in step (4) is obtained by further adding a Notch signaling pathway inhibitor to the NRM2 medium in step (3c). The Notch signaling pathway inhibitor is preferably DAPT. The concentration of the DAPT can be 5-20 μM, preferably 7.5-15 μM, and most preferably 10 μM.

[0124] In some embodiments, step (4) uses the same factors as the previous step, i.e., step (3c). In further embodiments, the RPCM medium of step (4) has exactly the same composition as the NRM1 medium or NRM2 medium of step (3c).

[0125] In a specific embodiment, the RPCM culture medium in Example 1, 3 or 5 is used in step (4).

[0126] To achieve suspension culture, in one embodiment, the RPC suspension aggregates are cultured in a culture container such as a culture plate or a culture bottle, preferably a low-adsorption culture plate, more preferably a low-adsorption 6-well culture plate.

[0127] For example, the amount of cells initially inoculated into a low-adsorption 6-well culture plate in step (4) is the amount of cells of the NR-like structure from a 6-well culture plate.

[0128] Typically, the culturing in step (4) is performed at 37° C. The RPC suspension aggregates are cultured for about 12-72 hours and then proceed to the next step.

[0129] At the end of step (4), RPC aggregates are formed, which have spherical and smooth surface morphological characteristics. In the method of the present application, RPC aggregates are mainly composed of RPCs. In the method of the present application, RPC aggregates are usually formed 30-60 days after the start of differentiation, that is, after the RPCs are cultured in the culture medium of step (4) of the present invention for about 1 day.

[0130] 2.5 Step (5): Further cultivation of RPC aggregates

[0131] This step is intended to further direct the differentiation of the RPCs formed in step (4) and to further improve the purity of the RPCs.

[0132] In some embodiments, after step (4), RPCs can continue to be cultured in the same culture medium to allow them to continue to differentiate into RPCs.

[0133] In some embodiments, compared to the culture medium used in step (4), the Notch signaling pathway inhibitor, such as DAPT, originally contained in the culture medium can be removed at the beginning of step (5) or during step (5).

[0134] In a preferred embodiment, this step is performed by adherent culture, so that the RPC aggregates form a monolayer of RPCs. In order to perform adherent culture, the RPC aggregates obtained in step (4) are preferably dispersed into single cells and counted for inoculation. The dispersion can be performed by enzyme treatment or by other methods such as mechanical means. The cells are cultured at a rate of about 0.5-4.0×10 6 cells / cm 2 , preferably 0.8-3.5×10 6 cells / cm 2 , more preferably 1.0×10 6 cells / cm 2 The density of the cells is inoculated into a culture container coated with a matrix. The matrix can be any matrix described in the present application.

[0135] Generally speaking, the culture in step (5) is performed at 37° C. In the case of adherent culture, the RPCs can be observed to reach 80%-100% confluence the next day. Step (5) can be continued for up to 30 days.

[0136] 2.6 Step (6): Differentiation of RPCs into PPCs

[0137] The method of the present invention can optionally be continued with step (6) to further differentiate RPCs into PPCs. The culture medium used in step (6) is referred to as "PPCM".

[0138] In some embodiments, after step (5), RPCs can continue to be cultured in the same culture medium to allow them to continue to differentiate into PPCs.

[0139] In some embodiments, the factors in the culture medium used in step (6) include one or more of pyruvic acid, retinoic acid, and taurine, preferably all of them; or consist of them. In the culture medium of step (6), the concentration of pyruvic acid may be 0.5-2mM, preferably 0.75-1.5mM, and most preferably 1mM; the concentration of retinoic acid may be 0.05-2μM, preferably 0.1-1μM, and most preferably 0.1μM; the concentration of taurine may be 50-200μM, preferably 75-150μM, and most preferably 100μM; and / or the concentration of L-DOPA may be 0.1-1μM, preferably 0.25-0.75μM, and most preferably 0.5μM.

[0140] In a preferred embodiment, the culture medium of step (6) further comprises T3. For example, the culture medium of step (6) is obtained by adding triiodothyronine (T3) to the culture medium of step (5). T3 can be added in an amount of 0.05-0.5 mM, preferably 0.08-0.2 mM, and more preferably about 0.1 nM.

[0141] In a specific embodiment, the PPCM in Example 3 is used in step (6).

[0142] In a preferred embodiment, this step is performed by adherent culture. In order to perform adherent culture, the PPCs obtained in step (5) are preferably dispersed into single cells and counted for inoculation. The dispersion can be performed by enzyme treatment or by other methods such as mechanical means. The cells are cultured at a rate of about 0.5-4.0×10 6 cells / cm 2 , preferably 0.8-3.5×10 6 cells / cm 2 , more preferably 1.0×10 6 cells / cm 2 The density of the cells is inoculated into a culture container coated with a matrix. The matrix can be any matrix described in the present application.

