Method for differentiating ocular cells and its use

Abstract

Provided herein are methods for producing stem cell-derived photoreceptor precursor (PRP) cell populations. Also provided herein are methods for using the PRP cell populations, e.g., for therapy.

Description

[Technical Field]

[0001] This application claims the benefits of U.S. Provisional Patent Application No. 62 / 660,899, filed on April 20, 2018, which is incorporated herein by reference in its entirety.

[0002] This disclosure relates to the field of stem cell biology in general. More specifically, this disclosure relates to methods for differentiating pluripotent stem cells into various ophthalmic cells (e.g., photoreceptor precursor (PRP) cells). [Background technology]

[0003] The retina is a photosensitive tissue layer that covers the inner surface of the eye. Photoreceptor cells (either rods or cones) in the retina directly respond to light, converting chemical photosignals into electrical events that trigger nerve impulses. Impairment or complete loss of photoreceptor cell function is one of the causes of irreversible blindness in retinal diseases (e.g., hereditary retinal degeneration and age-related macular degeneration (AMD)). Death of retinal ganglion cells (RGCs) in glaucoma also leads to irreversible blindness. Rescuing degenerated retinas is a major challenge, and cell replacement is one of the most promising approaches (Pearson et al., 2012).

[0004] The production of induced pluripotent stem cells (iPSCs) from somatic cells of adult mice in 2006 represented a significant breakthrough in stem cell research, drug discovery, disease modeling, and cell therapy (Takahashi et al., 2006). Human iPSCs can differentiate into specific cell types and have the potential to be patient-specific immunocompatible cells for regenerative medicine (Yu et al., 2007). The use of human pluripotent stem cells, germinal stem (ES) cells, and iPSCs opens new avenues for treating human retinal degenerative diseases. [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] Human ES (hES) cells and human iPS (hiPS) cells, which possess the ability to proliferate indefinitely in culture while maintaining pluripotency, have the potential to be used as an inexhaustible source of photoreceptor cells for tissue transplantation (Comyn et al., 2010). Converging data are increasingly demonstrating the potential involvement of hES cells in the neuroretina after embryoid body formation and their ability to further differentiate into cells expressing photoreceptor markers (Zhong et al., 2014; Meyer et al., 2009). While the various methods already developed represent actual progress, they still suffer from the drawbacks generally associated with the highly specialized differentiation of pluripotent stem cells into specific cell types. These protocols for photoreceptor-directed differentiation of hES or hiPS cells require several steps, the addition of several molecules, and are rather inefficient. Current methods for differentiation into photoreceptor cell lines require embryoid body formation and / or manual selection of retinal cells from cultures. Therefore, in this field, there is a need for efficient, reliable, and large-scale methods to obtain substantially pure cultures of certain human neuroepithelial cells (particularly photoreceptor precursor (PRP) cells). [Means for solving the problem]

[0006] This embodiment provides methods and compositions for producing ocular cells, as well as their use. In the first embodiment, a method is provided for producing a population of neuroretinal progenitor cells (NRPs), comprising: obtaining a starting population of human induced pluripotent stem cells (iPSCs); culturing the iPSCs in retinal induction medium (RIM) to initiate differentiation of these cells into anterior neuroectoderm cells; further culturing these cells in a first retinal differentiation medium (RD1) containing a BMP inhibitor to further differentiate these cells into anterior neuroectoderm cells; inducing retinal differentiation of these cells to form retinal progenitor cells (RPCs) by culturing the anterior neuroectoderm cells in a second retinal differentiation medium (RD2) essentially free of a BMP inhibitor; and culturing these RPCs in retinal maturation (RM) medium to produce NRPs. In further embodiments, a method is provided for producing a population of NRPs, comprising: obtaining a starting population of human iPSCs; culturing these cells in RD1 containing a BMP inhibitor to further differentiate them into anterior neuroectoderm cells; inducing retinal differentiation of the anterior neuroectoderm cells by culturing them in RD2 essentially free of a BMP inhibitor to form RPCs; and culturing these RPCs in RM medium to produce NRPs. In certain embodiments, culturing is further defined as adhesive two-dimensional culture.

[0007] In some embodiments, the method further includes culturing iPSCs in retinal induction medium (RIM) before culturing the cells in RD1 to initiate differentiation of these cells into anterior neuroectoderm cells. In certain embodiments, the method further includes culturing a population of NRPs as suspension aggregates in a medium containing a γ-secretase inhibitor and a ROCK inhibitor.

[0008] In some aspects of the above embodiments, the method further comprises culturing the NRPs in FDSC medium containing a γ-secretase inhibitor and FGF for a period of time sufficient to produce a population of NRPs.

[0009] In certain embodiments, RIM comprises a BMP inhibitor, a TGFβ inhibitor, and / or IGF-1. In certain embodiments, RIM comprises a BMP inhibitor, a TGFβ inhibitor, a Wnt inhibitor, and / or IGF-1. In certain embodiments, RIM is essentially free of activin A. In certain embodiments, RIM is essentially free of a Wnt inhibitor such as CKI-7. In some embodiments, culturing iPSCs in RIM lasts for 1 to 3 days (e.g., 1, 2, or 3 days).

[0010] In some embodiments, RD1 medium further comprises a TGFβ inhibitor, a Wnt inhibitor, IGF-1, and a MEK inhibitor. In certain embodiments, RD1 medium further comprises a TGFβ inhibitor, a Wnt inhibitor, IGF-1, and a MEK inhibitor. In certain embodiments, RD1 medium does not contain CKI-7. In some embodiments, culturing in RD1 lasts for 1 to 3 days (e.g., 1, 2, or 3 days).

[0011] In some embodiments, RD2 medium comprises a TGFβ inhibitor, a Wnt inhibitor, IGF-1, and a MEK inhibitor. In certain embodiments, an increase in VSX2 expression in anterior neuroectodermal cells is measured for differentiation potential. In certain embodiments, RD2 medium is essentially free of LDN193189. In some embodiments, culturing in RD2 lasts for 5 to 9 days (e.g., 5, 6, 7, 8, or 9 days). In certain embodiments, after culturing in RD2, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the cells express PMEL17. In certain embodiments, after culturing in RD2, at least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, or 100% of the cells express VSX2. In certain embodiments, RPCs express PAX6, MITF, and / or PMEL. In some embodiments, RPCs do not express or are essentially free of expression of TRYP1, CRALBP, and / or BEST1.

[0012] In some embodiments, culturing from obtaining iPSCs to culturing in RM is further defined as adherent two-dimensional culture. In some embodiments, culturing from obtaining iPSCs to culturing in RM essentially does not contain aggregates.

[0013] In some embodiments, the RM medium contains nicotinamide and ascorbic acid. In certain embodiments, the RM medium further contains FGF and a TGFβ inhibitor. In some embodiments, the RM medium further contains FGF, SB431542, CKI-7, and IGF-1. In some embodiments, the RM medium further contains a γ-secretase inhibitor. In certain embodiments, culturing in the RM medium to produce NRP takes 3 to 7 days. In some embodiments, NRP expresses PAX6 and VSX2. In certain embodiments, after culturing in the RM medium to produce NRP, at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 95%, or 100% of the cells express PAX6 and VSX2. In certain embodiments, RPC expresses PAX6, MITF, and / or PMEL.

[0014] In another embodiment, a method for producing a population of photoreceptor precursor cells (PRP) is provided, which includes obtaining a starting population of NRP according to the embodiment, and further culturing this NRP in a photoreceptor precursor induction medium (FDSC) medium containing a γ-secretase inhibitor and FGF for a period sufficient to produce a population of PRP. In some embodiments, culturing is further defined as adherent two-dimensional culture.

[0015] In some embodiments, culturing NRP in an FDSC medium containing a γ-secretase inhibitor and FGF for a period sufficient to produce a population of PRP takes 10 to 20, 20 to 30, 30 to 40, or 40 to 50 days (e.g., 11, 12, 13, 14, 15, 16, 17, 18, or 19 days, or more). FDSC further contains a TGFβ inhibitor and a WNT inhibitor. In some embodiments, the RM medium further contains FGF and a TGFβ inhibitor.

[0016] In some embodiments, a method for producing PRP further comprises maturing a population of PRP as a suspension aggregate in nicotinamide-containing RM medium or photoreceptor maturation (PM) medium, thereby producing a population of mature PRP aggregates. In some embodiments, maturation is carried out over 6 to 10 days (e.g., 6, 7, 8, 9, or 10 days). In additional embodiments, the method further comprises cryopreserving the mature PRP aggregates. In some embodiments, the PM medium further comprises a γ-secretase inhibitor.

[0017] In certain embodiments, a method for producing PRP further includes dissociating mature PRP aggregates into essentially single cells in PM medium. In some embodiments, the method includes cryopreserving the mature PRP as single cells.

[0018] In some embodiments, PRP is cultured as adherent cells in PM medium for 5 to 12 days (e.g., 5, 6, 7, 8, 9, 10, 11, or 12 days). In certain embodiments, the PM medium further comprises a γ-secretase inhibitor. In certain embodiments, the dissociated cells are re-aggregated. In some embodiments, the PM medium further comprises a CDK inhibitor. In certain embodiments, at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 95%, or 100% of the cells express Recoverin (RCVRN).

[0019] In some embodiments, the method further includes purifying the PRP. In certain embodiments, the purification includes selecting cells that are positive for CD171, thereby obtaining a purified PRP cell population. In some embodiments, the purification includes selecting cells that are positive for CD171 and / or SUSD2, thereby obtaining a purified PRP cell population. In certain embodiments, the purification includes selecting cells that are positive for CD171, SUSD2, CD56 (NCAM), CD57 (LAMP-3), CD81, CD111 (Nectin-1), CD133, CD147, CD184 (CXCR4), CD200, CD230, CD276, CD298, CD344 (Frizzled), PSA-NCAM, and / or PTK7, thereby obtaining a purified PRP cell population. In specific embodiments, purification includes selecting cells positive for CD111, CD133, CD230, and / or CD344, thereby obtaining a purified PRP cell population. In some embodiments, purification includes selecting cells positive for CD344, thereby obtaining a purified PRP cell population. In some embodiments, purification is performed on day 65 or day 75. In certain embodiments, purification includes depleting cells positive for two or more markers selected from the group consisting of CD9, CD49f, CD340, podoplanin, CD29, CD63, and CD298.

[0020] In some embodiments, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the cells in the purified PRP cell population express class III β-tubulin (TUBB3). In certain embodiments, at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the cells in the purified PRP cell population express RCVRN. In some embodiments, at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 95%, or 100% of the cells in the purified PRP cell population express RCVRN. In some embodiments, the PRP expresses one or more markers selected from the group consisting of OTX2, CRX, BLIMP1, Neurod1, RCVRN, TUBB3, and CD171 / L1CAM. In certain embodiments, the PRP does not express, or essentially does not express, TRYP1, CRALBP, BEST1, Ki67, MITF, and / or PMEL17. In some embodiments, the cells have low or essentially no expression of PAX6, CHX10 (also referred to herein as VSX2; both terms are used interchangeably herein), and / or Onecut1. In certain embodiments, less than 15%, 10%, or 5% (e.g., less than 4%, 3%, 2%, or 1%) of the cells in the purified PRP population express PAX6.

[0021] Further embodiments provide a method for producing a population of PRPs, comprising obtaining a starting population of NRPs according to an embodiment, and further culturing the NRPs in a PRP maturation medium (PM) containing a cyclin-dependent kinase inhibitor for a period sufficient to produce a population of PRPs.

[0022] In some embodiments, at least 70% of the cells (e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 95%, or 100%) are positive for PAX6 and CHX10 before culturing in PM medium. In certain embodiments, NRP is cultured as aggregates in the presence of a γ-secretase inhibitor and a ROCK inhibitor. In some embodiments, PM further comprises a γ-secretase inhibitor. In certain embodiments, PM further comprises a MEK inhibitor.

[0023] In additional embodiments, the method further includes dissociating the PRP into essentially single cells in PM medium. In some embodiments, the PRP is cultured as adherent cells in PM medium.

[0024] In some embodiments, the method further includes enriching PRP by selecting cells that are positive for CD171, SUSD2, CD56 (NCAM), CD57 (LAMP-3), CD81, CD111 (Nectin 1), CD133, CD147, CD184 (CXCR4), CD200, CD230, CD276, CD298, CD344 (Frizzled), PSA-NCAM, and / or PTK7, and / or removing cells that are positive for CD9, CD49f, CD340, podoplanin CD29, CD63, and / or CD298. In some embodiments, at least 90% of the cells (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) are positive for TUBB3, and / or at least 70% of the cells (e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 95%, or 100%) are positive for RCVRN.

[0025] In yet another embodiment, a method is provided for producing a population of optic vesicles (OVs), comprising obtaining a starting population of PRP according to the embodiment, and further culturing the PRP as a suspension aggregate in RM medium or PRP maturation (PM) medium for a period of time sufficient to produce a population of OVs.

[0026] In some embodiments, PRP is cultured as a suspension aggregate in RM medium or PRP maturation (PM) medium for a period sufficient to produce a population of OV for at least 20 days (e.g., at least 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more days). In some embodiments, the RM medium further comprises a γ-secretase inhibitor. In certain embodiments, the RM medium further comprises FGF and SB431542. In some embodiments, at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the cells in the population of OV express VSX2. In some embodiments, at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 40%, 50%, 60%, or more of the cells in the OV population express RCVRN. In some embodiments, the OV express gamma-synuclein (SNCG), opsin, RCVRN, and rhodopsin. In certain embodiments, the FDSC further comprises a TGFβ inhibitor and a Wnt inhibitor.

[0027] In some embodiments, NRPs are cultured in RM medium containing a γ-secretase inhibitor and FGF for a period sufficient to produce a population of PRPs, for 10–20 days (e.g., 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more). In some embodiments, at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more of the cells in the population of PRPs express TUBB3. In some embodiments, at least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, or 100% of the cells in the population of PRPs express RCVRN.

[0028] In additional embodiments, the method further comprises culturing a population of NRPs as suspension aggregates in a medium containing a γ-secretase inhibitor and a ROCK inhibitor. In some embodiments, the medium further comprises ascorbic acid and nicotinamide. In certain embodiments, at least 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 95%, or 100% of the cells in the population of NRPs express VSX2.

[0029] In another embodiment, a pharmaceutical composition is provided comprising PRP, OV, or NRP manufactured according to the embodiment, and a pharmaceutically acceptable carrier.

[0030] Further embodiments provide a method for treating damage or degeneration of retinal neurons in a subject, comprising administering an effective amount of the pharmaceutically acceptable composition of the embodiment (e.g., PRP, OV, or NRP manufactured herein) to the eye of the subject. In some embodiments, the retinal neurons are photoreceptors.

[0031] Eye cells (e.g., PRP cells) produced by the methods described herein can be used in any currently known methods and applications in the art for photoreceptor cells. For example, a method for evaluating a compound can be provided, comprising assaying the pharmacological or toxicological properties of the compound to photoreceptor cells. A method for evaluating a compound with respect to its effects on PRP cells can also be provided, comprising a) contacting the PRP cells provided herein with the compound, and b) assaying the effects of the compound on the PRP cells.

[0032] In certain embodiments of the above embodiments, RIM comprises LDN193189, SB431542, CKI-7, and IGF-1. In some embodiments, RD1 medium further comprises a TGFβ inhibitor, a Wnt inhibitor, IGF-1, and a MEK inhibitor. In certain embodiments, RD2 medium comprises a TGFβ inhibitor, a Wnt inhibitor, IGF-1, and a MEK inhibitor. In some embodiments, the BMP inhibitor is LDN193189. In some embodiments, TGFβ is further defined as a type I receptor activin receptor-like kinase (ALK5) inhibitor. In certain embodiments, the TGFβ inhibitor is SB431542, 6SB525334, SB-505124, Lefty, A 83-01, D 4476, GW 788388, LY 364847, R 268712, or RepSox. In some embodiments, the WNT inhibitor is CKI-7, IWP2, IWP4, PNU 74654 XAV939, TAK 715, DKK1, or SFRP1. In certain embodiments, the MEK inhibitor is PD0325901. In some embodiments, the RD2 medium contains SB431542, CKI-7, IGF-1, and PD0325901. In specific embodiments, the RM medium further contains FGF, SB431542, CKI-7, and IGF-1. In some embodiments, the γ-secretase inhibitor is DAPT, Begacestat, Compound W, JLK6, L-685,485, Flurizan, DBZ, MRK560, PF3084014 hydrobromide, or BMS299897. In some embodiments, cycling-dependent kinase (CDK) inhibitors are CDK4 / 6 inhibitors, for example. PD0332991 (Palbociclib)

[0033] In another embodiment, a composition is provided comprising an NRP population, wherein at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of the cells in the NRP population express PAX6, at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of the cells in the NRP population express PMEL17, and / or at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 95%, or 100% of the cells in the NRP population express VSX2. In some embodiments, at least 95% of the cells in the NRP population express PAX6, at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of the cells in the NRP population express PMEL17, and / or at least 75% of the cells in the NRP population express VSX2. In some specific embodiments, at least 95% of the cells in the NRP population express PAX6, at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of the cells in the NRP population express PMEL17, and at least 75% of the cells in the NRP population express VSX2. In some embodiments, the cells in the NRP population further express Ki67.

[0034] In another embodiment, a composition is provided comprising a PRP population, wherein at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of the cells in the PRP population express TUBB3, at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the cells in the PRP cell population express RCVRN, and / or less than 15% of the cells in the PRP population express PAX6. In some embodiments, at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 95%, or 100% of the cells in the PRP population express RCVRN. In certain embodiments, less than 10% or 5% of the cells in the PRP population express PAX6. In some embodiments, at least 90% of the cells in the PRP population express TUBB3, at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the cells in the PRP cell population express RCVRN, and less than 15% of the cells in the PRP population express PAX6. In some embodiments, at least 90% of the cells in the PRP population express TUBB3, at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 95%, or 100% of the cells in the PRP cell population express RCVRN, and / or less than 5% of the cells in the PRP population express PAX6. In certain embodiments, the PRP expresses one or more markers selected from the group consisting of OTX2, IRBP, SUSD2, CRX, BLIMP1, Neurod1, RCVRN, TUBB3, and CD171 / L1CAM. In some embodiments, the PRP does not express, or essentially does not express, TRYP1, CRALBP, BEST1, Ki67, MITF, and / or PMEL17.

[0035] Further embodiments provide compositions comprising a population of OV cells, wherein at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of these cells express VSX2, and / or at least 20% of these cells express RCVRN. In some embodiments, at least 65% of the cells express VSX2, and / or at least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, or 100% of the cells express RCVRN. In certain embodiments, at least 65% of cells express VSX2, and at least 30% of cells express RCVRN.