[0143] Generally, the culturing in step (6) is performed at 37° C. After the PPCs are formed in step (6), they may be continuously cultured in the same or other appropriate culture medium.

[0144] 2.7 Other Notes

[0145] No serum is added to the culture medium of the present invention. Preferably, the culture medium of the present invention is a chemically defined medium. No supporting cells such as trophoblast cells are introduced into the culture method of the present invention. Therefore, the method of the present invention is a method with repeatability, robustness and safety.

[0146] 3. Purpose

[0147] The method of the present invention can be used to produce PPCs suitable for high purity and having directed differentiation ability, and high-purity cone cells can be further obtained by differentiating the PPCs.

[0148] The PPCs produced by the methods of the present invention can also be used as starting cells to differentiate into one or more different types of other cells.

[0149] The resulting PPCs and cones can be used in drug development, disease modeling, preclinical and clinical research, and in both existing therapies and new therapies in development.

[0150] The method of the present invention can be adapted to the production of clinical-grade cells, for example, by using clinical-grade corresponding reagents, such as culture medium, nutritional supplements, factors, enzymes, substrates. The clinical grade also includes GMP grade, cGMP grade or CTSTM grade.

[0151] The present invention relates to the following specific embodiments:

[0152] 1. A method for generating retinal progenitor cells (RPCs) from human pluripotent stem cells (hPSCs), comprising:

[0153] (1) providing a culture comprising human pluripotent stem cells,

[0154] (2) culturing the culture containing human pluripotent stem cells in step (1) to form embryoid bodies (EBs);

[0155] (3) culturing the embryoid bodies obtained in step (2) in a culture medium supplemented with the following factors: one or more Wnt signaling pathway inhibitors, one or more BMP signaling pathway inhibitors, and one or more TGF-β signaling pathway inhibitors;

[0156] (4) forming aggregates containing retinal progenitor cells (RPCs) by suspension culture; and

[0157] (5) Continuing to culture the aggregates obtained in step (4) to obtain a culture rich in retinal progenitor cells (RPCs).

[0158] 2. The method of embodiment 1, wherein the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells.

[0159] 3. The method described in embodiment 1 or 2, wherein the Wnt signaling pathway inhibitor in step (3) is selected from one or more of the following groups: XAV939, DDK1, IwR1e and CKI-7.

[0160] 4. The method described in embodiments 1 to 3, wherein the BMP signaling pathway inhibitor in step (3) is selected from one or more of the following groups: Noggin, DMH-1, Dorsomorphin, COCO and LDN-193189.

[0161] 5. The method according to any one of embodiments 1 to 4, wherein the TGF-β signaling pathway inhibitor in step (3) is selected from one or more of the following groups: SB431542, A83-01, Repsox.

[0162] 6. The method of any one of embodiments 1 to 5, wherein the factor in step (3) comprises or consists of a combination of Dorsomorphin, SB431542 and IwR1e.

[0163] 7. The method of any one of embodiments 1 to 5, wherein the factor in step (3) comprises or consists of a combination of Dorsomorphin, Noggin, XAV939, SB431542 and IwR1e.

[0164] 8. The method of any one of embodiments 1 to 5, wherein the factor in step (3) comprises or consists of a combination of Dorsomorphin, XAV939, SB431542 and IwR1e.

[0165] 9. The method according to any one of embodiments 1 to 8, wherein step (3) comprises a suspension culture step (3a) and an adherent culture step (3b), and step (3b) uses the same factors as (3a).

[0166] 10. The method described in embodiment 9 further comprises a step (3c) after step (3b), wherein in step (3c), the culturing is carried out in a culture medium comprising the following factors: retinoic acid, taurine and pyruvate.

[0167] 11. In the method described in embodiment 10, the culture medium in step (3c) further contains one or more factors selected from the following group: Notch signaling pathway inhibitors and Hedgehog / Smoothened signaling pathway agonists.

[0168] 12. The method described in embodiment 11, wherein the Hedgehog / Smoothened signaling pathway agonist is SAG or SHH.

[0169] 13. The method described in embodiment 11 or 12, wherein the Notch signaling pathway inhibitor is DAPT.

[0170] 14. The method according to any one of embodiments 10-13, wherein the factor added to the culture medium in step (3c) is selected from any one of the following groups:

[0171] (i) pyruvic acid, retinoic acid and taurine;

[0172] (ii) pyruvate, retinoic acid, taurine, and DAPT;

[0173] (iii) pyruvate, retinoic acid, taurine and SHH; and

[0174] (iv) pyruvic acid, retinoic acid, taurine and SAG.

[0175] 15. The method according to any one of embodiments 10-14, wherein step (3c) is adherent culture.

[0176] 16. The method of any one of embodiments 1-9, wherein step (4) uses the same factors as step (3), preferably the same culture medium.

[0177] 17. The method of any one of embodiments 10-15, wherein step (4) uses the same factors as step (3c), preferably the same culture medium.

[0178] 18. The method according to any one of embodiments 10-15, wherein in step (4), a Notch signaling pathway inhibitor is further added on the basis of the factors used in step (3c).

[0179] 19. The method described in embodiment 18, wherein the Notch signaling pathway inhibitor is DAPT.

[0180] 20. The method according to any one of embodiments 1 to 19, wherein the culture in step (5) is adherent culture.

[0181] 21. The method of any one of embodiments 1-20, wherein step (5) uses the same factors as step (4), preferably the same culture medium.

[0182] 22. The method according to any one of embodiments 1-20, wherein step (5) uses the same factors as step (4), preferably the same culture medium, but the Notch signaling pathway inhibitor contained therein is removed at least during a portion of the time period of step (5).

[0183] 23. The method of any one of embodiments 9-22, wherein the adherent culture is carried out in a culture container coated with a matrix.

[0184] 24. The method according to any one of embodiments 1 to 23, wherein in step (2), the pluripotent stem cells are seeded into an ultra-low attachment culture vessel and culture medium supplemented with a ROCK inhibitor is used.

[0185] 25. The method of embodiment 24, wherein the ROCK inhibitor is Y-27632.

[0186] 26. A culture comprising retinal progenitor cells obtained by the method described in any one of embodiments 1-25.

[0187] 27. The culture comprising retinal progenitor cells of embodiment 26, wherein VSX2 + The cell ratio is greater than 75%.

[0188] 28. A method for preparing photoreceptor precursor cells (PPCs), comprising further culturing a culture containing retinal progenitor cells obtained by the method of any one of embodiments 1 to 25 into photoreceptor precursor cells.

[0189] 29. The method of embodiment 28, wherein the further culturing is carried out in the culture medium used in the previous step.

[0190] 30. The method of embodiment 29, wherein the culture medium does not contain retinoic acid, taurine and pyruvate.

[0191] 31. The method of embodiment 28, wherein the further culturing is carried out in a medium comprising the following factors: retinoic acid, taurine and optionally pyruvic acid. 32. The method of embodiment 31, wherein T3 is further added during the culturing.

[0192] 33. A culture comprising photoreceptor precursor cells obtained by the method of any one of embodiments 28-32.

[0193] 34. The culture comprising photoreceptor precursor cells of embodiment 33, wherein CRX + The percentage of cells is greater than 75%, preferably greater than 80%, more preferably greater than 85%.

[0194] 35. A method for preparing photoreceptor cells, comprising further culturing a culture comprising retinal progenitor cells obtained by the method of any one of embodiments 1 to 25 into photoreceptor cells, or comprising further culturing a culture comprising photoreceptor progenitor cells obtained by the method of any one of embodiments 28 to 32 into photoreceptor cells.

[0195] 36. The method of embodiment 35, wherein the photoreceptor cells are cone cells.

[0196] 37. The method of embodiment 35 or 36, wherein the culturing is carried out in a culture medium comprising the following factors: retinoic acid and taurine.

[0197] 38. A culture comprising photoreceptor cells obtained by the method of any one of embodiments 35-37.

[0198] 39. The culture comprising photoreceptor cells of embodiment 38, wherein ARR3 + 、CRX + and RCVRN + The cells are greater than 90%.

[0199] 40. Use of the culture comprising retinal progenitor cells as described in embodiment 26 or 27 for the preparation of a medicament.

[0200] 41. Use of a culture comprising photoreceptor precursor cells as described in embodiment 33 or 34 for the preparation of a medicament.

[0201] 42. Use of a culture comprising photoreceptor cells as described in embodiment 38 or 39 for the preparation of a medicament. Example

[0202] Example 1. Differentiation of RPCs from iPSCs

[0203] The process steps of the method of the present invention and the culture medium used at different stages are summarized in Figure 1 The detailed steps and materials are described below.