[0036] Another embodiment provides a method for obtaining a PRP-enriched population of PRP cells, comprising obtaining a starting cell population containing PRP cells, and enriching the starting cell population with respect to PRP cells by selecting cells positive for CD171, SUSD2, CD56 (NCAM), CD57 (LAMP-3), CD81, CD111 (Nectin 1), CD133, CD147, CD184 (CXCR4), CD200, CD230, CD276, CD298, CD344 (Frizzled), PSA-NCAM, and / or PTK7, and / or removing cells positive for CD9, CD49f, CD340, podoplanin, CD29, CD63, and / or CD298, thereby obtaining a PRP-enriched cell population in which PRP cells are enriched compared to the starting cell population. In some embodiments, the method further comprises determining the level of enrichment of PRP cells in the PRP-enriched population. In some embodiments, the level of enrichment is determined by the use of a cellular marker selected from the group consisting of TUBB3, RCVRN, OTX2, IRBP, SUSD2, CRX, BLIMP1, Neurod1, and CD171 / L1CAM. In certain embodiments, the PRP-enriched cell population is enriched with PRP cells compared to the starting cell population, as determined by RCVRN sorting. In some embodiments, the PRP-enriched population consists of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% PRP cells. In certain embodiments, the PRP-enriched population consists of at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 95%, or 100% PRP cells. In some embodiments, the PRP-enriched population consists of essentially pure PRP cells. In certain embodiments, the PRP cells are human PRP cells. In some embodiments, the starting cell population is prepared from iPSCs. In some embodiments, selection and / or removal is performed by sorting based on magnetic beads or by sorting based on fluorescence.In some embodiments, this method includes selecting cells that are positive for CD171, SUSD2, CD111, CD133, CD230, and / or CD344.

[0037] Another embodiment provides a method for performing quality control during the manufacture of an NRP cell product, comprising detecting the expression of a cellular marker selected from the group consisting of PAX6, CHX10(VSX2), Ki67, and PMEL. In some embodiments, at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 95%, or 100% of the cells are positive for PAX6, CHX10(VSX2), Ki67, and PMEL. In certain embodiments, at least 90% of the cells are positive for PAX6, CHX10(VSX2), Ki67, and PMEL.

[0038] In further embodiments, a method is provided for performing quality control during the manufacture of a PRP cell product, comprising detecting the expression of an off-target cell marker selected from the group consisting of PAX6, ONECUT1, HNCHF6, CHX10, and Ki67. In some embodiments, the off-target cells are positive for PAX6 and ISL1. In some embodiments, the PRP cell product passing through quality control contains less than 10% or 5% PAX6-positive cells. In certain embodiments, the PRP cell product passing through quality control contains less than 10% PAX6-positive cells, less than 0.05% Ki67-positive cells, less than 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, or 100% CHX10-positive cells, and / or less than 2% ONECUT1-positive cells. In some embodiments, the PRP cell product passing through quality control contains less than 5% PAX6-positive cells, less than 0.04% Ki67-positive cells, less than 15% CHX10-positive cells, and / or less than 1% ONECUT1-positive cells. In certain embodiments, the PRP cell product passing through quality control contains less than 5% PAX6-positive cells, less than 0.04% Ki67-positive cells, less than 15% CHX10-positive cells, and less than 1% ONECUT1-positive cells.

[0039] Further embodiments provide a method for performing quality control during the manufacture of an OV cell product, comprising detecting the expression of one or more cell markers selected from the group consisting of RCVRN, CHX10, PAX6, and Ki67. In certain embodiments, the cell markers are RCVRN and CHX10. In certain embodiments, at least 60% of the cells in the OV cell product are positive for RCVRN, and at least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, or 100% of the cells in the OV cell product are positive for CHX10. In some embodiments, the cell markers are PAX6 and Ki67. In some embodiments, the method further includes detecting the absence of TYRP1. In some embodiments, the method further includes detecting the expression of one or more markers selected from the group consisting of MITF, CRALBP, BEST1, OTX2, CRX, BLIMP1, Neurod1, TUBB3, ONECUT1, and CD171 / L1CAM.

[0040] Other purposes, features, and advantages of this disclosure will become apparent from the detailed description below. However, it should be understood that the detailed description and specific examples are given only as examples, illustrating preferred embodiments of the invention, for various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0041] The following drawings form part of this specification and are intended to further illustrate certain aspects of the invention. The invention can be better understood by referring to one or more of these drawings in conjunction with the detailed description of the specific embodiments presented herein. [Brief explanation of the drawing]

[0042] [Figure 1]Candidate Overview. An overview of iPSC-derived ophthalmic cell differentiation, including neuroretinal progenitor cells (NRP), photoreceptor precursor cells (PRP), and optic vesicles (OV). Candidate 1 (C-1) represents PRP differentiated under adhesive two-dimensional (2-D) conditions (2-D PRP). Candidate 2 (C-2) represents PRP differentiated under both 2-D and suspension aggregate three-dimensional (3-D) conditions (hybrid PRP). Candidate 3 (C-3) represents OV differentiated under both 2-D and 3-D conditions (hybrid OV). Candidate 4 (C-4) represents NRP differentiated under 2-D conditions.

[0043] [Figure 2] An overview of the 2-D PRR differentiation process, including the duration of the culture medium phase.

[0044] [Figure 3A-3C] 2-D PRP MACS enrichment. Flow cytometry analysis of 2-D PRP regarding the expression of βIII-tubulin (TUBB3) and recabin (RCVRN) both before (Figure 3A) and after (Figure 3B) MACS enrichment. (Figure 3C) Summary of flow cytometry analysis before and after MACS for 2-D PRP differentiation (n=5).

[0045] [Figure 4A-4D] 2-D PRP characterization. (Figure 4A) Image of 2-D PRP aggregates after thawing on day 2 (10× objective lens). (Figure 4B) Immunofluorescence staining of 2-D PRP after thawing on day 1 for TUBB3 and RCVRN (20× objective lens). (Figure 4C) Immunofluorescence staining of 2-D PRP after thawing on day 1 for CRX and RCVRN (20× objective lens). (Figure 4D) Single-cell gene expression analysis of 2-D PRP aggregates after thawing on day 2.

[0046] [Figure 5] An overview of the hybrid PRP differentiation process.

[0047] [Figure 6]Input iPSC density optimization. Flow cytometry analysis of 15-day hybrid PRP differentiation for Pax6 and Vsx2 cells with input densities ranging from 1 × 10⁴ (10K) to 1 × 10⁶ (1000K) cells per well in a 6-well plate.

[0048] [Figure 7] RM medium optimization. Flow cytometry analysis of 18-day RPCs grown in RM medium with or without FGF2 and SB431542.

[0049] [Figures 8A-8B] PM medium optimization. (Figure 8A) Morphology of late-seeded PRP in PM medium with or without PD0332991 or activin A. (Figure 8B) Flow cytometry analysis of late-seeded C-2 PRP in PM medium with or without PD0332991 or activin A.

[0050] [Figure 9] Hybrid PRP MACS enrichment. Flow cytometry analysis of TUBB3, nestin, and RCVRN expression of hybrid PRP both before and after MACS enrichment.

[0051] [Figure 10A-10D] Hybrid PRP characterization. (Figure 10A) Image of hybrid PRP aggregates after thawing on day 2 (10× objective lens). (Figure 10B) Immunofluorescence staining of hybrid PRP after thawing on day 1 for TUBB3 and RCVRN (20× objective lens). (Figure 10C) Immunofluorescence staining of hybrid PRP after thawing on day 1 for CRX and RCVRN (20× objective lens). (Figure 10D). Single-cell gene expression analysis of hybrid PRP aggregates after thawing on day 2.

[0052] [Figure 11] An overview of the hybrid OV differentiation process.

[0053] [Figures 12A-12D]Morphology and characterization of hybrid OV. (Figure 12A) Image of differentiated hybrid OV at day 68. (Figure 12B) Time course of immunofluorescence staining of differentiated hybrid OV for VSX2 and RCVRN. (Figure 12C) Immunofluorescence staining of late hybrid OV for gamma-synuclein (SNCG; retinal ganglion cell marker), RCVRN (PRP and photoreceptor marker), green-sensitive opsin (OPN1MW; cone marker), cone arrestin (ARR3; cone marker), and rhodopsin (RHO; rod marker). (Figure 12D) Flow cytometry analysis of time course of differentiated hybrid OV for VSX2 and RCVRN.

[0054] [Figure 13] Outline of the 2-D NRP differentiation process.

[0055] [Figure 14A-14C] 2-D NRP characterization. (Figure 14A) Image of NRP aggregates after thawing on day 2. (Figure 14B) Immunofluorescence staining of adherent NRP after thawing on day 1 for PAX6 (left) and VSX2 (right). (Figure 14C) Flow cytometry analysis of NRP after thawing on day 2 for PAX6, PMEL17, and VSX2.

[0056] [Figures 15A-15B] Flow analysis of co-expression of surface antigens and recoverins corresponding to Table 4 was determined using quadrant flow plots after double labeling with recoverins and antibodies against each surface antigen. In addition, the percentage population of cells expressing only recoverins (in FITC, x axis) or only surface antigens (in APC, y axis) was evaluated. All plots were gated to unstained (empty) cells and corresponding isotype controls (REA IgG1, MsIgG1, Ms IgG2a, MsIgG2b, MsIgM).

[0057] [Figure 16]Time course of recabinin expression after enrichment with surface antigens. Recabinin expression is highest at D65 for CD344-enriched PRP. Higher recabinin expression rates are also obtained with CD111 and CD230 enrichment compared to pre-enrichment (pre-MACS) cells.

[0058] [Figure 17] Evaluation of post-enrichment expression of on-target PRP and off-target retinal cells after enrichment with surface antigens CD111, CD230, and CD344 at D55.

[0059] [Figure 18] Evaluation of post-enrichment expression of on-target PRP and off-target retinal cells enriched with surface antigens CD111, CD230, and CD344 at D65.

[0060] [Figure 19] Evaluation of post-enrichment expression of on-target and off-target retinal cells enriched with surface antigens CD111, CD230, and CD344 in D75 cells.

[0061] [Figure 20] Evaluation of post-enrichment expression of on-target and off-target retinal cells enriched with CD133 in D75 cells.

[0062] [Figure 21] Flow analysis of depletion markers.

[0063] [Figure 22] Flow cytometry analysis of PAX6 and CHX10 expression induced or uninduced by CKI-7 at days 15 and 25 of PRP differentiation.

[0064] [Figure 23] Expression rates of PAX6 and CHX10 at days 15 and 25 of PRP differentiation, with or without CKI-7.

[0065] [Figures 24A-24B] (Figure 24A) Flow cytometry analysis of Ki67 and CHX10 expression with and without CKI-7 at days 15 and 25 of PRP differentiation. *The use of conjugated CHX10 shifted the plot (made it higher), but in this case the expression level decreased, which may be due to steric hindrance preventing the antibody from labeling the maximum epitope. (Figure 24B) Expression rates of Ki67 and CHX10 with and without CKI-7 at days 15 and 25 of PRP differentiation.

[0066] [Figures 25A-25B] (Figure 25A) Flow cytometry analysis of TYRP1 and PMEL expression induced or uninduced by CKI-7 at days 15 and 25 of PRP differentiation. (Figure 25B) Expression rates of TYRP1 and PMEL induced or uninduced by CKI-7 at days 15 and 25 of PRP differentiation.

[0067] [Figures 26A-26B] Comparison of initial eye field and RPE markers in the presence (Figure 26A) and absence (Figure 26B) of RIM.

[0068] [Figures 27A-27D] The effects of RIM-enabled versus RIM-absent expression of PAX6 (Figure 27A), CHX10 (Figure 27B), TRYP10 (Figure 27C), and PMEL (Figure 27D) on early (D15) neuroretinal differentiation across different lineages.

[0069] [Figures 28A-28D] Comparison of PRP's meta-stage differentiation (approximately D30) in the presence or absence of RIM in the expression of PAX6 (Figure 28A), CHX10 (Figure 28B), TRYP10 (Figure 28C), and PMEL (Figure 28D).

[0070] [Figures 29A-29B] Expression of on-target PRP characterization markers at day 75 in the presence (Figure 29A) and absence (Figure 29B) of RIM.

[0071] [Figure 30] Expression of off-target retinal cell markers in D75 differentiation in the presence or absence of RIM.

[0072] [Figure 31A-31B] Recoverin expression is over 90% in PRP cells enriched with SUSD2 compared to CD171. (Figure 31A) RCVRN-positive population enriched with SUSD2. (Figure 31B) RCVRN-positive population enriched with CD171.

[0073] [Figures 32A-32E] Maximum expression of recabilin (Figure 32A) and SUSD2 (Figure 32B) was observed between days 55 and 65 of differentiation, with recabilin peaking immediately after SUSD2. Co-expression of recabilin and SUSD2 (Figure 32C) mimicked SUSD2 monoexpression, suggesting that cells expressing SUSD2 also express recabilin, but not all recabilin-positive cells express SUSD2. Overlaying the three graphs clearly shows parallel expression of dual recabilin / SUSD2 versus single SUSD2 strains (Figure 32D). SUSD2 was labeled with recabilin after fixation. Independent time-course studies of co-expression of recabilin and SUSD2 from early D55 to late D105 (Figure 32E) support peak SUSD2 expression around D65, but also show similarly elevated expression at D55.

[0074] [Figure 33A-33C]Recabilin expression rates further increased after SUSD2 enrichment at D65, D75, and D85 (Figure 33A), with the highest expression at D65 corresponding to the initial peak SUSD2 and recabilin expression time. While the percentage of recabilin-positive cell population decreased from D65 to D75 to D85 (B), at D85, SUSD2 enrichment still increased the proportion of recabilin-positive cells compared to CD171 enrichment (Figure 33B). However, the limit of SUSD2 enrichment was low cellular output, less than 25% of the initial cellular input recovered after enrichment (Figure 33C). This was true for cells enriched with either SUSD2 or CD171 at D85, where only 3% of total input cells were enriched with SUSD2 and only 18% were enriched with CD171. The flow-through cells (labeled SUSD2- and CD171-) appear to contain a large portion of the cells, suggesting that the expression of both surface markers may decrease during the later stages of differentiation.

[0075] [Figures 34A-34E] At D65, SUSD2 expression before MACS and recabinet expression before and after MACS were high, while CHX10 expression was lowest, but significantly increased at D75 and D85 (Figure 34A). Co-expression of recabinet-CHX10 was less than 3% at D65, and increased and decreased at D75 and D85, respectively, suggesting a short-lived bipolar cell population (Figure 34B). NeuroD1 expression was highest at D65 before and after enrichment compared to D75 and D85 (Figure 34C), and NeuroD1 co-expression was also highest at D65 and further enhanced after SUSD2 enrichment (Figure 34D). Additional experiments (Figure 34E) confirmed low CHX10 at D55 and showed a significant decrease in CHX10 after SUSD2 MACS enrichment at D75. [Modes for carrying out the invention]

[0076] I. Description of Exemplary Embodiments In certain embodiments, this disclosure provides a method for producing ocular cells (e.g., photoreceptor precursor (PRP) cell populations). PRP cells may be derived from pluripotent stem cells such as ES cells or iPS cells in a defined 2D cell culture without requiring embryoid body formation or selection of cell colonies. Alternatively, PRP cells may be obtained in a hybrid culture of adherent 2D and suspension aggregate 3D. Briefly, PSCs can be differentiated into anterior neuroectoderm cells, these anterior neuroectoderm cells can be cultured for a short period in a retinal differentiation medium containing a BMP inhibitor, and then further cultured in a retinal differentiation medium without a BMP inhibitor. Interestingly, the inventors have found that the neuroretinal differentiation potential of anterior neuroectoderm cells is enhanced by the removal of the BMP inhibitor. These cells can then be further differentiated into retinal progenitor cells (RPCs) in the presence of nicotinamide and in the absence of activin A, and these cells can be led to the photoreceptor lineage rather than other retinal lineages such as retinal pigment epithelial cells. Finally, RPCs can be differentiated in the presence of a TGFβ inhibitor, a WNT inhibitor, basic FGF, and a γ-secretase inhibitor to produce neuroretinal progenitor cells (NRPs), which can then be differentiated into PRP cells. Therefore, this disclosure provides a highly efficient and reproducible method for differentiating PSCs into PRP cells.

[0077] This disclosure also provides a method for producing PRP cells or optic vesicles by a combination of 2D aggregate culture and 3D aggregate culture. PRP aggregates can be produced by culturing RPCs as aggregates in activin A-free retinal maturation medium for a certain period of time, and then the PRP aggregates can be dissociated and cultured as monolayers. Alternatively, PRP aggregates can be produced by culturing RPCs in activin A-free retinal maturation medium for a long period of time, and finally optic vesicles can be produced.

[0078] Further embodiments of this disclosure provide a method for purifying a population of PRP cells obtained by the above method. This purification method may include positive selection and / or negative selection. For example, cells expressing CD171 can be selected by cell sorting. Thus, this purification process yields a PRP-enriched cell population with a higher proportion of PRP cells compared to the population obtained after differentiation from RPC.

[0079] Therefore, this method is more time- and cost-efficient and may enable the production of PRP-enriched cell populations for therapeutics from a regenerative source of stem cells on a clinical scale. Using this method, it may be possible to elucidate mechanisms, novel genes, soluble factors, or membrane-binding factors that are important for the development, differentiation, maintenance, survival, and function of photoreceptor cells.

[0080] PRP cells and photoreceptor cells provided herein can be used in a variety of in vivo and in vitro methods. For example, PRP cells can be used in vivo to treat retinal conditions (e.g., macular degeneration and retinitis pigmentosa, but not limited to these). Similarly, PRP cells and photoreceptor cells can be used in vitro in screening assays to identify putative therapeutic or prophylactic candidates. Further embodiments and advantages of this disclosure are described below.

[0081] I. Definition The term "purified" does not require absolute purity; rather, it is intended as a relative term. Therefore, a purified population of cells is approximately 90% or more, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% pure, and most preferably essentially free of other cell types.

[0082] As used herein, “essentially” or “essentially not” with respect to a characteristic component is used herein to mean that the particular component is not intentionally included in the composition and / or is present only as a contaminant or in trace amounts. Accordingly, the total amount of this particular component due to any unintentional contamination of the composition is well below 0.05%, preferably less than 0.01%. Most preferably, the amount of this particular component in the composition is undetectable by standard analytical methods.

[0083] As used herein, "a" or "an" may mean one or more. As used herein in the claims, when used with the word "comprising," the word "a" or "an" may mean one or more.

[0084] The use of the term “or” in the claims supports the definitions of “and / or” as defined herein, but unless expressly indicated as referring only to options, or unless the options are mutually exclusive, it is used to mean “and / or.” As used herein, “another” may mean at least the second and subsequent options.