[0204] (1) Preparation of human pluripotent stem cells (before D0)

[0205] iPSCs were seeded into culture plates coated with Laminin-521 (BioLamina, MX521 or CT521), with each well containing 2.5 mL hPSC XF medium (BI), the cells were cultured in a 37°C incubator with 5% CO2. When the confluence reached 80% to 90%, the medium was discarded and 2 mL of CTS was added to each well. TM Wash the cells with DPBS (Gibco). Add 1 mL CTS to each well. TM TrypLE TM Select enzyme (Gibco), digest for 6 to 10 min in a 37°C incubator with 5% CO2. When some cells begin to fall off the culture plate, add 3 mL of Digestion was terminated with hPSC XF medium (BI). After centrifugation, cells were incubated with 10 μM Rock inhibitor ( Y-27632, Wako The cells were resuspended in hPSCXF medium (BI) and the viable cells were counted.

[0206] (2) Preparation of embryoid bodies (EBs) (D0-D2)

[0207] iPSCs were seeded at a density of 10,000 cells per well in ultra-low attachment 96-well culture plates, each well of which contained 10 μM Rock inhibitor ( Y-27632, Wako) 200 μL hPSC XF medium (BI) was added to formally start differentiation (D0). The cells were cultured in a 37°C incubator containing 5% CO2.

[0208] The next day, the formation of embryoid bodies (EBs) was observed in each well, and these EBs were of the same size.

[0209] (3) Directed differentiation of EBs into neural aggregates (D2-D30)

[0210] Suspension culture (D2-D7)

[0211] EBs were transferred to a low-adsorption 6-well culture plate at a density of 28 to 32 per well, and the old medium was discarded. 2.5 mL of a medium having the composition shown in Table 1 below was added to each well as EB medium (EB medium; EBM) for suspension culture.

[0212] Table 1 Formula of EBM

[0213]

[0214] EBs were cultured in a 37°C incubator containing 5% CO 2 for 5 days, and the medium was completely replaced with fresh EBM every 2 days.

[0215] Adherent culture (D7-D21)

[0216] On the 7th day after the start of differentiation, EBs were transferred to a 6-well culture plate coated with Laminin-521 to allow the EBs to fully adhere. NR first-stage medium (NRM1) with the same EBM formula as shown in Table 1 was used for adherent culture, 5 mL per well, and half of the old medium was replaced with fresh NRM1 every two days (every third day):

[0217] Adherent culture (D21-D30)

[0218] On day 21 after the start of differentiation, NR second-stage medium (NRM2) having the composition shown in Table 2 below was used for adherent culture at 5 mL per well, and half of the old medium was replaced with fresh NRM2 every two days.

[0219] Table 2 Formulation of NRM2

[0220]

[0221] From the first day when EBs were seeded on the culture plate coated with Laminin-521, EBs attached to the culture plate and gradually formed adherent cell aggregates. After that, typical pie-shaped specific structures were formed, indicating the formation of NR ( Figure 2 ).

[0222] (4) Formation of RPC aggregates from NR-like structures (D30-D31)

[0223] On the 30th day of differentiation, NRs rich in RPCs were selected and separated under a stereomicroscope, and the selected NRs were transferred to a low-adsorption 6-well culture plate and formed RPC aggregates through suspension culture. The RPC first stage culture medium (RPCM1) having the composition shown in Table 3 below was used for suspension culture, 2.5 mL per well.

[0224] Table 3 Formulation of RPCM1

[0225]

[0226]

[0227] (5) Transformation of RPC aggregates into RPC monolayer culture (D31-D60)

[0228] Adherent culture (D31-D45)

[0229] On day 31 of differentiation, RPC aggregates in the low-adhesion 6-well plate were selected and transferred to a 24-well plate. After discarding the old culture medium, 0.5 mL of Accutase enzyme (Gibco) was added to each well to digest the RPC aggregates for 5 to 10 minutes. When the outer layer of cells in the RPC aggregates began to become loose, 1.5 mL of CTS-grade RPC first stage medium (RPCM1) with the composition shown in Table 3 was added to terminate the digestion.

[0230] The RPC aggregates were transferred to a 15 mL centrifuge tube and allowed to settle naturally for 2 to 5 min. After the supernatant was removed, 1 mL of RPCM1 was added. RPC aggregates were broken into single cells and centrifuged at 1.0 × 10 6 cells / cm 2The density of 500 μg / mL was inoculated into a 6-well culture plate coated with clinical grade Laminin-521 (BioLamina, MX521 or CT521), wherein each well contained 5 mL RPCM1. The next day, the isolated RPCs were observed to grow in an adherent form with a confluence of 90% to 100%. Half of the old culture medium was replaced with fresh RPCM1 culture medium every two days.

[0231] Adherent culture (D45-D60)

[0232] On the 45th day after the start of differentiation, RPC second stage medium (RPCM2) having the composition shown in Table 4 below was used for adherent culture, 5 mL per well, and half of the old medium was replaced with fresh RPCM2 every two days to promote the differentiation of RPCs.

[0233] Table 4 Formulation of RPCM2

[0234]

[0235] Example 2. Characterization of RPCs differentiated from iPSCs

[0236] In this example, RT-qPCR and immunofluorescence staining were used to identify the cells obtained by the method shown in Example 1 to confirm whether RPCs were successfully differentiated.

[0237] The cells obtained in Example 1 were subjected to RT-qPCR detection. Figure 4 As shown in A, on day 34, the expression levels of RPC markers PAX6 and RAX increased significantly, while the increase of VSX2 was smaller; Figure 4 As shown in B, on day 45, the expression levels of RPC markers PAX6 and RAX decreased compared with day 34, and the expression level of PPC marker CRX increased, indicating that PPCs may have been present.

[0238] The cells obtained in Example 1 were identified by immunofluorescence staining. Figure 4 As shown in C, most of the differentiated cells expressed VSX2, indicating that a large number of high-purity RPCs were successfully differentiated. Specifically, in the differentiated cell population, VSX2 + The cell ratio is greater than 80%.

[0239] Example 3. Effects of Notch and Hedgehog / Smoothened signaling pathways on PPCs differentiated from iPSCs

[0240] In this example, the effects of a Notch signaling pathway inhibitor and a Hedgehog / Smoothened signaling pathway agonist on differentiation of PPCs were compared. The detailed steps and materials are described below.

[0241] Steps (1) and (2) are consistent with those in Example 1.

[0242] (3) Directed differentiation of EBs into neural aggregates (D2-D30)

[0243] Suspension culture (D2-D7)

[0244] Cells were grown in the presence of 10 μM Rock inhibitor. After culturing in an ultra-low-adsorption 96-well plate in hPSC XF medium (BI) for 2 days (differentiation day 0 to day 2), EBs were transferred to a low-adsorption 6-well plate at a density of 28 to 32 EBs per well, and the old medium was discarded. 2.5 mL of EBM having the composition shown in Table 5 below was added to each well for suspension culture.

[0245] Table 5 Formulation of EBM

[0246]

[0247] EBs were cultured in a 37°C incubator containing 5% CO 2 for 5 days, and the medium was completely replaced with fresh EBM every 2 days.

[0248] Adherent culture (D7-D21)

[0249] On the 7th day after the start of differentiation, EBs were transferred to a 6-well culture plate coated with Laminin-521 to allow the EBs to fully adhere. NRM1 (same formula as EBM in Table 5) was used for adherent culture, and half of the old culture medium was replaced with fresh NRM1 every third day.

[0250] Adherent culture (D21-D30)

[0251] On day 21 after differentiation started, three different NRM2s with the compositions shown in Table 6 were used for adherent culture. Among the three NRM2s, one group was supplemented with 50 ng / mL SHH, another group was supplemented with 100 nM SAG, or another group was supplemented with 10 μM DAPT, and the other components were the same. Half of the old culture medium was replaced with fresh NRM2 every two days.

[0252] Table 6 Formulation of NRM2

[0253]

[0254] (4) Formation of RPC aggregates from NR-like structures (D30-D31)

[0255] On the 30th day of differentiation, NRs rich in RPCs were selected and separated under a stereomicroscope, and the selected NRs were transferred to a low-adsorption 6-well culture plate and formed RPC aggregates through suspension culture. The amount of cells used to inoculate a low-adsorption 6-well culture plate was the amount of NRs from a 6-well culture plate. Three RPCMs (the same as the NRM2 formula in Table 6) were used for suspension culture, in which 50 ng / mL of SHH, or 100 nM SAG, or 10 μM DAPT were added corresponding to the culture medium formula in step (3). Half of the old culture medium was replaced with fresh RPCM every two days.

[0256] (5) Transformation of RPC aggregates into RPC monolayer culture (D31-D60)

[0257] Adherent culture (D31-D60)

[0258] On the 31st day of differentiation, the RPC aggregates in the low-adsorption 6-well plate were selected and transferred to a 24-well plate, and the old culture medium was discarded. The RPC aggregates were digested for 5 to 10 minutes by adding 0.5 mL of Accutase enzyme (Gibco) to each well. When the outer cells of the RPC aggregates began to become loose, 1.5 mL of RPCM (the same as the NRM2 formula in Table 6) was added to terminate the digestion, wherein 50 ng / mL of SHH, or 100 nM SAG, or 10 μM DAPT were added corresponding to the culture medium formula in steps (3) and (4). Half of the old culture medium was replaced with fresh RPCM every two days.