[0085] Throughout this application, the term “approximately” is used to indicate that a value includes the inherent error variability of the device, the method used to determine this value, or the variability present among the subjects of study.

[0086] The term “cell population” is used herein to refer to a group of cells (typically of a common type). A cell population may originate from a common progenitor cell or may include multiple cell types. A “enriched” cell population refers to a cell population derived from a starting cell population (e.g., an unfractionated heterogeneous cell population) in which the proportion of a particular cell type is higher than the proportion of that cell type in the starting population. A cell population may be enriched with one or more cell types and depleted with one or more cell types.

[0087] The term “stem cell” as used herein refers to a cell that, under appropriate conditions, can differentiate into a variety of specific cell types, and under other appropriate conditions, can self-replicate and remain essentially undifferentiated in a pluripotent state. The term “stem cell” also encompasses pluripotent cells, multipotent cells, precursor cells, and progenitor cells. Exemplary human stem cells can be obtained from hematopoietic or mesenchymal stem cells obtained from bone marrow tissue, embryonic stem cells obtained from embryonic tissue, or embryonic germ cells obtained from fetal reproductive tissue. Exemplary pluripotent stem cells can also be produced from somatic cells by reprogramming them to a pluripotent state by expressing certain transcription factors associated with pluripotency; these cells are called “induced pluripotent stem cells” or “iPSCs.”

[0088] The term "pluripotency" refers to the property of cells to differentiate into all other cell types of an organism, except for extraembryonic or placental cells. Pluripotent stem cells can differentiate into all three germ layer cell types (e.g., ectoderm, mesoderm, and endoderm cell types) even after long-term culture. In some embodiments, pluripotent stem cells are embryonic stem cells derived from the inner cell mass of a blastocyst. In other embodiments, pluripotent stem cells are induced pluripotent stem cells obtained by reprogramming somatic cells.

[0089] The term "differentiation" refers to the process by which undifferentiated cells become more differentiated, involving changes in their structural and / or functional properties. Mature cells typically possess altered cellular structures and tissue-specific proteins.

[0090] As used herein, “undifferentiated” refers to cells that exhibit characteristic markers and morphological features that clearly distinguish them from terminally differentiated cells of embryonic or adult origin.

[0091] Embryoid bodies (EBs) are aggregates of pluripotent stem cells that can differentiate into cells of the endoderm, mesoderm, and ectoderm. Spheroid structures arise when pluripotent stem cells aggregate under non-adherent culture conditions, thus forming EBs in suspension.

[0092] "Isolated" cells are actually isolated or purified from other cells in an organism or culture. Isolated cells may be, for example, at least 99%, at least 98%, at least 95%, or at least 90% pure.

[0093] An "embryo" refers to a zygote with an artificially reprogrammed nucleus, or a mass of cells obtained from one or more divisions of an activated oocyte.

[0094] Embryonic stem (ES) cells are undifferentiated pluripotent cells obtained from early stages of the embryo, such as the inner cell mass at the blastocyst stage, or undifferentiated pluripotent cells produced by artificial means (e.g., nuclear transfer), and can give rise to any differentiated cell type of the embryo or adult, such as germ cells (e.g., sperm and eggs).

[0095] Induced pluripotent stem cells (iPSCs) are cells produced by reprogramming somatic cells by expressing or inducing the expression of a combination of factors (referred to herein as reprogramming factors). iPSCs can be produced using fetal, postnatal, neonatal, juvenile, or adult somatic cells. In certain embodiments, factors that may be used to reprogram somatic cells into pluripotent stem cells include, for example, Oct4 (sometimes referred to as Oct 3 / 4), Sox2, c-Myc, and Klf4, Nanog, and Lin28. In some embodiments, somatic cells are reprogrammed by expressing at least two, at least three, or four reprogramming factors to reprogram somatic cells into pluripotent stem cells.

[0096] An "allele" refers to one of two or more forms of a gene. Diploid organisms such as humans have two copies of each chromosome, and therefore each chromosome contains one allele.

[0097] The term "homozygous" is defined as having two of the same alleles at a particular locus. The term "heterozygous" refers to having two different alleles at a particular locus.

[0098] A "haplotype" refers to a combination of alleles at multiple loci along a single chromosome. Haplotypes can be based on a set of single nucleotide polymorphisms (SNPs) on a single chromosome and / or alleles in the major histocompatibility complex.

[0099] As used herein, the term “haplotype-matched” is defined as a cell (e.g., iPS cells) and the subject being treated sharing one or more major histocompatibility locus haplotypes. The haplotype of the subject can be readily determined using assays known in the art. Haplotype-matched iPS cells may be autologous or allogeneic. Autologous cells grown in tissue culture and differentiated into PRP cells are inherently haplotype-matched to the subject.

[0100] "Substantially identical HLA types" indicates that the donor's human leukocyte antigen (HLA) type and the patient's HLA type match to a degree that the transplanted cells, obtained by inducing differentiation of iPSCs derived from the donor's somatic cells, can engraft when transplanted into the patient.

[0101] In this specification, "superdonor" refers to an individual that is homozygous for certain MHC class I and II genes. These homozygous individuals can function as superdonors, and their cells (including tissues or other materials containing these cells) can be transplanted into individuals that are homozygous or heterozygous for this haplotype. Superdonors may be homozygous for HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, or HLA-DQ locus / locular alleles, respectively.

[0102] In this specification, "feeder-free" or "feeder-independent" refers to cultures in which cytokines and growth factors (e.g., TGFβ, bFGF, LIF) are supplemented as a substitute for the feeder cell layer. Therefore, pluripotent cells can be cultured and maintained in an undifferentiated growth state using "feeder-free" or feeder-independent culture systems and media. In some cases, feeder-free cultures utilize animal-based matrices (e.g., MATRIGEL®) or are grown on substrates such as fibronectin, collagen, or vitronectin. These approaches make it possible to maintain human stem cells in an essentially undifferentiated state without requiring a mouse fibroblast "feeder layer."

[0103] In this specification, the “feeder layer” is defined, for example, as a coating layer of cells on the bottom of a culture dish. Feeder cells can release nutrients into the culture medium and provide a surface to which other cells, such as pluripotent stem cells, can adhere.

[0104] The terms “defined” or “fully defined,” when used in relation to culture media, extracellular matrices, or culture conditions, refer to culture media, extracellular matrices, or culture conditions in which the chemical composition and quantities of virtually all components are known. For example, a defined medium does not contain undefined factors such as fetal bovine serum, bovine serum albumin, or human serum albumin. Generally, a defined medium includes a basic medium supplemented with recombinant albumin, chemically distinct lipids, and recombinant insulin (e.g., Dulbecco’s Modified Eagle Medium (DMEM), F12, or Roswell Park Memorial Laboratory Medium (RPMI) 1640, which contains amino acids, vitamins, inorganic salts, buffers, antioxidants, and energy sources). An example of a fully defined medium is Essential 8® medium.

[0105] With regard to culture media, extracellular matrices, or culture systems used with human cells, the term "xeno-free (XF)" refers to a state in which the substances used are not of non-human animal origin.

[0106] "Preconfluent" refers to a cell culture in which approximately 60-80% of the culture's surface is covered by cells. Typically, preconfluent refers to a culture in which approximately 70% of the culture's surface is covered by cells.

[0107] The term "retinal progenitor cells" is also known as "retinal precursor cells" or "RPCs" and includes cells capable of generating all cell types of the retina (e.g., neuroretinal cells (e.g., rods, cones, photoreceptor precursor cells), and cells that can differentiate into RPEs).

[0108] The term "neuroretinal progenitor cells" or "NRP" refers to cells whose ability to differentiate into neuroretinal cells is limited.

[0109] The terms "photoreceptor precursor cells" and "PRP" cells refer to cells differentiated from embryonic stem cells or induced pluripotent stem cells that can differentiate into photoreceptor cells expressing rhodopsin or one of the three cone opsins as a cell marker, and optionally expressing rod or cone cGMP. The photoreceptors may be rod photoreceptors and / or cone photoreceptors.

[0110] The terms "optic vesicle" or "OV" refer to cell aggregates or organoids (e.g., PRP cell aggregates) that have a morphology containing optic vesicle structures.

[0111] The "retinal pigment epithelium" refers to the layer of pigment cells between the choroid, which is a layer filled with blood vessels, and the neuroretina.

[0112] "Retinal Induction Medium (RIM)" as used herein refers to a growth medium containing a WNT pathway inhibitor and a BMP pathway inhibitor that can induce differentiation of PSCs into retinal lineage cells. RIM may also contain a TGFβ pathway inhibitor, and may contain IGF-1 and ascorbic acid.

[0113] "Retinal differentiation medium (RD)" is defined herein as a medium comprising a WNT inhibitor, a TGFβ inhibitor, and a MEK inhibitor, and for differentiating anterior neuroectoderm cells. RDM may or may not contain a BMP inhibitor (i.e., RD1) (i.e., RD2), and may contain IGF-1 and ascorbic acid.

[0114] "Retinal maturation medium (RM)" is defined as a growth medium for culturing retinal cells, comprising nicotinamide and ascorbic acid. RM preferably does not contain activin A. RM may or may not contain a γ-secretase inhibitor (e.g., DAPT) or a TGFβ inhibitor (e.g., SB431542) (i.e., RM1) (i.e., RM2), and may also contain IGF-1 and ascorbic acid.

[0115] "PRP Maturation Medium (PM)" is referred to herein as a growth medium for culturing PRP cells, comprising nicotinamide and a γ-secretase inhibitor (e.g., DAPT). PM is a CDK inhibitor (e.g., PD0332991 The following may or may not contain a TGF-β pathway activator (e.g., activin A) or a mitogen (e.g., retinoic acid) (i.e., PM1) (i.e., PM2).

[0116] "Photoreceptor precursor induction medium (FDSC)" refers to a growth medium containing a TGFβ inhibitor, a WNT inhibitor, and a γ-secretase inhibitor. FDSC may also contain basic FGF and ascorbic acid.

[0117] The term “retinal degeneration-related disorders” is intended to refer to any disorder resulting from congenital or postnatal degeneration or abnormality of the retina. Examples of retinal degeneration-related disorders include retinal dysplasia, retinal degeneration, age-related macular degeneration, diabetic retinopathy, retinitis pigmentosa, congenital retinal dystrophy, Leber congenital amaurosis, retinal detachment, glaucoma, optic neuropathy, and trauma.

[0118] As used herein, “therapeutically effective amount” means an amount of a compound sufficient to have an effect on a treatment when administered to a subject for the treatment of a disease or condition.

[0119] II. Pluripotent stem cells A. Embryonic stem cells ES cells originate from the inner cell mass of a blastocyst and have a high potential for differentiation in vitro. ES cells can be isolated by removing the trophectoderm layer outside the developing embryo and then culturing the inner mass cells on a non-proliferating cell feeder layer. The reseeded cells can proliferate and continue to produce new colonies of ES cells, which can be removed, dissociated, reseeded again, and proliferated. This process of "passaging" undifferentiated ES cells can be repeated many times to create cell lines containing undifferentiated ES cells (U.S. Patent No. 5,843,780; U.S. Patent No. 6,200,806; U.S. Patent No. 7,029,913).

[0120] Methods for producing mouse ES cells are known. In one method, preimplantation blastocysts from 129 mouse strains are treated with mouse antiserum to remove the trophectoderm, and the inner cell mass is cultured on a feeder cell layer of chemically inactivated mouse embryonic fibroblasts in a medium containing fetal bovine serum. Colonies of developing undifferentiated ES cells are subcultured on the mouse embryonic fibroblast feeder layer in the presence of fetal bovine serum to produce a population of ES cells. In some methods, mouse ES cells can be grown in the absence of a feeder layer by adding cytokine leukemia suppressor (LIF) to serum-containing culture medium (Smith, 2000). In other methods, mouse ES cells can be grown in serum-free medium in the presence of osteomorphonectomy proteins and LIF (Ying et al., 2003).

[0121] Human ES cells can be produced from or derived from zygote or blastocyst-stage mammalian embryos produced by sperm-oocyte fusion, nuclear transfer, disease development, or chromatin reprogramming and subsequent integration of the reprogrammed chromatin into the plasma membrane, in order to produce embryonic cells by methods already described (Thomson and Marshall, 1998; Reubinoff et al., 2000). In one method, human blastocysts are exposed to anti-human serum to lyseed trophectoderm cells and remove them from the inner cell mass cultured on a feeder layer of mouse embryonic fibroblasts. Furthermore, aggregates of cells derived from the inner cell mass are chemically or mechanically dissociated and reseeded, and colonies with undifferentiated morphology are selected with a micropipette, dissociated, and reseeded (U.S. Patent No. 6,833,269). In some methods, human ES cells can be grown without serum by culturing them on a fibroblast feeder layer in the presence of basic fibroblast growth factor (Amit et al., 2000). In other methods, human ES cells can be grown without a feeder cell layer by culturing them on a protein matrix such as MATRIGEL® or laminin in the presence of a "conditioned" medium containing basic fibroblast growth factor (Xu et al., 2001).

[0122] ES cells can also be derived from other organisms such as rhesus monkeys and marmosets by methods already described (Thomson and Marshall, 1998; Thomson et al., 1995; Thomson and Odorico, 2000), as well as from established mouse and human cell lineages. For example, established human ES cell lineages include MAOI, MA09, ACT-4, HI, H7, H9, H13, H14, and ACT30. As a further example, an established mouse ES cell lineage is the CGR8 cell lineage, which was established from the inner cell mass of mouse strain 129 embryos, and cultures of CGR8 cells can be grown in the presence of LIF without a feeder layer.

[0123] ES stem cells can be detected using protein markers (e.g., transcription factor Oct4, alkaline phosphatase (AP), stage-specific embryonic antigen SSEA-1, stage-specific embryonic antigen SSEA-3, stage-specific embryonic antigen SSEA-4, transcription factor NANOG, tumor rejection antigen 1-60 (TRA-1-60), tumor rejection antigen 1-81 (TRA-1-81), SOX2, or REX1).

[0124] B. Induced pluripotent stem cells Pluripotency induction was first achieved in 2006 using mouse cells (Yamanaka et al., 2006) and in 2007 using human cells (Yu et al., 2007; Takahashi et al., 2007) by reprogramming somatic cells through the introduction of transcription factors associated with pluripotency. Pluripotent stem cells can be maintained in an undifferentiated state and can differentiate into any adult cell type.

[0125] Any somatic cell, except germ cells, can be used as a starting point for iPSCs. For example, the cell type may be keratinocytes, fibroblasts, hematopoietic cells, mesenchymal cells, hepatocytes, or gastric cells. T cells can also be used as a source of somatic cells for reprogramming (U.S. Patent No. 8,741,648). The degree of cell differentiation or the age of the animal from which the cells are collected is not limited, and even undifferentiated progenitor cells (e.g., somatic stem cells) and finally differentiated mature cells can be used as a source of somatic cells in the methods disclosed herein. In one embodiment, the somatic cell is itself a PRP cell (e.g., human PRP cell). This PRP cell may be an adult PRP cell or an embryonic PRP cell. The iPSC can grow under conditions known to differentiate human ES cells into specific cell types and can express human ES cell markers (e.g., SSEA-1, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81).

[0126] Induced pluripotent stem cells (iPSCs) can be produced by reprogramming somatic cells using methods known to those skilled in the art. Those skilled in the art can easily produce induced pluripotent stem cells, see, for example, U.S. Patent Application Publication No. 20090246875, No. 2010 / 0210014; No. 20120276636; U.S. Patent No. 8,058,065; No. 8,129,187; No. 8,278,620; PCT International Publication No. 2007 / 069666A1; and U.S. Patent No. 8,268,620 (these are incorporated herein by reference). Generally, nuclear reprogramming factors are used to produce pluripotent stem cells from somatic cells. In some embodiments, at least two, at least three, or at least four of Klf4, c-Myc, Oct3 / 4, Sox2, Nanog, and Lin28 are utilized. Other embodiments utilize Oct3 / 4, Sox2, c-Myc, and Klf4.

[0127] These cells are treated with a nuclear reprogramming agent, which is generally one or more factors capable of inducing iPSCs from somatic cells, or nucleic acids encoding these substances (e.g., in a form incorporated into a vector). Common examples of nuclear reprogramming agents include at least Oct3 / 4, Klf4, and Sox2, or nucleic acids encoding these molecules. Additional nuclear reprogramming agents that can be used include functional inhibitors of p53, L-myc or nucleic acids encoding L-myc, and Lin28 or Lin28b or nucleic acids encoding Lin28 or Lin28b. Nanog can also be used for nuclear reprogramming.As disclosed in U.S. Patent Application Publication No. 20120196360, exemplary reprogramming factors for the production of iPSCs include: (1) Oct3 / 4, Klf4, Sox2, L-Myc (Sox2 may be replaced with Soxl, Sox3, Soxl5, Soxl7, or Soxl8, and Klf4 may be replaced with Klfl, Klf2, or Klf5); (2) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, SV40 large T antigen (SV40LT); (3) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, human papillomavirus (HPV) 16 E6; (4) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, HPV16 E7;(5)Oct3 / 4, Klf4, Sox2, L-Myc, TERT, HPV16 E6, HPV16 E7;(6)Oct3 / 4, Klf4, Sox2, L-Myc, TERT, Bmil;(7)Oct3 / 4, Klf4, Sox2, L-Myc, Lin28;(8)Oct3 / 4 , Klf4, Sox2, L-Myc, Lin28, SV40LT;(9)Oct3 / 4, Klf4, Sox2, L-Myc, Lin28, TERT, SV40LT;(10)Oct 3 / 4, Klf4, Sox2, L-Myc, SV40LT; (11) Oct3 / 4, Esrrb, Sox2, L-Myc (Esrrb can be replaced with Esrrg); (12) Oct3 / 4, Klf4, Sox2; (13) Oct3 / 4, Klf4, Sox2, TERT, SV40LT; (14) Oct3 / 4, Klf4, Sox2, TERT, HP VI 6 E6;(15)Oct3 / 4, Klf4, Sox2, TERT, HPV16 E7;(16)Oct3 / 4, Klf4, Sox2, TERT, HPV16 E6, HPV16 E7;(17)Oct3 / 4, Klf4, Sox2, TERT, Bmil;(18)Oct3 / 4, Klf4, Sox2, Lin28;(19)Oct3 / 4, Klf4, Sox2, Lin28, SV40LT;(20)Oct3 / 4, Klf4, Sox2, Lin28, TERT, SV40LT;(21)Oct3 / 4, Klf4, Sox2, SV40LT;or(22)Oct3 / 4, Esrrb, Sox2 (Esrrb can be replaced with Esrrg).In one non-limiting example, Oct3 / 4, Klf4, Sox2, and c-Myc are used. In other embodiments, Oct4, Nanog, and Sox2 are used; see, for example, U.S. Patent No. 7,682,828, incorporated herein by reference. Examples of these factors include, but are not limited to, Oct3 / 4, Klf4, and Sox2. In other examples, these factors include, but are not limited to, Oct3 / 4, Klf4, and Myc. In some non-limiting examples, Oct3 / 4, Klf4, c-Myc, and Sox2 are used. In other non-limiting examples, Oct3 / 4, Klf4, Sox2, and Sal 4 are used. Factors such as Nanog, Lin28, Klf4, or c-Myc can enhance reprogramming efficiency and can be expressed from several different expression vectors. For example, embedded vectors such as EBV element-based systems can be used (U.S. Patent No. 8,546,140). In a further embodiment, the reprogramming protein may be directly introduced into somatic cells by protein transduction. Reprogramming may further involve contacting the cell with one or more signaling receptors, such as glycogen synthase kinase 3 (GSK-3) inhibitors, mitogen-activated protein kinase kinase (MEK) inhibitors, transforming growth factor beta (TGF-β) receptor inhibitors or signaling inhibitors, leukemia suppressor (LIF), p53 inhibitors, NF-kappa B inhibitors, or combinations thereof. The regulators may be small molecules, inhibitory nucleotides, expression cassettes, or protein factors. It is expected that substantially any iPS cells or cell lineages can be used.