[0259] The RPC aggregates were transferred to a 15 mL centrifuge tube and allowed to settle naturally for 2 to 5 min. The supernatant was removed and 1 mL of RPCM was added to break the RPC aggregates into single cells. 6 cells / cm 2 The cells were seeded at a density of 500 μg / mL into a 6-well culture plate coated with Laminin-521, with each well containing 5 mL of RPCM. The next day, the isolated RPCs were observed to grow in an adherent form with a confluence of 90% to 100%. Half of the old culture medium was replaced with the corresponding fresh RPCM every two days.

[0260] (6) Differentiation of RPCs to PPCs (D60-D90)

[0261] Adherent culture (D60-D90)

[0262] On day 60 after the start of differentiation, a PPC differentiation medium (PPCM) having the composition shown in the following Table 7 was used for adherent culture. Half of the old medium was replaced with fresh PPCM every two days to promote differentiation of PPCs.

[0263] Table 7 Formulation of PPCM

[0264]

[0265] Example 4. Characterization of PPCs differentiated from iPSCs

[0266] In order to confirm whether PPCs were successfully differentiated by the method shown in Example 3, the obtained cells were identified based on the detection of cell-specific biomarkers.

[0267] At four time points, namely, day 7, day 21, day 30, and day 45, the expression trends of the RPC marker RAX and the PPC marker CRX in the cells cultured according to the method of Example 3 were analyzed. Figure 5 As shown in A, compared with the control group (no addition), the addition of SHH can significantly increase the expression of RPC molecular marker RAX on the 21st day, and the addition of SHH, SAG and DAPT can significantly increase the expression of PPC molecular marker CRX on the 45th day, among which the expression level of CRX in the SHH group was the highest.

[0268] The PPCs obtained by the method described in Example 3 were identified by immunofluorescence staining and the expression levels of the markers were determined. Figure 5 As shown in B, after adding SHH, SAG or DAPT, most of the differentiated cells expressed CRX, indicating that a large number of high-proportion PPCs were successfully differentiated. Figure 6 As shown, in the differentiated cell population, CRX + The proportion of cells was greater than 75% in the SHH-treated group and the SAG-treated group. + The cell ratio is greater than 85%.

[0269] Example 5. Differentiation of PPCs from iPSCs

[0270] This example describes a simple method that optimizes the operating method, reduces the use of factors, and does not require the early differentiation of iPSCs or frequent changes in culture medium formulations.

[0271] Steps (1) and (2) are consistent with those in Example 1, and the subsequent steps are as follows.

[0272] (3) Directed differentiation of EBs into neural aggregates (D2-D30)

[0273] This stage uses a CTS-grade EBM / NRM having the composition shown in Table 8 below.

[0274] Table 8 Formulation of EBM / NRM

[0275]

[0276] Suspension culture (D2-D7)

[0277] Cells were cultured in the presence of 10 μM Rock inhibitor (Wako). After culturing in an ultra-low attachment 96-well plate in hPSC XF medium (BI) for 2 days, EBs were transferred to a low attachment 6-well plate at a density of 28 to 32 EBs per well, and the old medium was discarded. 2.5 mL of EBM was added to each well for suspension culture.

[0278] EBs were cultured in a 37°C incubator containing 5% CO 2 for 5 days, and the medium was completely replaced with fresh EBM every 2 days.

[0279] Adherent culture (D7-D30)

[0280] On the 7th day after the start of differentiation, EBs were transferred to a 6-well culture plate coated with Laminin-521 to allow the EBs to attach completely. NRM with the composition shown in Table 8 was used for adherence culture, and half of the old culture medium was replaced with fresh NRM every third day to promote the differentiation of RPCs.

[0281] (4) Formation of RPC aggregates from NR-like structures (D30-D31)

[0282] On the 30th day of differentiation, NRs rich in RPCs were selected and separated under a stereomicroscope, and the selected NRs were transferred to a low-adsorption 6-well culture plate and formed into RPC aggregates through suspension culture. The amount of cells used to inoculate a low-adsorption 6-well culture plate was the amount of NRs from a 6-well culture plate. RPCMs having the composition shown in Table 9 below were used for suspension culture.