[0128] The mouse and human cDNA sequences of this nuclear reprogramming material are available by reference to the NCBI accession numbers mentioned in International Publication No. 2007 / 069666, which is incorporated herein by reference. Methods for introducing one or more reprogramming materials or nucleic acids encoding these reprogramming materials are known in the art and are disclosed, for example, in U.S. Patent Application Publication No. 2012 / 0196360 and U.S. Patent No. 8,071,369, both of which are incorporated herein by reference.

[0129] Once induced, iPSCs can be cultured in a medium sufficient to maintain pluripotency. As described in U.S. Patent No. 7,442,548 and U.S. Patent Application Publication No. 2003 / 0211603, iPSCs can be used with various media and techniques developed for culturing pluripotent stem cells (more specifically, embryonic stem cells). In the case of mouse cells, culture is carried out by adding leukemia suppressor (LIF) as a differentiation inhibitor to the standard medium. In the case of human cells, it is preferable to add basic fibroblast growth factor (bFGF) instead of LIF. Other methods for culturing and maintaining iPSCs, as known to those skilled in the art, may also be used.

[0130] In certain embodiments, undefined conditions may be used, for example, to maintain stem cells in an undifferentiated state, pluripotent cells may be cultured on fibroblast feeder cells or on a medium exposed to fibroblast feeder cells. In some embodiments, these cells are cultured in the presence of mouse embryonic fibroblasts treated with radiation or antibiotics to terminate cell division, acting as feeder cells. Alternatively, pluripotent cells may be cultured and maintained in an essentially undifferentiated state using defined feeder-independent culture systems (e.g., TESR® medium (Ludwig et al., 2006a; Ludwig et al., 2006b) or E8® medium (Chen et al., 2011)).

[0131] In some embodiments, iPSCs may be modified to express exogenous nucleic acids, for example, to include an enhancer operably linked to a promoter and a nucleic acid sequence encoding a first marker. Suitable promoters include, but are not limited to, any promoter expressed in photoreceptor cells (e.g., rhodopsin kinase promoters). The construct may also include other elements (e.g., a ribosome binding site for translation initiation (internal ribosome binding sequence), and a transcription / translation terminator). Generally, it is advantageous to transfect cells with this construct. Suitable vectors for stable transfection include, but are not limited to, retroviral vectors, lentiviral vectors, and Sendai viruses.

[0132] In some embodiments, the plasmid encoding the marker comprises: (1) a high copy number origin; (2) a selection marker for antibiotic selection by kanamycin (e.g., a neogene, but not limited to these); (3) a transcription termination sequence (e.g., a tyrosinase enhancer); and (4) a multicloning site for the incorporation of various nucleic acid cassettes; and (5) a nucleic acid sequence encoding the marker operably linked to a tyrosinase promoter. Numerous plasmid vectors for inducing protein-encoding nucleic acids are known in the art. These include, but are not limited to, the vectors disclosed in U.S. Patent No. 6,103,470; No. 7,598,364; No. 7,989,425; and No. 6,416,998 (these are incorporated herein by reference).

[0133] Viral gene delivery systems may be RNA-based or DNA-based viral vectors. Episome gene delivery systems may be plasmids, Epstein-Barr virus (EBV)-based episome vectors, yeast-based vectors, adenovirus-based vectors, simian virus 40 (SV40)-based episome vectors, bovine papillomavirus (BPV)-based vectors, or lentiviral vectors.

[0134] Markers may include, but are not limited to, fluorescent proteins (e.g., green fluorescent protein or red fluorescent protein), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, firefly / sea mushroom luciferase, or nanoluc), or other proteins. Markers may be proteins (e.g., secreted cell surface proteins or cellular proteins; synthesized or taken up by cells); nucleic acids (e.g., mRNA, or enzymatically active nucleic acid molecules), or polysaccharides. These may include antibodies, lectins, probes, or determinants of any such cellular component detectable by nucleic acid amplification reactions specific to the marker of the cell type of interest. Markers can also be identified by biochemical assays or enzymatic assays or biological reactions that depend on the function of the gene product. Nucleic acid sequences encoding these markers can be operably ligated to tyrosinase enhancers. In addition, other genes (e.g., genes that may affect stem cells for PRP differentiation, or photoreceptor function, or for physiological or pathological effects) may be included.

[0135] 1. MHC haplotype matching Major histocompatibility complex (MHC) is the primary cause of immune rejection in allogeneic organ transplantation. There are three major class I MHC haplotypes (A, B, and C) and three major class II MHC haplotypes (DR, DP, and DQ). HLA loci exhibit high polymorphism and are distributed over 4 Mb on chromosome 6. The ability to haplotype HLA genes within this region is clinically important because this region is associated with autoimmune and infectious diseases, and HLA haplotype compatibility between donor and recipient can affect the clinical outcome of transplantation. HLA corresponding to MHC class I presents peptides from within the cell, while HLA corresponding to MHC class II presents antigens to T lymphocytes from outside the cell. MHC haplotype compatibility between the graft and host triggers an immune response against the graft, leading to its rejection. Therefore, patients can be treated with immunosuppressants to prevent rejection. HLA-matched stem cell lines may overcome the risk of immune rejection.

[0136] Because HLA is important in transplantation, HLA loci are typically classified by serology and polymerase chain reaction (PCR) to identify preferred donor-recipient pairs. Serological detection of HLA class I and II antigens can be achieved using complement-mediated lymphotoxicity testing with purified T or B lymphocytes. This procedure is primarily used to match HLA-A and HLA-B loci. Molecular-based histology compatibility testing can often be more accurate than serological testing. HLA antigens can be identified using low-resolution molecular methods (e.g., SSOP (sequence-specific oligonucleotide probe) methods) in which PCR products are tested against a series of oligonucleotide probes, and this method is currently the most common method used for class II-HLA classification. High-resolution techniques (e.g., SSP (sequence-specific primer) methods) that utilize allele-specific primers for PCR amplification can identify specific MHC alleles.

[0137] When donor cells are HLA homozygous (i.e., contain the same locus for each antigen-presenting protein), MHC compatibility between the donor and recipient is significantly increased. While most individuals are heterozygous for MHC class I and MHC class II genes, certain individuals are homozygous for these genes. These homozygous individuals can act as superdonors, and grafts produced from these cells can be transplanted into all individuals that are homozygous or heterozygous for this haplotype. Furthermore, if homozygous donor cells have a haplotype that is frequently found in a particular population, these cells can be used in transplant therapy for a large number of individuals.

[0138] Therefore, iPSCs can be produced from somatic cells of the subject being treated, or from somatic cells of another subject having the same or substantially the same HLA type as this patient. For example, the donor's major HLA (e.g., the three major loci of HLA-A, HLA-B, and HLA-DR) is identical to the recipient's major HLA. In some cases, a somatic cell donor may be a superdonor, and thus PRP cells can be generated using iPSCs derived from an MHC homozygous superdonor. Thus, iPSCs derived from a superdonor can be transplanted into a subject that is homozygous or heterozygous with respect to the haplotype. For example, an iPSC may be homozygous for two HLA alleles, such as HLA-A and HLA-B. Therefore, iPSCs produced from a superdonor can be used in the manner disclosed herein to produce PRP cells that can potentially "match" a large number of potential recipients.

[0139] 2. Episome vectors In certain embodiments, reprogramming factors are expressed from expression cassettes contained within one or more exogenous episomal gene elements (see U.S. Patent Application Publication 2010 / 0003757, incorporated herein by reference). Therefore, iPSCs may not inherently contain exogenous gene elements (e.g., those derived from retroviral or lentiviral vector elements). These iPSCs are prepared using an extrachromosomal replication vector (i.e., an episomal vector), which is capable of episomal replication to produce iPSCs that are essentially free of exogenous vector or viral elements (see U.S. Patent No. 8,546,140, ​​incorporated herein by reference; see Yu et al., 2009). Many DNA viruses (e.g., adenovirus, monkey vacuolar virus 40 (SV40), or bovine papillomavirus (BPV)) or budding yeast ARS (autonomous replication sequence)-containing plasmids replicate extrachromosomally or episomatically in mammalian cells. This episomal plasmid essentially eliminates all of these drawbacks associated with vector integration (Bode et al., 2001). For example, lymphotrophic herpes virus-based inclusion, as defined above, or Epstein-Barr virus (EBV), can replicate extrachromosomally to facilitate the delivery of reprogramming genes to somatic cells. Useful EBV elements are OriP and EBNA-1, or their variants or functional equivalents. A further advantage of episomal vectors is that exogenous elements are lost over time after introduction into cells, resulting in self-persistent iPSCs that essentially do not contain these elements.

[0140] Other extrachromosomal vectors include other lymphotropic herpesvirus-based vectors. Lymphotropic herpesviruses are herpesviruses that replicate in lymphoblasts (e.g., human B lymphoblasts) and become plasmids as part of their natural life cycle. Herpes simplex virus (HSV) is not a "lymphotropic" herpesvirus. Exemplary lymphotropic herpesviruses include, but are not limited to, EBV, Kaposi's sarcoma herpesvirus (KSHV); herpesvirus thymili (HS); and Marek's disease virus (MDV). Similarly, other sources of episome-based vectors (e.g., yeast ARS, adenovirus, SV40, or BPV) are considered.

[0141] C. Somatic cell nuclear transfer Pluripotent stem cells can be prepared by somatic cell nuclear transfer. Somatic cell nuclear transfer involves the transplantation of donor nuclei into spindle-free oocytes. In one method, donor fibroblast nuclei derived from rhesus monkey dermal fibroblasts are introduced into the cytoplasm of spindle-free mature metaphase II rhesus monkey oocytes by electrofusion (Byrne et al., 2007). The fused oocytes are activated by exposure to ionomycin and then incubated until the blastocyst stage. The inner cell mass of the selected blastocysts is then cultured to produce an embryonic stem cell lineage. This embryonic stem cell lineage exhibits normal ES cell morphology, expresses various ES cell markers, and differentiates into multiple cell types both in vitro and in vivo.

[0142] III. Photoreceptor Progenitor Cells In some embodiments, neuroretinal progenitor (NRP) cells, photoreceptor precursor (PRP) cells, or optic vesicles (OVs) are produced by the methods disclosed herein. Cells in the retina that respond directly to light are photoreceptor cells. Photoreceptors are photosensitive neurons in the outer part of the retina and can be either rods or cones. In the process of light transmission, photoreceptor cells convert the incident light energy focused by a lens into an electrical signal, which is then transmitted to the brain via the optic nerve. Vertebrates have two types of photoreceptor cells (e.g., cones and rods). Cones are adapted to detect fine detail, central vision, and color vision and function well in bright light. Rods are involved in peripheral vision and scotopic vision. Nerve signals from rods and cones are processed by other neurons in the retina.

[0143] PRP cells can express markers (e.g., OTX2, CRX, PRDM1 (BLIMP1), Neurod1, RCVRN, TUBB3, and L1CAM (CD171)). PRP cells express several proteins that can function as markers for detection using methodologies (e.g., immunocytochemistry, Western blotting, flow cytometry, and enzyme-linked immunosorbent assay (ELISA)). For example, one characteristic PRP marker is RCVRN. PRP cells may not express embryonic stem cell markers OCT-4, NANOG, or REX-1 (at any detectable level). Specifically, the expression of these genes is approximately 100 to 1000 times lower in PRP cells compared to ES cells or iPSC cells, as assessed by quantitative RT-PCR.

[0144] For example, PRP cell markers can be detected at the mRNA level by reverse transcriptase polymerase chain reaction (RT-PCR), Northern blot analysis, or dot blot hybridization analysis using sequence-specific primers with standard amplification methods using publicly available sequence data (GENBANK®). The expression of tissue-specific markers detected at the protein or mRNA level is considered positive if this level is at least 2, 3, 4, 5, 6, 7, 8, or 9 times higher than that of control cells (e.g., undifferentiated pluripotent stem cells or other unrelated cell types), more specifically if it is 10, 20, 30, 40, 50 times, or higher.

[0145] Dysfunction, damage, and loss of photoreceptor cells are the cause of many eye diseases and disorders (e.g., age-related macular degeneration (AMD), hereditary macular degenerations such as Best's disease, and retinitis pigmentosa). A potential treatment for such diseases is the transplantation of PRP or photoreceptor cells into the retina of those requiring such treatment. It is hypothesized that supplementation with PRP or photoreceptor cell transplantation may delay, halt, or reverse deterioration, improve retinal function, and prevent blindness resulting from such conditions. However, directly obtaining PRP or photoreceptor cells from human donors and human embryos is difficult.

[0146] In some embodiments, a method is provided for producing PRP cells from a suspension of essentially single cells of PSCs, such as human iPSCs. In some embodiments, the PSCs are cultured to preconfluence to prevent any cell aggregates. In certain embodiments, the PSCs are dissociated by incubation with a cell dissociation solution or cell dissociation enzyme (e.g., exemplified by Versene, Trypsin, ACCUTASE®, or TRYPLE®). The PSCs can also be dissociated into a suspension of essentially single cells by pipetting.

[0147] In addition, adding brevistatin (e.g., approximately 2.5 μM) to the culture medium can increase PSC viability after single-cell dissociation without cells adhering to the culture vessel. Alternatively, a ROCK inhibitor can be used instead of brevistatin to increase PSC viability after single-cell dissociation.

[0148] To efficiently differentiate PRP cells from single-cell PSCs, accurate counting of the input density can increase PRP differentiation efficiency. Therefore, single-cell suspensions of PSCs are generally counted before seeding. For example, single-cell suspensions of PSCs are counted using a hemocytometer or automated cell counter (e.g., VICELL® or TC20). These cells can then be diluted to cell densities of approximately 10,000 to 500,000 cells / mL, 50,000 to 200,000 cells / mL, or 75,000 to 150,000 cells / mL. In non-limiting examples, single-cell suspensions of PSCs are diluted to a density of approximately 100,000 cells / mL in a fully defined culture medium such as ESSENTIAL 8® (E8®) medium.

[0149] Once a single-cell suspension of PSCs is obtained at a known cell density, the cells are generally seeded into a suitable culture vessel, such as a tissue culture plate (e.g., a flask, 6-well plate, 24-well plate, or 96-well plate). Culture vessels that can be used to culture these cells include, but are not limited to, flasks, tissue culture flasks, petri dishes, petri dishes, tissue culture dishes, multi-petri dishes, microplates, microwell plates, multi-plates, multi-well plates, microslides, chamber slides, tubes, trays, CELLSTACK® chambers, culture bags, and roller bottles, as long as stem cells can be cultured inside. These cells may be cultured in the following volumes, depending on the need for culture: at least or about 0.2, 0.5, 1, 2, 5, 10, 20, 30, 40, 50 ml, 100 ml, 150 ml, 200 ml, 250 ml, 300 ml, 350 ml, 400 ml, 450 ml, 500 ml, 550 ml, 600 ml, 800 ml, 1000 ml, 1500 ml, or any range that can be derived from these. In certain embodiments, the culture vessel may be a bioreactor, which may refer to any ex vivo device or system that supports a biologically active environment capable of growing cells. The bioreactor may have a volume of at least 2, 4, 5, 6, 8, 10, 15, 20, 25, 50, 75, 100, 150, 200, 500 liters, 1, 2, 4, 6, 8, 10, 15 cubic meters, or any range that can be derived from these.

[0150] In certain configurations, PSCs such as iPSCs are seeded at a cell density suitable for efficient differentiation. Generally, these cells are seeded at a density of approximately 1,000 to 75,000 cells / cm³. 2 (For example, approximately 5,000 to 40,000 cells / cm²) 2Seed the cells at a cell density of approximately 50,000 to 400,000 cells per well in a 6-well plate. In an exemplary method, the cells are seeded at a cell density of approximately 100,000, 150,000, 200,000, 250,000, 300,000, or 350,000 cells per well (for example, approximately 200,000 cells per well).

[0151] PSCs, such as iPSCs, are generally cultured on culture plates coated with one or more cell adhesion proteins to promote cell adhesion while maintaining cell viability. For example, preferred cell adhesion proteins include extracellular matrix proteins (e.g., vitronectin, laminin, collagen, and / or fibronectin), and these extracellular matrix proteins can be used to coat the culture surface as a means of providing a solid support for the proliferation of pluripotent cells. The term "extracellular matrix (ECM)" is recognized in the art. Components of the ECM may include, but are not limited to, one or more of the following proteins: fibronectin, laminin, vitronectin, tenascin, entactin, thrombospondin, elastin, gelatin, collagen, fibrillin, merosine, ancholine, chondronectin, binding proteins, bone sialoproteins, osteocalcin, osteopontin, epinectin, hyaluronectin, undulin, epiligrin, and kalinin. Other ECM components may include synthetic peptides for adhesion (e.g., RGD motif or IKVAV motif), synthetic hydrogels (e.g., PEG, PLGA, etc.), or natural hydrogels (e.g., alginates). In an exemplary method, PSCs are grown on a culture plate coated with vitronectin. In some embodiments, the cell adhesion protein is a human protein.

[0152] Extracellular matrix proteins may be of natural origin and purified from human or animal tissue, or ECM proteins may be genetically engineered recombinant proteins or naturally synthesized. ECM proteins may be whole proteins or in the form of natural or engineered peptide fragments. Examples of ECM proteins that may be useful in the matrix for cell culture include laminin, collagen I, collagen IV, fibronectin, and vitronectin. In some embodiments, the matrix composition is heterogeneous. For example, a heterogeneous matrix for culturing human cells may use matrix components of human origin and exclude any non-human animal components.

[0153] In some embodiments, the total protein concentration in the matrix composition may be about 1 ng / mL to about 1 mg / mL. In some preferred embodiments, the total protein concentration in the matrix composition is about 1 μg / mL to about 300 μg / mL. In more preferred embodiments, the total protein concentration in the matrix composition is about 5 μg / mL to about 200 μg / mL.

[0154] Cells such as PRP cells or PSCs can be cultured with nutrients necessary to support the growth of each specific population of cells. Generally, cells are cultured in a growth medium containing a carbon source, a nitrogen source, and buffers to maintain pH. This medium may also contain fatty acids or lipids, amino acids (e.g., non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, pyruvate, buffers, pH indicators, and inorganic salts. Exemplary growth media include minimal essential media (e.g., Dulbecco's Modified Eagle Medium (DMEM) or ESSENTIAL 8® (E8®) medium) supplemented with various nutrients (e.g., non-essential amino acids and vitamins) to enhance stem cell growth. Examples of minimal essential media include, but are not limited to, Minimum Essential Medium Eagle® Alpha, Dulbecco's Modified Eagle Medium (DMEM), RPMI-1640 medium, 199 medium, and F12 medium. In addition, minimal essential media can be supplemented with additives (e.g., horse, calf, or fetal bovine serum). Alternatively, the medium may be serum-free. In other cases, the growth medium may include serum-free formulations, referred herein as “Knockout Serum Substitutes,” which are optimized for growing and maintaining undifferentiated cells (e.g., stem cells) during culture. KNOCKOUT® serum substitutes are disclosed, for example, in U.S. Patent Application Publication No. 2002 / 0076747, incorporated herein by reference. Preferably, PSCs are cultured in a fully defined and feeder-free medium.

[0155] Therefore, single-cell PSCs are generally cultured in a fully defined culture medium after seeding. In certain embodiments, the medium is aspirated and fresh medium (e.g., E8® medium) is added to the culture approximately 18–24 hours after seeding. In certain embodiments, single-cell PSCs are cultured in a fully defined culture medium for approximately 1, 2, or 3 days after seeding. Preferably, single-cell PSCs are cultured in a fully defined culture medium for approximately 2 days before the differentiation process can proceed.

[0156] In some embodiments, the culture medium may or may not contain any serum substitute. Examples of serum substitutes include albumin (e.g., lipid-rich albumin, albumin substitutes (e.g., recombinant albumin), vegetable starch, dextran, and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thioglycerol, or substances appropriately containing these equivalents. Serum substitutes can be prepared, for example, by the methods disclosed in International Publication No. 98 / 30679. Alternatively, any commercially available material can be used more conveniently. Examples of commercially available materials include KNOCKOUT® serum substitute (KSR), chemically defined lipid concentrates (Gibco), and GLUTAMAX® (Gibco).

[0157] Other culture conditions can be appropriately defined. For example, the culture temperature may be about 30-40°C, and may be, for example, at least or about 31, 32, 33, 34, 35, 36, 37, 38, or 39°C, but is not particularly limited thereto. In one embodiment, cells are cultured at 37°C. The CO2 concentration may be about 1-10% (e.g., about 2-5%), or any range that can be derived within this range. The oxygen partial pressure may be at least, at most, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20%, or any range that can be derived within this range.

[0158] A. Differentiation medium Retinal induction medium After single-cell PSCs have attached to a culture plate, these cells are preferably cultured in retinal induction medium (RIM) to initiate the differentiation process into retinal lineage cells. RIM contains a WNT pathway inhibitor and can induce differentiation of PSCs into retinal lineage cells. RIM further contains a TGFβ pathway inhibitor and a BMP pathway inhibitor. An exemplary RIM medium is shown in Table 1.

[0159] RIM may contain DMEM and F12 in a ratio of approximately 1:1. Exemplary methods include RIM containing a WNT pathway inhibitor (e.g., CKI-7), a BMP pathway inhibitor (e.g., LDN193189), and a TGFβ pathway inhibitor (e.g., SB431542). For example, RIM may contain approximately 5 nM to 50 nM (e.g., approximately 10 nM) of LDN193189, approximately 0.1 μM to 5 μM (e.g., approximately 0.5 μM) of CKI-7, and approximately 0.5 μM to 10 μM (e.g., approximately 1 μM) of SB431542. In addition, RIM may contain knockout serum substitutes (e.g., approximately 1% to 5%), MEM non-essential amino acids (NEAAs), sodium pyruvate, N-2 supplements, B-27 supplements, ascorbic acid, and insulin growth factor 1 (IGF1). Preferably, IGF1 is animal-free IGF1 (AF-IGF1) and is included in the RIM at a concentration of approximately 0.1 ng / mL to approximately 10 ng / mL (e.g., approximately 1 ng / mL). The culture medium is aspirated daily and replaced with fresh RIM. Cells are cultured in the RIM for approximately 1 to 5 days (e.g., approximately 1, 2, 3, 4, or 5 days, e.g., approximately 2 days) to produce anterior neuroectoderm cells.

[0160] Retinal differentiation medium Next, anterior neuroectoderm cells can be cultured in retinal differentiation medium (RD) for further differentiation. RD comprises a WNT pathway inhibitor, a TGFβ pathway inhibitor, and a MEK inhibitor. In one embodiment, RD comprises a WNT pathway inhibitor (e.g., CKI-7), optionally a BMP pathway inhibitor (e.g., LDN193189), a TGFβ pathway inhibitor (e.g., SB431542), and a MEK inhibitor (e.g., PD0325901). Generally, the concentrations of the WNT pathway inhibitor, BMP pathway inhibitor, and TGFβ pathway inhibitor are higher in RDM compared to RIM, for example, about 9 to 11 times higher, and for example, about 10 times higher. In an exemplary method, the RD includes LDN193189 at approximately 50 nM to approximately 200 nM (e.g., approximately 100 nM), CKI-7 at approximately 1 μM to approximately 10 μM (e.g., approximately 5 μM), SB431542 at approximately 1 μM to approximately 50 μM (e.g., approximately 10 μM), and PD0325901 at approximately 0.1 μM to approximately 10 μM (e.g., approximately 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, or 9 μM). An exemplary RD is shown in Table 1.

[0161] In some embodiments, cells can be initially differentiated in the presence of a BMP inhibitor (e.g., LDN1913189) for a certain period before differentiation in the absence of the BMP inhibitor. First, anterior neuroectoderm cells are cultured in RD1 containing a BMP pathway inhibitor (e.g., LDN193189) for approximately 1–3 days (e.g., 2 days). Next, these cells are cultured in RD2 without the BMP pathway inhibitor. The second step can be extended to approximately 5–10 days (e.g., approximately 7 days) to continue the differentiation of the anterior neuroectoderm cells. This method has been shown to increase VSX2 expression in subsequently produced PRP cells. VSX2 is the earliest specific marker of neural RPCs in the optic vesicle and goblet cells (Rowan et al., 2004). VSX2 + Retinal progenitor cells can give rise to all cell types of the neuroretina (cones, rods, ganglion cells, amacrine cells, bipolar cells, horizontal cells, and Müller glia).

[0162] Generally, RD contains DMEM and F12 in a ratio of approximately 1:1, knockout serum substitute (e.g., approximately 1% to approximately 5%, e.g., approximately 1.5%), MEM NEAA, sodium pyruvate, N-2 supplement, B-27 supplement, ascorbic acid, and IGF1 (e.g., approximately 1 ng / mL to approximately 50 ng / mL, e.g., approximately 10 ng / mL). In specific methods, cells are given fresh RD daily after aspirating the culture medium from the previous day. Generally, cells are cultured in RDM for approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 days (e.g., approximately 7 days) to differentiate anterior neuroectoderm cells into RPCs.

[0163] Retinal maturation culture medium Next, anterior neuroectoderm cells can be further differentiated and expanded by culturing these cells in retinal maturation medium (RM) to produce RPCs. RM may contain nicotinamide. RM may contain about 1 mM to about 50 mM (e.g., about 10 mM) of nicotinamide. RM may further contain ascorbic acid (e.g., 50 to 500 μm, particularly about 100 to 300 μm, e.g., about 200 μm). Preferably, RM does not contain activin A or is essentially activin A-free. Exemplary RM media are shown in Table 1. RM (e.g., RM2) may further contain γ-secretase inhibitors (e.g., DAPT, basic FGF) and / or TGFβ pathway inhibitors (e.g., SB431542).

[0164] The RM may contain DMEM and F12 in a ratio of approximately 1:1, knockout serum substitute in approximately 1% to 5% (e.g., approximately 1.5%), MEM non-essential amino acids (NEAAs), sodium pyruvate, N-2 supplement, B-27 supplement, and ascorbic acid. This medium can be replaced daily with RM at room temperature. These cells are generally cultured in the RM for approximately 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 days (e.g., approximately 10 days) to induce expanded RPCs.

[0165] PRP maturation medium (PM) PRP can be matured in PRP maturation medium (PM). An example of PM medium is shown in Table 1. PM medium contains ascorbic acid, nicotinamide, and a γ-secretase inhibitor (e.g., DAPT (e.g., about 1 μM to about 10 μM (e.g., about 5 μM))). PM (e.g., PM2) may also contain a CDK inhibitor, e.g., a CDK4 / 6 inhibitor, e.g., PD0332991 (e.g., about 1 μM to about 50 μM, e.g., about 10 μM of PD0332991).

[0166] PM medium may contain DMEM and F12 in a ratio of approximately 1:1, knockout serum substitute in approximately 1% to 5% (e.g., approximately 1.5%), MEM non-essential amino acids (NEAAs), sodium pyruvate, N-2 supplement, B-27 supplement, and ascorbic acid. This medium may be replaced daily with PM medium at room temperature. These cells are generally cultured in RM medium for approximately 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days (e.g., approximately 10 days) to induce mature PRP cells.

[0167] Photoreceptor precursor induction medium (FDSC) For further differentiation of RPCs, cells are cultured, preferably in FDSC medium. Exemplary FDSC media are shown in Table 1. FDSC medium contains a WNT pathway inhibitor, a γ-secretase inhibitor, and a TGFβ pathway inhibitor. In one embodiment, FDSC contains a WNT pathway inhibitor (e.g., CKI-7), a TGFβ pathway inhibitor (e.g., SB431542), and a γ-secretase inhibitor (e.g., DAPT). In an exemplary method, FDSC medium contains about 1 μM to about 10 μM (e.g., about 5 μM) of CKI-7, about 1 μM to about 50 μM (e.g., about 10 μM) of SB431542, and about 1 μM to about 10 μM (e.g., about 5 μM) of DAPT. FDSC may also contain basic FGF.

[0168] FDSC medium may contain DMEM and F12 in a ratio of approximately 1:1, knockout serum substitute in approximately 1% to 5% (e.g., approximately 1.5%), MEM non-essential amino acids (NEAAs), sodium pyruvate, N-2 supplement, B-27 supplement, and ascorbic acid. In addition, this medium may contain basic FGF (e.g., approximately 5 ng / mL to approximately 15 ng / mL (e.g., approximately 10 ng / mL)). This medium can be replaced daily with FDSC medium at room temperature. These cells are generally cultured in FDSC for approximately 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days (e.g., approximately 15 days) to induce PRP cells. [Table 1] JPEG0007898257000002.jpg255170JPEG0007898257000003.jpg255168JPEG00078982570 00004.jpg254170JPEG0007898257000005.jpg255169JPEG0007898257000006.jpg218170

[0169] In addition, by adding brevistatin (e.g., approximately 2.5 μM) to this medium to promote aggregate formation, the PRP viability can be increased while maintaining purity. Alternatively, the PRP viability after dissociation into single cells may be increased by using a ROCK inhibitor instead of brevistatin, such as TRYPLE®.

[0170] PRP aggregates can be cultured to produce hybrid photoreceptor cells or optic vesicles.

[0171] B. Cryopreservation of PRP cells Photoreceptor precursor cells produced by the methods disclosed herein can be cryopreserved; see, for example, PCT International Publication No. 2012 / 149484A2, incorporated herein by reference. These cells can be cryopreserved with or without a substrate. In some embodiments, the storage temperature ranges from about -50°C to about -60°C, about -60°C to about -70°C, about -70°C to about -80°C, about -80°C to about -90°C, about -90°C to about -100°C, and overlapping ranges therebetween. In some embodiments, lower temperatures are used for preserving (e.g., maintaining) the cryopreserved cells. In some embodiments, liquid nitrogen (or other similar coolant) is used to preserve the cells. In further embodiments, these cells are preserved for more than about 6 hours. In additional embodiments, these cells are preserved for about 72 hours. In some embodiments, these cells are preserved for 48 hours to about 1 week. In yet another embodiment, these cells are preserved for about 1, 2, 3, 4, 5, 6, 7, or 8 weeks. In further embodiments, these cells are stored for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These cells can be stored for longer periods of time. These cells can be cryopreserved separately or on a substrate such as any of the substrates disclosed herein.

[0172] In some embodiments, additional cryoprotectants may be used. For example, these cells can be cryopreserved in a cryopreservation solution containing one or more cryoprotectants (e.g., DM80, serum albumin (e.g., human serum albumin or bovine serum albumin)). In certain embodiments, the solution contains about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% DMSO. In other embodiments, the solution contains about 1% to about 3%, about 2% to about 4%, about 3% to about 5%, about 4% to about 6%, about 5% to about 7%, about 6% to about 8%, about 7% to about 9%, or about 8% to about 10% dimethyl sulfoxide (DMSO) or albumin. In certain embodiments, the solution contains 2.5% DMSO. In yet another particular embodiment, the solution contains 10% DMSO.

[0173] Cells can be cooled at approximately 1°C / min, for example, during cryopreservation. In some embodiments, the cryopreservation temperature is approximately -80°C to approximately -180°C or approximately -125°C to approximately -140°C. In some embodiments, cells are cooled to 4°C and then cooled at approximately 1°C / min. Cryopreserved cells can be transferred to the vapor phase of liquid nitrogen before thawing for use. In some embodiments, for example, when the cells reach approximately -80°C, they are transferred to the liquid nitrogen storage area. Cryopreservation can also be performed using a freezer with a controlled rate. Cryopreserved cells can be thawed at a temperature of approximately 25°C to approximately 40°C, typically at approximately 37°C.

[0174] Alternatively, these cells can be cryopreserved as aggregates without dissociating into single-cell suspensions. For example, after PRP enrichment, single cells can be re-aggregated in minimal medium (RMN) in a tissue culture flask for two days. The aggregates can be pooled to obtain sample aliquots for cell counting. After a series of washes, the aggregates can be resuspended in CryoSTOR CS10 Freeze Medium, and this aggregate suspension can be transferred to a liquid nitrogen storage vial with, for example, 25 × 10^6 aggregated cell products / vial.

[0175] C. Inhibitors WNT pathway inhibitors WNTs are a highly conserved family of secretory signaling molecules that regulate intercellular interactions and are associated with the Drosophila segment polarity gene wingless. In humans, the WNT family of genes encodes 38-43 kDa cysteine-rich glycoproteins. WNT proteins possess a hydrophobic signaling sequence, a conserved asparagine-binding oligosaccharide consensus sequence (see, e.g., Shimizu et al. Cell Growth Differ 8:1349-1358 (1997)), and 22 conserved cysteine ​​residues. Due to their ability to promote cytoplasmic beta-catenin stabilization, WNT proteins can act as transcriptional activators and inhibit apoptosis. Overexpression of certain WNT proteins has been shown to be associated with certain cancers.

[0176] In this specification, WNT inhibitors (also referred to as WNT pathway inhibitors) generally refer to WNT inhibitors. Therefore, a WNT inhibitor refers to any inhibitor of a member of the WNT family protein (e.g., Wnt1, Wnt2, Wnt2b, Wnt3, Wnt4, Wnt5A, Wnt6, Wnt7A, Wnt7B, Wnt8A, Wnt9A, Wnt10a, Wnt11, and Wnt16). Certain embodiments of this method relate to WNT inhibitors in differentiation media. Examples of suitable WNT inhibitors known in the art include: N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide dihydrochloride (CKI-7), N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidine-2-yl)thio]acetamide (IWP2), N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-3-(2-methoxyphenyl)-4-oxothieno[3,2-d]pyrimidine-2-yl)thio]acetamide (IWP4), 2-phenoxybenzoic acid-[( [5-methyl-2-furanyl)methylene]hydrazide (PNU74654), 2,4-diaminoquinazoline, quercetin, 3,5,7,8-tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidine-4-one (XAV939), 2,5-dichloro-N-(2-methyl-4-nitrophenyl)benzenesulfonamide (FH535), N-[4-[2-ethyl-4-(3-methylphenyl)-5-thiazolyl]-2-pyridinyl]benzamide (TAK715), Dickkopf-related protein 1 (DKK1), and secreted frizzled-related protein (SFRP1) 1. In addition, WNT inhibitors may include antibodies against WNT, dominant-negative variants of WNT, and siRNAs and antisense nucleic acids that suppress WNT expression. WNT inhibition can also be achieved using RNA-mediated interference (RNAi).

[0177] BMP pathway inhibitors Bone morphogenetic proteins (BMPs) are multifunctional growth factors belonging to the transforming growth factor beta (TGFβ) superfamily. BMPs are considered to constitute a crucial group of morphogenetic signaling pathways, organizing structures throughout the body. The important physiological functions of BMP signaling are highlighted by their numerous roles in dysregulation of BMP signaling in pathological processes.

[0178] BMP pathway inhibitors (also referred to herein as BMP inhibitors) may generally include inhibitors of BMP signaling, or may include inhibitors specific to BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10, or BMP15. Examples of BMP inhibitors include: 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidine-3-yl)quinoline hydrochloride (LDN193189), 6-[4-[2-(1-piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine dihydrochloride (dolsomorphine), 4-[6-(4 -(1-methylethoxy)phenyl]pyrazolo[1,5-a]pyrimidine-3-yl]quinoline (DMH1), 4-[6-[4-[2-(4-morpholinyl)ethoxy]phenyl]pyrazolo[1,5-a]pyrimidine-3-yl]quinoline (DMH-2), and 5-[6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidine-3-yl]quinoline (ML347).

[0179] TGFβ pathway inhibitors Transforming growth factor beta (TGFβ) is a secreted protein that regulates proliferation, cell differentiation, and other functions in most cells. TGFβ is a type of cytokine that plays a role in immunity, cancer, bronchial asthma, pulmonary fibrosis, heart disease, diabetes, and multiple sclerosis. There are at least three isoforms of TGFβ, called TGFβ1, TGFβ2, and TGFβ3. The TGFβ family is part of a superfamily of proteins known as the transforming growth factor beta superfamily (e.g., inhibin, activin, anti-Müllerian hormone, bone morphogenetic protein, decapentaplesic, and Vg-1).