[0283] Table 9 Formulation of RPCM

[0284]

[0285] (5) RPC aggregates were transformed into RPC culture and differentiated into PPC (D31-D60)

[0286] Adherent culture (D31-D60)

[0287] On day 31 of differentiation, select the RPC aggregates in one well of the low-adsorption 6-well plate and transfer them to one well of a 24-well cell culture plate. Carefully discard the old culture medium. Digest the RPC aggregates for 5 to 10 minutes by adding 0.5 mL of Accutase enzyme (Gibco) to each well. When the outer layer of cells of the RPC aggregates begins to become loose, add 1.5 mL of the RPCM shown in Table 9 to terminate the digestion.

[0288] Transfer the RPC aggregates to a 15 mL centrifuge tube and allow to settle naturally for 2 to 5 min. Remove the supernatant and add 1 mL of RPCM to break up the RPC aggregates into single cells, and count the live cells. Single cells were counted at 1.0 × 10 6 cells / cm 2 The cells were seeded at a density of 500 μg / mL into a 6-well culture plate coated with Laminin-521, with each well containing 5 mL of RPCM. The next day, the isolated RPCs were observed to grow in an adherent form with a confluence of 90% to 100%. Half of the old culture medium was replaced with fresh RPCM every two days to promote the differentiation of PPCs.

[0289] Example 6. Characterization of PPCs differentiated from iPSCs

[0290] In order to confirm whether PPCs were successfully differentiated from pluripotent stem cells by the method shown in Example 5, the obtained cells were identified based on the detection and identification of cell-specific biomarkers.

[0291] At three time points, namely, the 30th day, the 45th day, and the 60th day, the expression levels of the RPC markers RAX, VSX2, and the PPC marker CRX in the cells obtained in Example 5 were measured, and their changing trends were analyzed. Figure 7 As shown in A, the expression levels of RPC molecular markers RAX and VSX2 were significantly increased on the 60th day, and the expression level of PPCs molecular marker CRX was significantly increased on the 60th day, indicating that the cultured cells differentiated into PPCs on the 60th day.

[0292] The PPCs obtained by the method described in Example 5 were identified by immunofluorescence staining. Figure 7 As shown in B, most of the differentiated cells expressed CRX, indicating that a large number of high-purity PPCs were successfully differentiated. Specifically, in the differentiated cell population, CRX + The cell ratio is greater than 80%.

[0293] Example 7. Differentiation of PPCs into Cones

[0294] In order to verify the ability of the PPCs obtained in the present invention to further differentiate into cone cells, the PPCs were further cultured.

[0295] Based on the PPCs differentiated in Example 5, cone cells were cultured in CTS-grade Cone medium (ConeM) having the composition shown in Table 10, wherein each well contained 5 mL of ConeM, and half of the old medium was replaced with fresh medium every two days. High-purity cone cells can be obtained by continuous culture for 10-120 days.

[0296] Table 10 ConeM formulation

[0297]

[0298]

[0299] Example 8. Characterization of cone cells differentiated from PPCs

[0300] In order to confirm whether cone cells were successfully differentiated by the method shown in Example 7, the obtained cells were identified based on the detection and identification of cell-specific biomarkers.

[0301] The cone cells obtained by the method described in Example 7 were identified by immunofluorescence staining. Figure 8 As shown in the figure, most of the differentiated cells expressed RCVRN, indicating that a large number of high-purity photoreceptor cells were successfully differentiated. Specifically, in the differentiated cell population, RCVRN + The cell ratio is greater than 95% ( Figure 8 ).like Fig. 9 As shown in the figure, most of the differentiated cells expressed ARR3 and CRX, indicating that a large number of high-purity cone cells were successfully differentiated. Specifically, in the differentiated cell population, CRX + The proportion of cells is greater than 90%, ARR3 + The cell ratio is greater than 90% ( Fig. 9 ).

[0302] Fig.10 ARR3 is shown in the figure. + Diagram of the morphology of cone cells.

Claims

1. A method for generating retinal progenitor cells (RPCs) from human pluripotent stem cells (hPSCs), comprising: (1) Providing a culture comprising human pluripotent stem cells; (2) culturing the culture containing human pluripotent stem cells in step (1) to form embryoid bodies (EBs); (3) culturing the embryoid bodies obtained in step (2) in a culture medium supplemented with the following factors: one or more Wnt signaling pathway inhibitors, one or more BMP signaling pathway inhibitors, and one or more TGF-β signaling pathway inhibitors; (4) forming aggregates containing retinal progenitor cells (RPCs) by suspension culture; and (5) Continuing to culture the aggregates obtained in step (4) to obtain a culture rich in retinal progenitor cells (RPCs).

2. The method of claim 1, wherein The Wnt signaling pathway inhibitor in step (3) is selected from one or more of the following groups: XAV939, DDK1, IwR1e and CKI-7; The BMP signaling pathway inhibitor in step (3) is selected from one or more of the following groups: Noggin, DMH-1, Dorsomorphin, COCO and LDN-193189; and / or The TGF-β signaling pathway inhibitor in step (3) is selected from one or more of the following groups: SB431542, A83-01, and Repsox.

3. The method of claim 1 or 2, wherein The factor in step (3) comprises a combination of Dorsomorphin, SB431542 and IwR1e, or consists of the combination; The factor in step (3) comprises a combination of Dorsomorphin, Noggin, XAV939, SB431542 and IwR1e, or consists of the combination; or The factor in step (3) comprises a combination of Dorsomorphin, XAV939, SB431542 and IwR1e, or consists of the combination.

4. The method according to any one of claims 1 to 3, wherein the step (3) comprises a suspension culture step (3a) and an adherent culture step (3b), and the step (3b) uses the same factors as (3a).

5. The method of claim 4, further comprising a step (3c) after step (3b), wherein in step (3c), the culture is performed in a medium containing the following factors: retinoic acid, taurine and pyruvate.

6. The method according to claim 5, wherein the culture medium in step (3c) further contains one or more factors selected from the group consisting of a Notch signaling pathway inhibitor and a Hedgehog / Smoothened signaling pathway agonist.

7. The method of claim 6, wherein the Hedgehog / Smoothened signaling pathway agonist is SAG or SHH; and / or the Notch signaling pathway inhibitor is DAPT.

8. The method according to any one of claims 5 to 7, wherein step (3c) is adherent culture.

9. The method according to any one of claims 1 to 4, wherein step (4) uses the same factors as step (3), preferably the same culture medium.

10. The method according to any one of claims 5 to 8, wherein step (4) uses the same factors as step (3c), preferably the same culture medium.

11. The method according to any one of claims 5-8, wherein in step (4), a Notch signaling pathway inhibitor is further added on the basis of the factors used in step (3c); in particular, the Notch signaling pathway inhibitor is DAPT.

12. The method according to any one of claims 1 to 11, wherein step (5) uses the same factors as step (4), preferably the same culture medium.

13. The method of any one of claims 1-12, wherein step (5) uses the same factors as step (4), preferably the same culture medium, but the Notch signaling pathway inhibitor contained therein is removed at least during a portion of the time period of step (5).

14. A culture comprising retinal progenitor cells obtained by the method of any one of claims 1 to 13.

15. The culture comprising retinal progenitor cells of claim 14, wherein VSX2 + The cell ratio is greater than 75%.

16. A method for preparing photoreceptor precursor cells (PPCs), comprising further culturing a culture containing retinal precursor cells obtained by the method of any one of claims 1 to 13 into photoreceptor precursor cells.

17. The method of claim 16, wherein the further culturing is performed in the culture medium used in the previous step.

18. The method of claim 17, wherein the culture medium is free of retinoic acid, taurine and pyruvate.

19. The method of claim 16, wherein the further culturing is carried out in a medium comprising retinoic acid, taurine, optionally pyruvate, and optionally T3.

20. A culture comprising photoreceptor precursor cells obtained by the method of any one of claims 16 to 19.

21. The culture comprising photoreceptor precursor cells of claim 20, wherein CRX + The percentage of cells is greater than 75%, preferably greater than 80%, more preferably greater than 85%.

22. A method for preparing photoreceptor cells, comprising further culturing a culture comprising retinal progenitor cells obtained by the method of any one of claims 1 to 13 into photoreceptor cells, or comprising further culturing a culture comprising photoreceptor progenitor cells obtained by the method of any one of claims 16 to 19 into photoreceptor cells.

23. The method of claim 22, wherein the photoreceptor cells are cone cells.

24. The method of claim 22 or 23, wherein the culturing is carried out in a medium comprising the following factors: retinoic acid and taurine.

25. A culture comprising photoreceptor cells obtained by the method of any one of claims 22-24.

26. The culture comprising photoreceptor cells of claim 25, wherein ARR3 + 、CRX + and RCVRN + The cells are greater than 90%.

27. Use of a culture comprising retinal progenitor cells as claimed in claim 14 or 15 for the preparation of a medicament.

28. Use of a culture comprising photoreceptor precursor cells as claimed in claim 20 or 21 for the preparation of a medicament.

29. Use of a culture comprising photoreceptor cells as claimed in claim 25 or 26 for the preparation of a medicament.

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