[0180] TGFβ pathway inhibitors (also referred to herein as TGFβ inhibitors) generally include any inhibitor of TGFβ signaling. For example, TGFβ inhibitors include 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazole-2-yl]benzamide (SB431542), 6-[2-(1,1-dimethylethyl)-5-(6-methyl-2-pyridinyl)-1H-imidazole-4-yl]quinoxaline (SB525334), and 2-(5-benzo[1,3]dioxol-5-yl-2-ieri-butyl-3H -Imidazole-4-yl)-6-methylpyridine hydrochloride hydrate (SB-505124), 4-(5-Benzol[1,3]dioxol-5-yl-4-pyridine-2-yl-1H-imidazole-2-yl)-benzamide hydrate, 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazole-2-yl]-benzamide hydrate, left-right determinant (Lefty), 3-(6-methyl-2-pyridinyl) Dinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide (A83-01), 4-[4-(2,3-dihydro-1,4-benzodioxin-6-yl)-5-(2-pyridinyl)-1H-imidazole-2-yl]benzamide (D4476), 4-[4-[3-(2-pyridinyl)-1H-pyrazole-4-yl]-2-pyridinyl]-N-(tetrahydro-2H-pyran-4-yl)-benzamide These are Mido (GW788388), 4-[3-(2-pyridinyl)-1H-pyrazole-4-yl]quinoline (LY364847), 4-[2-fluoro-5-[3-(6-methyl-2-pyridinyl)-1H-pyrazole-4-yl]phenyl]-1H-pyrazole-1-ethanol (R268712), or 2-(3-(6-methylpyridinyl-2-yl)-1H-pyrazole-4-yl)-1,5-naphthiridine (RepSox).

[0181] MEK inhibitors MEK inhibitors are chemicals or drugs that inhibit the mitogen-activated protein kinase enzymes MEK1 or MEK2. These chemicals and drugs can be used to affect the MAPK / ERK pathway. Examples of MEK inhibitors include: N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-benzamide (PD0325901), N-[3-[3-cyclopropyl-5-(2-fluoro-4-iodoanilino)-6,8-dimethyl-2,4,7-trioxopyrido[4,3-d]pyrimidine-1-yl]phenyl]acetamide (GSK1120212), 6-(4-bromo-2-fluoroanilino)- 7-Fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide (MEK162), N-[3,4-difluoro-2-(2-fluoro-4-iodoanilino)-6-methoxyphenyl]-1-(2,3-dihydroxypropyl)cyclopropane-1-sulfonamide (RDEA119), and 6-(4-bromo-2-chloroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide (AZD6244).

[0182] Gamma-secretase inhibitors Gamma-secretase is a multi-subunit protease complex (which is itself an endogenous membrane protein) that cleaves single-pass transmembrane proteins at residues within the transmembrane domain. This type of protease is known as an endometrial protease. The most well-known substrate of gamma-secretase is amyloid precursor protein, a large endogenous membrane protein. When this endogenous membrane protein is cleaved by both gamma-secretase and beta-secretase, it produces a short amino acid peptide called amyloid-beta. The abnormally folded fibril form of this amino acid peptide is a major component of amyloid plaques found in the brains of Alzheimer's disease patients.

[0183] The term "gamma-secretase inhibitor" as used herein generally refers to a γ-secretase inhibitor. Examples of γ-secretase inhibitors include, but are not limited to, the following: N-[(3,5-difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethyl ester (DAPT), 5-chloro-N-[(1S)-3,3,3-trifluoro-1-(hydroxymethyl)-2-(trifluoromethyl)propyl]-2-thiophenesulfonamide (Vegas Estate), MDL-28170, 3,5-bis(4-) Trophenoxy)benzoic acid (compound W), 7-amino-4-chloro-3-methoxy-1H-2-benzopyran (JLK6), (5S)-(tert-butoxycarbonylamino)-6-phenyl-(4R)-hydroxy-(2R)-benzylhexanoyl)-L-leucine-L-phenylalaninamide (L-685,485), (R)-2-fluoro-α-methyl[1,1'-biphenyl]-4-acetic acid ((R)-flurbiprofen; flurizan), N-[ (1S)-2-[[(7S)-6,7-dihydro-5-methyl-6-oxo-5H-dibenzo[b,d]azepine-7-yl]amino]-1-methyl-2-oxoethyl]-3,5-difluorobenzeneacetamide (dibenzazepine; DBZ), N-[cis-4-[(4-chlorophenyl)sulfonyl]-4-(2,5-difluorophenyl)cyclohexyl]-1,1,1-trifluoromethanesulfonamide (MRK560), (2S)-2-[[(2S )-6,8-difluoro-1,2,3,4-tetrahydro-2-naphthalenyl]amino]-N-[1-[2-[(2,2-dimethylpropyl)amino]-1,1-dimethylethyl]-1H-imidazole-4-yl]pentanamide hydrobromide (PF3084014 hydrobromide), and 2-[(1R)-1-[[(4-chlorophenyl)sulfonyl](2,5-difluorophenyl)amino]ethyl-5-fluorobenzenebutanoic acid (BMS299897).

[0184] Cyclin-dependent kinase inhibitors Cyclin-dependent kinases (CDKs) are a family of serine kinases first discovered for their role in controlling the cell cycle. CDKs are also involved in the control of neuronal transcription, mRNA processing, and differentiation. In many human cancers, CDKs are overactive or CDK inhibitor proteins are non-functional. CDK inhibitors can be CDK1 inhibitors, CDK2 inhibitors, CDK3 inhibitors, CDK4 inhibitors, CDK5 inhibitors, CDK6 inhibitors, CDK7 inhibitors, CDK8 inhibitors, and / or CDK9 inhibitors. In certain embodiments, the CDK inhibitor is a CDK4 / 6 inhibitor.

[0185] Examples of CDK inhibitors include, but are not limited to: palbociclib (PD-‌0332991) HCl, roscovitine (seliciclib, CYC202), SNS-032 (BMS-387032), dinaciclib (SCH727965), flavopiridol (alvocidib), MSC2530818, JNJ-7706621, AZD5438, MK-8776 (SCH 900776), PHA-793887, BS-181 HCl, A-674563, abemaciclib (LY2835219), BMS-265246, PHA-767491, or milciclib (PHA-848125).

[0186] IV. Use of photoreceptor precursor cells Certain embodiments provide methods for producing PRP or PRP-enriched cell populations that can be used for many important research, development, and commercial purposes.

[0187] In some embodiments, by the methods disclosed herein, at least or about 50% (e.g., at least or about 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or any range derivable therein) of the PRP cells in at least or about 10 6 、10 7 、10<000********** 8 、5×10<00********** 8 、10<00********** 9 、10<00********** 10A population of individual cells (or any range that can be derived from them) is obtained.

[0188] In certain embodiments, the starting cells for this method are at least or about 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 This may include the use of individual cells or any range that can be derived from them. This starting cell population is at least or about 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 It may have a seeding density of individual cells / mL or any range that can be derived from these.

[0189] PRP cells or photoreceptor cells produced by the methods disclosed herein can be used in any method and application currently known in the art with respect to PRP or photoreceptor cells. For example, a method for evaluating a compound can be provided, comprising assaying the pharmacological or toxicological properties of the compound to PRP or photoreceptor cells. A method for evaluating a compound with respect to its effect on PRP cells can also be provided, comprising a) contacting the compound with the PRP cells provided herein, and b) assaying the effect of the compound on the PRP cells.

[0190] PRP cells, or cells derived from PRP cells, can be used for transplantation in cell rescue therapy or total tissue replacement therapy. The cells of this embodiment can also be used to create retinal disease models for studying pathophysiology, and can also be used for drug screening.

[0191] A. Screening of test compounds PRP cells can be commercially used to screen for factors (e.g., solvents, small molecule drugs, peptides, oligonucleotides) or environmental conditions (e.g., culture conditions or handling) that affect the properties of such cells and their various offspring. For example, the test compound may be a compound, small molecule, polypeptide, growth factor, cytokine, or other biological agent.

[0192] In one embodiment, the method includes contacting PRP cells with a test drug and determining whether the test drug modulates the activity or function of PRP cells in a population. In some applications, screening assays are used to identify drugs that modulate PRP cell proliferation, alter PRP cell differentiation, or affect cell viability. Screening assays can be performed in vitro or in vivo. Methods suitable for screening and identifying ophthalmic or PRP drugs include those suitable for high-throughput screening. For example, PRP cells may be placed or mounted on a culture dish, flask, roller bottle, or plate (e.g., a single multi-well dish or plate, e.g., 8, 16, 32, 64, 96, 384, and 1536 multi-well plates or dishes) at a desired defined position for the identification of potential therapeutic molecules. Examples of libraries that can be screened include small molecule libraries, siRNA libraries, and adenovirus transfection vector libraries.

[0193] Other screening applications relate to testing pharmaceutical compounds for their effects on maintaining or repairing retinal tissue. Screening can be performed because the compound is designed to have a pharmacological effect on these cells, or because compounds designed to have effects elsewhere may have unintended side effects on cells of this tissue type.

[0194] B. Treatment and Transplantation Other embodiments may also provide the use of PRP cells to enhance the maintenance and repair of ocular tissue in relation to any desired condition (e.g., retinal degeneration or significant damage). Retinal degeneration may be associated with age-related macular degeneration (AMD), Stargardt macular dystrophy, retinitis pigmentosa, glaucoma, retinal vascular disease, viral infections of the eye, and other retinal / eye diseases. Photoreceptor precursor cells may constitute at least 50%, at least 75%, at least 85%, at least 95%, at least 99%, or about 100% of the cells in the culture.

[0195] In another aspect, the Disclosure provides a method for treating an individual in need, comprising administering to the individual a composition comprising photoreceptor precursor cells. The composition may be administered into the eye, subretinal space, or intravenously. Such an individual may have a macular degeneration, such as age-related macular degeneration, which may be in the early or late stages. Such an individual may have retinitis pigmentosa, Stargardt disease, retinal dysplasia, retinal degeneration, diabetic retinopathy, congenital retinal dystrophy, Leber congenital amaurosis, retinal detachment, glaucoma, or neuropathy.

[0196] To determine the suitability of a cell composition for therapeutic administration, the cells can be first tested in a suitable animal model. In one embodiment, PRP cells are evaluated in vivo for their viability and their ability to maintain their phenotype. The cell composition is administered to immunodeficient animals (e.g., nude mice, or animals immunodeficient by chemical or radiation). Tissue is collected after the proliferation period and evaluated for the presence of pluripotent stem cell-derived cells.

[0197] Many animals are available for testing the compatibility of PRP cell compositions. For example, the Royal College of Surgeons' (RCS) rat is a known model of retinal dystrophy (Lund et al., 2006). In addition, the compatibility and viability of PRP cells can be determined by transplantation in Matrigel (e.g., subcutaneously or retina-wise) in immunodeficient animals such as NOG mice (Kanemura et al., 2014). Other models that may be used include, but are not limited to, the S334ter rat model of retinal degeneration and the NIH Rowett nude (RNU) rat model.

[0198] Human PRP cells, or pharmaceutical compositions containing these cells, as described herein, can be used to manufacture drugs for treating specific patient conditions. PRP cells can be cryopreserved beforehand. In certain embodiments, the PRP cells of this disclosure are derived from iPSCs and can therefore be used to provide “personalized medicine” to patients with eye diseases. In some embodiments, somatic cells obtained from a patient can be genetically engineered to correct disease-causing mutations, differentiated into PRP, and manipulated to form PRP tissue. This PRP tissue can be used to replace the endogenous degenerated PRP of the same patient. Alternatively, iPSCs generated from a healthy donor or an HLA homozygous “superdonor” can be used.

[0199] Various eye conditions can be treated or prevented by the introduction of PRP cells obtained using the methods disclosed herein. These conditions include retinal diseases, or diseases generally associated with retinal dysfunction or decomposition, retinal damage, and / or loss of the retinal pigment epithelium. Conditions that can be treated include, but are not limited to, degenerative diseases of the retina (e.g., Stargardt macular dystrophy, retinitis pigmentosa, macular degeneration (e.g., age-related macular degeneration), glaucoma, and diabetic retinopathy). Further conditions include: Leber congenital amaurosis, hereditary or acquired macular degeneration, Best's disease, retinal detachment, gynostosis, total choroidal atrophy, pattern dystrophy, other dystrophys of photoreceptor cells, and retinal damage resulting from damage caused by any one of the following: light, laser, inflammation, infection, radiation, neovascularization, or trauma. In certain embodiments, a method is provided for treating or preventing a condition characterized by retinal degeneration, comprising administering an effective amount of a composition comprising PRP cells to the subject of interest. This method may include selecting a subject having one or more of these conditions, and administering a therapeutically effective amount of PRP cells sufficient to treat and / or alleviate the symptoms of the condition. PRP cells can be transplanted in various forms. For example, PRP cells may be introduced into the target site in the form of a cell suspension, attached as a monolayer to a matrix, extracellular matrix, or substrate (e.g., a biodegradable polymer), or in combination. Alternatively, PRP cells may be transplanted together with other retinal cells, such as retinal pigment epithelial cells (co-transplantation). In some embodiments, the PRP cells are produced from iPSCs derived from the subject being treated, and are therefore autologous. In other embodiments, the PRP cells are produced from MHC-matched donors.

[0200] Advantageously, the pharmaceutical compositions of this disclosure can be used to compensate for a lack or reduction in photoreceptor cell function. Examples of retinal dysfunctions that can be treated by the retinal cell populations and methods of the present invention include, but are not limited to, photoreceptor degeneration (e.g., those occurring in retinitis pigmentosa, cone dystrophy, cone-rod and / or rod-cone dystrophy, and macular degeneration); retinal detachment and trauma; photodamage caused by lasers or sunlight; macular holes; macular edema; night blindness and color vision deficiency; ischemic retinopathy caused by diabetes or vascular occlusion; retinopathy resulting from premature birth; infectious conditions, e.g., CMV, retinitis, and toxoplasmosis; inflammatory conditions, e.g., uveitis; tumors, e.g., retinoblastoma and intraocular melanoma; and replacement of intraretinal neurons affected by ophthalmic nerve disorders (e.g., glaucoma, traumatic optic neuropathy, and radiation optic neuropathy and retinopathy).

[0201] In one embodiment, these cells can treat or alleviate the symptoms of retinitis pigmentosa in patients requiring treatment. In another embodiment, these cells can treat or alleviate the symptoms of macular degeneration (e.g., age-related macular degeneration (wet or dry), Stargardt disease, myopic macular degeneration, or similar) in patients requiring treatment. For all of these treatments, these cells may be autologous or homogeneous to the patient. In a further embodiment, the cells of this disclosure may be administered in combination with other treatments.

[0202] In some embodiments, PRP cells can be used in autologous PRP grafts for subjects suitable for regenerative medicine. PRP cells can be transplanted in combination with other retinal cells such as photoreceptors. Transplantation of PRP cells produced by the methods disclosed herein can be carried out by various techniques known in the art. According to one embodiment, transplantation is carried out by transsquamative vitrectomy and subsequent delivery of cells through a small retinal opening into the subretinal space, or by direct injection. PRP cells can be introduced to a target site in the form of a cell suspension or cell aggregate, attached to a matrix such as an extracellular matrix, or provided on a substrate such as a biodegradable polymer. Similarly, PRP cells can be transplanted together with other cells (co-transplantation) (e.g., PRP cells and retinal pigment epithelial (PRE) cells). For this reason, compositions containing PRP cells obtained by the methods disclosed herein are provided.

[0203] PRP cells, and optionally photoreceptor cells differentiated from these PRP cells, can be used to generate neurosensory retinal structures. For example, this disclosure aims to generate a multilayer cell structure composed of RPE cells and photoreceptor cells (or PRP cells). This structure can be used for drug screening, as a disease model, or as a pharmaceutical product. In the latter case, the pharmaceutical product may be an RPE-photoreceptor graft, which can be placed on a biocompatible solid support or matrix (preferably a biocompatible matrix or support) that can be implanted like a "patch".

[0204] To further illustrate, the biocompatible support for cells may be a biodegradable synthetic (e.g., polyester) film support for retinal precursor cells. The biodegradable polyester may be any biodegradable polyester suitable for use as a substrate or scaffold to support the proliferation and differentiation of retinal precursor cells. The polyester should be able to form a thin film (preferably a film with a fine pattern on its surface) and should be biodegradable if used for tissue or cell transplantation. Suitable biodegradable polyesters for use in the present invention include: polylactic acid (PLA), polylactides, polyhydroxyalkanoates, both homopolymers and copolymers, e.g., polyhydroxybutyrate (PHB), polyhydroxybutyrate co-hydroxyvalate (PHBV), polyhydroxybutyrate co-hydroxyhexanoate (PHBHx), polyhydroxybutyrate co-hydroxyoctonoate (PHBO), and polyhydroxybutyrate co-hydroxyoctadecanoate (PHBOd), polycaprolactone (PCL), polyesteramide (PEA), aliphatic copolyesters, e.g., polybutylene succinate (PBS) and polybutylene succinate / adipate (PBSA), and aromatic copolyesters. Both high and low molecular weight polyesters, substituted and unsubstituted polyesters, blocks, branched or random, and mixtures and blends of polyesters can be used. Preferably, the biodegradable polyester is polycaprolactone (PCL).

[0205] A pharmaceutical composition of PRP cells prepared by the method disclosed herein. This composition contains at least about 1 × 10⁻⁶ cells. 3 Each PRP cell, approximately 1 x 10⁶ 4 Each PRP cell, approximately 1 x 10⁶ 5 Each PRP cell, approximately 1 x 10⁶ 6 Each PRP cell, approximately 1 x 10⁶ 7 Each PRP cell, approximately 1 x 10⁶ 8 A number of PRP cells, or approximately 1 x 10⁶ 9It may contain 100 PRP cells. In certain embodiments, the composition is a substantially purified preparation (with respect to non-PRP cells) comprising differentiated PRP cells produced by the method disclosed herein. Compositions are also provided comprising a scaffold (e.g., a polymer carrier and / or extracellular matrix) and an effective amount of PRP cells produced by the method disclosed herein. For example, the cells are provided as a monolayer of cells. The matrix material is generally physiologically acceptable and suitable for use in in vivo applications. Examples of physiologically acceptable materials include, but are not limited to, the following: absorbable and / or non-absorbable solid matrix materials, e.g., intestinal submucosa (SIS), cross-linked or uncross-linked alginates, hydrophilic colloids, foams, collagen gels, collagen sponges, polyglycolic acid (PGA) meshes, fleece, and bioadhesives.

[0206] Suitable polymer carriers include synthetic or natural polymers, and porous meshes or sponges formed from polymer solutions. For example, the matrix may be a polymer mesh or sponge, or a polymeric hydrogel. Natural polymers that can be used include proteins, such as collagen, albumin, and fibrin; and polysaccharides, such as alginates and hyaluronic acid polymers. Synthetic polymers include both biodegradable and non-biodegradable polymers. For example, biodegradable polymers include hydroxy acid polymers, such as polylactic acid (PLA), polyglycolic acid (PGA), and polylactic acid-glycolic acid (PGLA), polyorthoesters, polyanhydrides, polyphosphazenes, and combinations thereof. Non-biodegradable polymers include polyacrylates, polymethacrylates, ethylene vinyl acetates, and polyvinyl alcohols.

[0207] Polymers capable of forming malleable, ionically or covalently crosslinked hydrogels can be used. Hydrogels are substances formed when organic polymers (natural or synthetic) are crosslinked via covalent, ionic, or hydrogen bonds to create a three-dimensional open lattice structure that traps water molecules and forms a gel. Examples of materials that can be used to form hydrogels include ionically crosslinked polysaccharides (e.g., alginates, polyphosphazenes, and polyacrylates) or block copolymers (e.g., PLURON1CS® or TETRON1CS®, polyethylene oxide-polypropylene glycol block copolymers crosslinked by temperature or H, respectively). Other materials include proteins such as fibrin, polymers such as polyvinylpyrrolidone, hyaluronic acid, and collagen.

[0208] The pharmaceutical composition may be optionally packaged in a suitable container with written instructions for a desired purpose (e.g., reconstitution of PRP cell function to improve diseases or abnormalities of retinal tissue). In some embodiments, PRP cells produced by the method of this disclosure can be used to replace the degenerated photoreceptor cells of the target required.

[0209] C. Distribution for commercial, therapeutic, and research purposes. In some embodiments, a reagent system is provided that includes a set or combination of cells containing PRP or PRP-enriched cell populations, which are present in the manufacture, distribution, or use. This cell set includes any combination of the cell populations described herein and undifferentiated pluripotent stem cells or other differentiated cell types, often sharing the same genome. Each cell type can be packaged together or in separate containers, simultaneously or at different times, in the same facility or at different locations, under the control of the same entity or under the control of different entities sharing business relationships.

[0210] The pharmaceutical composition may be optionally packaged in a suitable container, along with written instructions for the desired purpose (e.g., reconstitution of PRP cell function to improve disease or damage of eye tissue).

[0211] V. Kit In some embodiments, a kit is provided that may contain, for example, one or more culture media and components for producing PRP cells. Such a formulation may contain a cocktail of retinal differentiation factors and / or trophic factors in a form suitable for combination with photoreceptor precursors or photoreceptor cells. This reagent system may, if necessary, be packaged in a lyophilized form in an aqueous medium. The container means of the kit generally includes at least one vial, test tube, flask, bottle, syringe, or other container means in which the components can be placed and preferably appropriately dispensed. If the kit contains multiple components, the kit also generally includes a second, third, or other additional container in which the additional components can be placed separately. However, a single vial may contain various combinations of components. The components of the kit may be provided as a dry powder. If the reagents and / or components are provided as a dry powder, this powder can be reconstituted by the addition of a suitable solvent. It is also conceivable that this solvent may be provided in a separate container means. The kit also typically includes means for containing the kit components in tight containment for commercial sale. Examples of such containers include injection-molded plastic containers or blow-molded plastic containers that hold the desired vials. The kit may also include instructions for use in electronic format, such as printed or digital. [Examples]

[0212] VI. Examples The following embodiments are included to demonstrate preferred embodiments of the present invention. Those skilled in the art should recognize that the techniques disclosed in the following embodiments represent techniques discovered by the inventors to function well in carrying out the present invention, and therefore may be considered to constitute a preferred model for carrying out the present invention. However, those skilled in the art should recognize that, in light of this disclosure, many modifications can still be made in the specific embodiments disclosed without departing from the spirit and scope of the present invention to obtain similar or equivalent results.

[0213] Example 1 - Preparation of a starting pluripotent stem cell population We developed a method for differentiating iSPCs into various stages of photoreceptor precursor (PRP) cells (Figure 1). Briefly, the population of retinal precursor cells (RPCs) originates from iPSCs, which are then further differentiated into neuroretinal progenitor cells (NRPs), and then into PRP cells.

[0214] First, iPSCs were grown in a fully defined culture medium (e.g., ESSENTIAL 8® (E8®) medium) on plates coated with vitronectin, without the use of a mouse or human feeder layer. The vitronectin stock was diluted 1:200 with calcium or magnesium-free DPBS, and the culture plates were coated with this diluted vitronectin solution and incubated at room temperature for approximately 1 hour. The iPSCs were divided when they were preconfluent to prevent overgrowth and the development of unhealthy and / or differentiated cells.

[0215] To obtain RPCs, iPSCs were first dissociated into single-cell suspensions. To obtain single-cell suspensions, these cells were washed with DPBS (calcium and magnesium-free) and incubated in cell dissociation enzymes (e.g., TRYPLE®) at 37°C for approximately 10 minutes. The cells were then detached by pipetting with a serological pipette, and the cell suspension was collected in a conical tube. If the cells did not detach by gentle pipetting, the culture was incubated for a longer period (e.g., an additional 2-3 minutes). To recover all cells, the culture vessel was washed with E8® medium at room temperature, and this medium was then added to the tube containing the cell suspension. In addition, brevistatin (e.g., 2.5 μM) was added to the E8® medium to increase the viability of PSCs after dissociation into single cells without the cells adhering to the culture vessel. To recover the cells, the vessel was centrifuged at 400 × g for approximately 5 minutes, the supernatant was aspirated, and the cells were resuspended in an appropriate volume of E8® medium.

[0216] To efficiently differentiate single-cell iPSCs into PRP cells, the input density of single-cell iPSCs is accurately counted using an automated cell counter such as VICELL®, and approximately 1 × 10⁶ cells are cultured in E8® medium at room temperature. 5 The cells were diluted to a cell suspension of 100 cells / mL. Once a single-cell suspension of iPSCs was obtained at a known cell density (e.g., by a hemocytometer), these cells were seeded into a suitable culture vessel, such as a 6-well plate coated with vitronectin. These cells were seeded at a cell density of approximately 200,000 cells per well and placed in a humidified incubator at 5% CO2 and 37°C. After approximately 18–24 hours, the medium was aspirated and fresh E8® medium was added to the culture. The cells were cultured in E8® medium for approximately 2 days after seeding for proper adhesion and iPSC expansion.

[0217] Example 2 - Differentiation of iSPC into RPC Single-cell iPSCs seeded at an appropriate cell density were cultured for approximately 2 days as in Example 1, and then cultured in various differentiation media to obtain RPCs. E8® medium was aspirated and retinal induction medium (RIM) at room temperature (e.g., Table 1) was added. Briefly, RIM contained DMEM and F12, knockout serum substitute, MEM non-essential amino acids (NEAAs), sodium pyruvate, N-2 supplement, B-27 supplement, and ascorbic acid in a ratio of approximately 1:1. In addition, RIM contained WNT pathway inhibitors, BMP pathway inhibitors, TGFβ pathway inhibitors, and insulin growth factor 1 (IGF1). The medium was aspirated daily and fresh RIM was added to the cells. These cells were cultured in RIM for approximately 2 days to generate anterior neuroectoderm cells.

[0218] These cells were then cultured in Retinal Differentiation Medium 1 (RD1) for approximately 1–4 days (particularly about 2 days). Briefly, RD1 (Table 1) contained DMEM and F12, knockout serum substitute, MEM NEAA, sodium pyruvate, N-2 supplement, B-27 supplement, and ascorbic acid in a ratio of approximately 1:1. In addition, RD1 contained a WNT pathway inhibitor (e.g., CKI-7), a BMP pathway inhibitor (e.g., LDN193189), a TGFβ pathway inhibitor (e.g., SB431542), a MEK inhibitor (e.g., PD325901), and IGF-1. The concentrations of the WNT pathway inhibitor, BMP pathway inhibitor, and TGFβ pathway inhibitor were 10 times higher in RDM compared to RIM. Differentiated retinal cells were produced daily by aspirating the medium and adding RD1 at room temperature to the cells. In particular, the BMP inhibitor may be removed after the first few days (e.g., after 2 days) to enhance VSX2 expression in the PRP cells. The RDM1 medium may be replaced with RD2 (Table 1) which does not contain a BMP inhibitor (e.g., LDN193189). The cells may be cultured in RD2 for about 5 to 10 days (e.g., about 7 days).

[0219] To obtain NRP cells, RPC cells were then cultured in retinal maturation medium (RM1 or RM2) (Table 1) for approximately 5 days to differentiate them into NRP cells. RM1 contained DMEM and F12 in a ratio of approximately 1:1, a knockout serum substitute, MEM NEAA, sodium pyruvate, N-2 supplement, B-27 supplement, and ascorbic acid. In addition, RM1 contained nicotinamide but did not contain activin A to prevent differentiation into RPE cells. RM1 medium may further contain a γ-secretase inhibitor (e.g., DAPT). RM2 may further contain a TGFβ inhibitor (e.g., SB431542) and / or bFGF. This medium was replaced daily with RM1 at room temperature. RPC cells were then cultured in photoreceptor precursor induction medium (FDSC) on days 15–18 to produce NRP cells. To analyze NRP cells, they were thawed in culture medium and assayed for PAX6 and VSX2 expression (Figure 14). NRP cells were found to be nearly 100% positive for PAX6, approximately 90% positive for PMEL17, and approximately 80% positive for VSX2 (Figure 14).

[0220] Example 3 - Differentiation of RPC into PRP cells To complete the differentiation process into PRP cells, the RPC cells from Example 2 were cultured in FDSC medium (Table 1). Briefly, FDSC medium contains DMEM and F12 in a ratio of approximately 1:1, a knockout serum substitute, MEM NEAA, sodium pyruvate, N-2 supplement, B-27 supplement, and ascorbic acid. In addition, FDSC medium may contain basic FGF, DAPT, SB431542, and CKI-7 to differentiate NRP into PRP cells. These cells were cultured in FDSC medium for approximately 10–20 days (particularly 15 days). PRP cells were analyzed for the expression of the markers Tuj1 / nestin and RCVRN (Figures 4A–4C).

[0221] At this stage, the obtained PRP cells can be dissociated using TRYPLE® and cryopreserved in heterogeneous component-free CS10 medium. Alternatively, the obtained PRP cells can be cultured for 1 to 5 days (e.g., 3 days) in a medium containing a ROCK inhibitor or brevistatin to promote aggregate formation, thereby improving cell viability and transplantation. Thus, the method disclosed herein provides pluripotent cell-derived PRP cells that can be reproduced on a large scale and consistently for clinical application.

[0222] Example 4 - Production of the ophthalmic vesicle RPC cells from Example 2 were cultured as aggregates in RM1 or RM2 medium for an extended period to produce optic vesicles. Brevistatin was used to promote aggregate formation. RM1 medium contained DMEM / F12, knockout serum substitute, sodium pyruvate, ascorbic acid, and nicotinamide. These optic vesicles were evaluated for marker expression at days 30, 40, 50, and 60. RCVRN expression increased from 3.3% at day 40 to over 30% at day 78 (Figure 12D).

[0223] Example 5 - Alternative method for producing PRP cells RPC cells from Example 2 were cultured as aggregates in RM1 medium or RM2 medium (Table 1) for approximately 40-70 days. Brevistatin was used during aggregate formation. RM2 medium contained DAPT in addition to the components of RM1 medium. The cells remained aggregated until approximately day 68, at which point the cells were dissociated, treated with BENZONASE®, and seeded in RM2 medium for 7 days to generate hybrid PRP cells. These hybrid PRP cells showed a phenotype similar to that of the PRP cells in Example 3. After magnetic activation cell sorting (MACS) purification, the hybrid PRP cell population was over 97% positive for TUBB3, negative for nestin (<3%), and nearly 80% positive for RCVRN (Figure 10).

[0224] Example 6 - Cryopreservation of NRP or PRP For cryopreservation of differentiated NRP or PRP, the culture medium was aspirated and the cells were washed with Dulbecco's phosphate-buffered saline (DPBS). These cells were then incubated with cell dissociation enzymes, and the cell suspension was pipetted into a conical tube. The cells were centrifuged, the supernatant was aspirated, and the cells were resuspended in room temperature culture medium. The cell suspension was then filtered through a STERIFLIP® cell strainer (20 μm), and the cells were counted. These cells were then centrifuged and stored in a cold CryoStor® CS10 at an appropriate density (e.g., 1 × 10⁶). 7 The cells were resuspended in (100 cells / mL). The cell suspension was divided into pre-labeled cryovials, placed in a freezer container, and stored in a -80°C freezer for 12–24 hours. The vials were then transferred to liquid nitrogen for storage.

[0225] Alternatively, these cells may be cryopreserved as aggregates instead of dissociating into a single-cell suspension. For example, after CliniMACS enrichment of D75 PRP, single cells were re-aggregated in minimal medium (RMN) in a tissue culture flask for 2 days. The aggregates were pooled in D77, and sample aliquots were obtained for cell counting. After a series of washes, the aggregates were resuspended in CryoSTOR CS10 cryopreservation medium, and 1 ml of the aggregate suspension was transferred to a liquid nitrogen storage vial with 25 × 10^6 aggregated cell products / vial.

[0226] Morphologically, the thawed aggregates remaining in the culture were indistinguishable from aggregates formed from single cells two days after thawing. The cell viability of the thawed aggregates was 67%, compared to 37% for thawed single cells. The following four conditions were used to test the expression of RCVRN (on-target PRP marker), CHX10 (early eye field cell), RCVRN+, CHX10 (off-target bipolar cell), Ki67 (off-target proliferating cell), and Pax6 (off-target neuroectoderm / early eye field): single cells thawed from cryopreserved cells and re-aggregated in RMN medium for two days (SC D77 2d post-thawed aggregates); single cells enriched with nutrients were aggregated in RMN for two days without cryopreservation (D77 pre-cryopreserved cultured aggregates); cryopreserved aggregates immediately after thawing (D77 aggregates at thawing); and cryopreserved aggregates thawed and cultured in RMN medium for two days (D79 thawed aggregate culture 2d). There were no differences in RCVRN expression among these four conditions. Off-target markers related to the early eye field (CHX10+ and Pax6+) remained low under all conditions, and proliferating cells (Ki67+) were negligible.

[0227] [Table 2]

[0228] Therefore, it was demonstrated that not only could aggregates be successfully cryopreserved, but the enriched PRP was not compromised during the cryopreservation or thawing stages. This was evident from the similar high expression of recavitin, an on-target PRP marker. Similarly, off-target markers and proliferating cell markers remained similarly low under all conditions. Cryopreserving aggregates allows the product to be delivered from the laboratory to the patient with minimal handover time, because the product being transplanted is in the form of aggregates. By eliminating the two-day "interval" in the culture step, concerns about contamination and cell loss are minimized.

[0229] Example 7 - MACS purification of PRP cells A population of PRP cells may contain residual contaminating non-PRP cells (collectively referred to as "contaminating cells"), such as PRE cells or other non-neuronal types. All of these contaminating cells can be isolated and removed to obtain a PRP-enriched cell population. These contaminating cells can be removed from the culture by various methodologies (e.g., magnetically activated cell sorting (MACS®), fluorescence-activated cell sorting (FACS), or single-cell sorting by positive and / or negative selection). Using the MACS® methodology, which is known in the art to isolate various cell populations depending on surface antigens, contaminating cells were isolated from the desired PRP cells.

[0230] Positive MACS selection for PRP cells may involve isolating cells expressing the neuronal marker CD171. To perform positive MACS selection for PRP cells, the entire population of PRP cells from Example 3 or 6 was dissociated into single-cell suspensions. The medium was aspirated, the cells were washed with DPBS, and TRYPLE® was added to dissociate the cells. The cells were then collected and centrifuged. The cells were washed with PRP medium and filtered using a 20 μm steriflip cell strainer. The cell suspension was counted using ViCell, and MACS buffer was added to this cell suspension in a 1 × 10⁶ solution. 7 Cells were added at a concentration of 100 cells / mL. Next, this cell suspension was stained with the primary antibody CD171-biotin (Miltenyi) at a 1:25 dilution. After incubation, 20 mL of MACS buffer was added, and the cells were centrifuged at 400 × g for 5 minutes. The cell pellet was resuspended in 20 mL of MACS buffer, mixed vigorously, and centrifuged at 400 × g for 5 minutes to remove any unbound antibodies. The cell pellet was then added to MACS buffer (e.g., 1.11 × 10⁶). 8 The cells were resuspended (at 10⁴ cells / mL), and microbeads coated with diluted (1:10) secondary antibody (e.g., anti-biotin) were added. The cells were incubated at 4°C for 20 minutes. After incubation, the cells were washed with MACS buffer to remove unbound microbeads, up to a maximum of 1.25 × 10⁶ 8The cells were resuspended in 500 μL of MACS buffer. The cell suspension was transferred to an LS column placed in a strong field, and the cells expressing antigen CD171, which were attached to the microbeads, remained in the column. This LS column was washed twice with MACS buffer. The enriched cell population was then washed out of the LS column and evaluated for sorted purity analysis. Images of cell staining for RCVRN are shown in Figures 3 and 9, demonstrating that MACS enrichment results in significant enrichment of PRP cells.

[0231] Further research identified additional surface proteins for enriching PRP cells. Surface protein evaluation using Miltenyi Marker Screen plates identified SUSD2 as a surface protein target for enriching photoreceptor precursor (PRP) products. Sushi domain-containing protein-2 (SUSD2) is a type I transmembrane protein that promotes cell-cell adhesion and cell-matrix adhesion. Overexpression of SUSD2 has been observed in cancer cells, and SUSD2 is an established enrichment marker for various mesenchymal stem cells. Assaying D75 PRP 2.3 CBP for surface proteins revealed elevated SUSD2 expression on cells positively labeled with recavitin, a neuron-specific calcium-binding protein primarily expressed at photoreceptors. This finding led to testing SUSD2 for use as an enrichment marker.

[0232] SUSD2 showed high co-expression with recabinin, a marker expressed by cells destined to become photoreceptors. In addition, SUSD2 was enriched in approximately 95% of recabinin-positive cells compared to approximately 83% of recabinin-positive cells after CD171 enrichment (Figure 31). PD0332991The study was conducted on cells seeded in LN521 containing [the specified compound]. Recabilin bound to CHX10, a transcription factor highly selective for early neuroretinal progenitor cells, can be considered a prominent characterizing marker for (cone) bipolar cells (Figure 34). Bipolar cells are late-developing retinal neurons that interact with photoreceptors and ganglion cells to facilitate signal transduction. CHX10-positive (recabilin-negative) cells may also express this compound in a subset of Müller glial cells.

[0233] SUSD2 expression was examined concurrently with RCVRN expression for differentiation endpoints D55–D105 before MACS enrichment (Figure 32). The precision with which SUSD2 targeted only PRP and not other retinal neurons was important for the purity of the final product. Despite the low cell yield after SUSD2 enrichment, the SUSD2 marker enriched recabinet-expressing cells at each time point examined (Table 3), suggesting that SUSD2 is a highly specific marker for PRP that may be recabinet-positive. Figure 33 further illustrates the target specificity of SUSD2.

[0234] As previously discussed, CHX10 and recabilin are prominent characterizing markers for (cone) bipolar cells. CHX10 is initially expressed in retinal progenitor cells and its expression is downregulated in postmittal PRP, but remains highly expressed in postmittal bipolar cells and some Müller glial cells. This study revealed that CHX10 levels were low at the earlier (D65) time point, both before and after SUSD2 MACS enrichment. In addition, recabilin-CHX10 expression was negligible at this point, suggesting that bipolar cells were not present in the product after SUSD2 enrichment at this stage.

[0235] Conversely, co-labeling with Recoverin and Neuronal Differentiation Factor 1 (NeuroD1, ND1), a transcription factor that is transiently expressed and plays a role in the final differentiation of photoreceptors, showed high expression levels before and after SUSD2 MACS enrichment in D65. Beyond D65, bipolar cells expressing RCVRN-CHX10 (and / or some Müller glial cells that may express CHX10 in some cases) were observed. RCVRN-ND1 expression was gradually downregulated, and this combination was expressed in less than half of the enriched D85 cells compared to D65 and D75, which may be consistent with the transient expression of ND1.

[0236]

Table 3

[0237] These studies, combined with the peak SUSD2 expression time, suggested that SUSD2 is a good enrichment marker at early time points such as D65. Furthermore, in D65, SUSD2 showed a high degree of selectivity for RCVRN-expressing cells and ND1-expressing cells, but not for CHX10-expressing (off-target or progenitor) cells. The selective nature of SUSD2 was emphasized by the enrichment of a highly Recoverin-positive pure population, even when the cell yield after enrichment was small. Overall, SUSD2 is a useful target marker for PRP enrichment on the condition that the final product is manufactured at an early time point with high SUSD2 expression (e.g., D55 and D65).

[0238] In addition to SUSD2 and CD171, alternative potential PRP enrichment markers were identified. This surface marker also co-localized, to a lesser extent, with Recoverin, a neuron-specific calcium-binding protein mainly expressed in photoreceptors and cone bipolar cells (Gunhan et al., 2003; Haverkamp et al., 2003). Table 4 lists 15 additional potential PRP enrichment markers excluding CD171 and SUSD2.

[0239]

Table 4

[0240] Research on these surface molecules has mainly focused on the expression and / or function of cells in the developing or adult retina. Table 4 lists the percentages of cell populations co-expressing surface antigens and recoverin, or expressing surface antigens only on off-target differentiated cells, respectively. The percent population expressing either of the two markers was determined by flow cytometry analysis of the double-stained D75 PRP product (Figure 15). In addition, the percent population of cells expressing only recoverin (in FITC, x-axis) or only surface antigen (in APC, y-axis) was evaluated. All plots were gated against unstained (empty) cells and corresponding isotype controls (REA IgG1, MsIgG1, Ms IgG2a, MsIgG2b, MsIgM). Surface molecules with high recoverin co-expression and low off-target expression (<20%) include CD11, CD133, CD230, and CD344.

[0241] This type of flow cytometry profile increases the likelihood that the antigen is expressed on more PRP cells and less on off-target cells. To determine if this is really the case, antibodies against the CD111 surface antigen, CD230 surface antigen, CD344 surface antigen, and CD133 surface antigen were used to enrich differentiated cells from multiple experiments for PRP. Figure 16 shows recoverin expression at D55, D65, and D75. Recoverin expression peaked at D65 under all conditions such as before MACS (single time point, diamond, CD133 enrichment was only performed for D75 PRP), and then decreased by D75. This data suggests that D65 may be the optimal time point for PRP enrichment by these specific surface antigens (except CD133).

[0242] Figures 17–22 show tables and graphs of the percentage expression of recabin (on-target PRP marker) and off-target cell markers Pax6 (expressed by retinal progenitor cells - RPC, amacrine cells - AC, and retinal ganglion cells - RGC), Onecut1 (expressed by RPC and horizontal cells), Ki67 (expressed by proliferating cells), and CHX10 (expressed by RPC and co-expressed with recabin on bipolar cells). The highest recabin expression occurred with CD344 enrichment. The eluted fractions of each enrichment showed some recabin-positive cells, but overall showed lower fractions compared to the enriched portion, while showing a higher proportion of the off-target marker Onecut1. Proliferating cells or CHX10+RPC / bipolar off-target cells were essentially absent in the evaluated cell population. Again, CD344-enriched cells showed the highest proportion of recabin-positive expression, as well as low levels of Pax6 and Onecut1. Proliferating cells were absent in this cell population.

[0243] An overall decrease in both on-target and off-target cells was observed by D75, suggesting that the expression of target markers may have significantly decreased by this point. Interestingly, CHX10 expression remained high, suggesting a second wave of potentially bipolar off-target and / or progenitor cells.

[0244] In all studies, recoverin expression was highest after CD344 enrichment, followed by CD230 enrichment. CD344 enrichment also reduced Pax6-positive and CHX10-positive off-target cell markers compared to MACS. Elutions or flow-throughs from all three enrichments were Pax6-positive. + Cells and CHX10 + It appears to contain the majority of the cells, and this suggests that enrichment is recovering +This suggests that the drug targets PRP and does not target off-target cells such as retinal ganglion cells (RGCs) and amacrine cells (ACs) (both of which express Pax6). The enriched region expressed moderate levels of CHX10 on day 75, indicating that some of the recabinet-labeled cells may also co-localize on CHX10-labeled cells, suggesting the presence of a population of bipolar cells (BPs) in the enriched fraction.

[0245] Enrichment with CD133 was performed on another cell line (31538.102) to test whether this line differentiated into PRP. CD133 is used in recovering + Cell enrichment resulted in an overall decrease in off-target cell marker expression after enrichment compared to before MACS. Ki67 also decreased after enrichment, but treatment with PD0332991 removed any residual proliferating cells present in the enriched fraction.

[0246] Therefore, the markers CD71, SUSD2, CD111, CD133, CD230, and CD344 could be used to enrich the PRP cell population and remove off-target cells.

[0247] Example 8 - PRP enrichment using CliniMACS® Further studies were conducted, for example, using the CliniMACS® instrument to evaluate the feasibility of PRP enrichment at a higher throughput scale. PRP enrichment using the CDC171 marker was tested using the CliniMACS® instrument, inputting C-1 or C-2 cells with LS tubes using the CD34.2 or Enrichment 1.1 program. PRP-enriched cells were then evaluated for TUBB3 / nestin and RCVRN. The CliniMACS high-throughput method exhibited similar output purity to the LS column method, where over 90% of the output cells were neurons, and over 50% of these cells were positive for RCVRN (Figures 3 and 9).

[0248] Example 9 - Materials and Methods A flow cytometry wash buffer was prepared by adding 20 mL of FBS or human serum albumin to 1000 mL of DPBS (i.e., calcium and magnesium-free). This buffer can be filtered and stored at 4°C for up to 4 weeks.

[0249] A flow cytometry permeabilization buffer was prepared by adding 20 mL of FBS to 1000 mL of DPBS (i.e., calcium and magnesium-free). 1 gram of saponin was added and thoroughly mixed. This buffer can be filtered and stored at 4°C for up to 4 weeks.

[0250] Flow cytometry Live-Dead Red stain was prepared by diluting Live-Dead Stain with DPBS (i.e., calcium and magnesium-free) at a ratio of 1:1000. 2 × 10⁶ samples were used for the assay. 6 One mL of this staining agent was prepared for each individual cell. This staining agent was freshly prepared before use.

[0251] Flow cytometry fixation buffer was prepared by adding 110 μL of 36.5% formaldehyde to 880 μL of DPBS (i.e., calcium and magnesium-free). 2 × 10⁶ samples were used for the assay. 6 One mL of staining agent was prepared for each individual cell. This buffer was freshly prepared before use.

[0252] Example 10 - PRP enrichment depletion marker In addition, we identified a surface molecule that could be used as a depletion marker to enrich the PRP cell population. This molecule did not show co-expression with recabinet, but it labeled off-target cells, making it a good depletion marker for unwanted cell types. Table 5 lists four possible depletion candidates that show very little co-expression with recabinet (<15%) but label other cell types (>30%). The expression profile of recabinet against depletion antibodies was evaluated by flow analysis. Figure 21 shows the low or near-absence of co-expression of depletion candidate surface antigens with recabinet.

[0253] [Table 5]

[0254] Example 11 - Optimization of PRP differentiation The PRP differentiation method was further evaluated with regard to the use of Wnt activators such as CKI-7 on days 0 and 1. Cells were differentiated in the absence of CKI-7, and the expression of PAX6, CHX10, Ki67, and PMEL was characterized on days 15 and 25 of the differentiation process. It was found that the cell population had similar expression of PAX6, CHX10, Ki67, and PMEL on days 15 and 25 without the use of CKI-7 on days 0 and 1 (Figures 22-25). Therefore, PRP cells can be differentiated in the absence of CKI-7 on days 0 and 1.

[0255] Alternatively, this differentiation method was performed without using RIM. Instead, cells were cultured in RD1 medium supplemented with LDN193189 for days 0-1, and then cultured in RD2 medium without LDN193189 (Figures 26-30).

[0256] The methods of Examples 1 to 3 were modified to remove the 2-day culture in RIM medium, and the initial neural retinal differentiation was evaluated. The cells were directly cultured in RD1 medium with high concentrations of LDN193189, SB431542, and CKI-7 from day 0 to 1, and then cultured in RD2 medium without LDN193189 from day 2 to 10. The cells were aggregated on day 15, and samples were processed for flow analysis at the time of aggregation, at the time of the intermediate process progression on day 30, and at the time of the final process progression on day 75.

[0257] From the D15 in-process flow analysis, it was found that the expression of PAX6, an initial human neuroectodermal fate determinant, was similar between the CBP 2.3 (by RIM) condition and the CBP 2.5 (without RIM) condition, but the expression of the initial eye field marker CHX10 was lower under the RIM condition (Figure 26). Removal of the RIM step by RD1 containing LDN was observed to increase CHX10 expression. Similarly, removal of RIM reduced the expression of the RPE marker Tyrp1 by nearly 50% compared to the condition containing RIM (Figures 27 and 28).

[0258] Multiple cell lines were tested to determine whether removal of RIM promotes PRP differentiation across various lines. Lines 31536.102, 31538.101, and 31538.102 were lines that consistently expressed low levels of CHX10 in the in-process analysis at D15, but CHX10 expression in both lines increased significantly when the RIM step was removed (Figure 27). Figure 27 compares the expression of the neuroectodermal marker Pax6, the eye field marker CHX10, and the RPE markers Tyrp1 and PMEL across four lines in the presence (CBP) or absence (-RIM) of RIM. Across the lines, Pax expression was similar in the presence or absence of RIM. CHX10 expression increased across the lines when RIM was removed, but in lines 8.101 and 8.102, it was dramatically improved by nearly 80% in both lines. At the same time, Tyrp1 expression decreased after removal of RIM.

[0259] Pigment cell-specific protein (PMEL) is generally associated with RPE pigmentation. In this differentiation line, PMEL expression remained high at D15, and low PMEL expression at D15 was found to typically result in poor PRP differentiation. Removal of RIM did not disrupt PMEL expression in either line 6.102 or line A, but significantly increased PMEL expression in lines 8.101 and 8.102. This demonstrates that RIM removal does not hinder the PRP generation process, but is beneficial for lines that require an extra boost toward PRP generation. In summary, the data suggest that removal of RIM may reduce the likelihood of cells differentiating into RPE at D15, but increase the likelihood of differentiation into PRP.

[0260] Based on past data, during the intermediate (in-process) PRP development (around D30), PAX6 and CHX10 expression peaks, while TYRP1 and PMEL expression further decreases. While the developmental process naturally drives these changes in protein expression, early removal of RIM provides additional support in lines previously considered unsuitable for PRP differentiation. Early removal of RIM affects D30 mid-stage PRP development in lines 8.101 and 8.102 by increasing PAX6 and CHX10 expression levels and decreasing TRYP1 and PMEL expression levels.

[0261] As the retina develops, heterogeneous populations of retinal cells emerge chronologically, and the characterization of these cell populations is based on a specific panel of antibodies targeting antigens expressed by off-target cell types. Current panels for identifying on-target PRP cells include recabilin (RCVRN) conjugated to either NeuroD1 (neuron differentiation factor 1, ND1; Figure 29A) or CHX10 (Figure 29B). Recabilin is a neuron-specific calcium-binding protein primarily expressed at photoreceptors, while NeuroD1 is a transiently expressed transcription factor that plays a role in the terminal differentiation of photoreceptors. CHX10 conjugated to RCVRN is a prominent characterization marker for (cone) bipolar cells. CHX10 is initially expressed in retinal progenitor cells and its expression is downregulated in postmittal PRP, but remains highly expressed in postmittal bipolar cells and some Müller glial cells. Bipolar cells express both CHX10 and RCVRN, while PRP expresses only RCVRN+ (positive) or only CHX10- (negative). Therefore, we used dual labeling with RCVRN and CHX10 to verify the absence or low presence of bipolar cells in the cell population and demonstrated that RCVRN+ cells primarily differentiate into PRP.

[0262] The effect of RIM removal on D75 PRP was most pronounced in strain A, and RCVRN and ND1 expression increased with RIM removal. This data indicates that RIM removal affects PRP development. Furthermore, CHX10 expression decreased or remained below 10% across strains with RIM removal, and RCVRN+ / CHX10+ double-positive cells also decreased.

[0263] Off-target cell markers include Pax6-Isl1 double positivity for retinal ganglion cells (RGCs), NHF6 for horizontal cells, and Ki67, a pan-proliferative cell marker. Pax6 is also expressed on other mature retinal cells, as described below (Figure 30). While Pax6 is a reliable marker for neural induction, in the retina, several post-mitotic off-target cells express Pax6 (e.g., RGCs, Müller glia, and amacrine cell subsets). Similarly, Isl1 is also expressed by several mature off-target retinal cells (e.g., ON bipolar cells, RGCs, and amacrine cell subsets). At this developmental stage, the RIM-free condition had little effect, except for a slight decrease in off-target marker expression. The double-positive Isl1 / Pax6 population represented a small percentage (≤10%) of the remaining RGC or amacrine cell subset, regardless of culture medium conditions. In addition, HNF6 was present across strains and conditions. + Horizontal cells were negligible, and proliferating cells were virtually nonexistent.

[0264] Therefore, this study revealed that removing RIM from the culture medium sequence used for differentiation significantly affects PRP differentiation and development, particularly in the early stages of development. The presence of RIM resulted in weakened CHX10 expression at D15 in some lines at D30 and across all lines. Removing RIM appeared to "rescue" the reduced CHX10 expression, and significant improvements were observed in lines 8.101 and 8.102 across lines at D15 and D30. Most importantly, while removing RIM increased RCVRN and ND1 expression levels and decreased CHX10 levels in some cases, no adverse effects were observed at any of the time points.

[0265] All methods disclosed and claimed herein can be assembled and performed without excessive experimentation in consideration of this disclosure. While the compositions and methods of the present invention are described in relation to preferred embodiments, it will be apparent to those skilled in the art that modifications can be made to the methods described herein, and to the steps or order of steps of these methods, without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that the agents described herein can be replaced with certain agents that are both chemical and physiological in nature, insofar as identical or similar results are achieved. All such similar substitutions and modifications that are apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims. References The following references are incorporated herein by reference insofar as they provide exemplary procedures or other details that supplement those described herein. Amit et al.,Dev.Bio.,227:271-278,2000. Buchholz et al.,Stem Cells,27:2427-2434,2009. Byrne et al.,Nature,450(7169):497-502,2007. Comyn et al.,Curr.Opin.Neurol.1,4-9,2010. Gunhan et al.,J Neurosci.23(4)1383-1389,2003. Haverkamp et al., J Comp Neurol. 455(4):463-476, 2003. Hirami et al., Neurosci. Lett., 48:126-131, 2009. Kanemura et al.,PLoS One,9,2014. Ludwig et al., Nat. Biotechnol., 24:185-187, 2006b. Ludwig et al., Nat. Methods, 3:637-646, 2006a. Meyer et al.,PNAS,106(39):16698-16703,2009. PCT International Publication No. 2007 / 069666A1 パンフレット PCT International Publication No. 2014 / 121077 パンフレット Pearson et al.,Nature 485,99-103,2012. Smith,In:Origins and Properties of Mouse Embryonic Stem Cells,2000. Strauss et al.,Physiological Reviews,85:845-881,2005. Takahashi et al.,Cell,126,663-676,2006. Takahashi et al.,Cell,131,861-872,2007. Thomson and Marshall, Curr.Top.Dev.Biol.,38:133-165,1998. Thomson and Odorico, Trends Biotechnol., 18(2):53-57, 2000. Thomson et al. Proc. Natl. Acad. Sci. USA, 92:7844-7848, 1995. U.S. Patent Application Publication Specification No. 2002 / 0076747 U.S. Patent Application Publication Specification No. 2009 / 0246875 U.S. Patent Application Specification No. 2010 / 0210014 U.S. Patent Application Publication Specification No. 2012 / 0196360 U.S. Patent Application Publication Details No. 2012 / 0276636 Specifications of U.S. Patent No. 5,843,780 Specifications of U.S. Patent No. 6,103,470 Specifications of U.S. Patent No. 6,200,806 Specifications of U.S. Patent No. 6,416,998 U.S. Patent No. 6,833,269 U.S. Patent No. 7,029,913 U.S. Patent No. 7,442,548 U.S. Patent No. 7,598,364 U.S. Patent No. 7,682,828 U.S. Patent No. 7,989,425 U.S. Patent No. 8,058,065 U.S. Patent No. 8,071,369 U.S. Patent No. 8,129,187 U.S. Patent No. 8,268,620 U.S. Patent No. 8,278,620 U.S. Patent No. 8,546,140 U.S. Patent No. 8,546,140 U.S. Patent No. 8,741,648 U.S. Patent Publication No. 2003 / 0211603 U.S. Patent Publication No. 2010 / 0003757 Xu et al., Nat. Biotechnol., 19:971-974, 2001. Ying et al., Cell, 115:281-292, 2003. Yu et al.,Science,318:1917-1920,2007. Zhong et.,Nature Communications,5:4047,2014.

Claims

1. A method for producing a population of photoreceptor precursor cells (PRPs), (a) Obtain a starting population of human induced pluripotent stem cells (iPSCs), (b) The iPSCs are cultured in a first retinal differentiation medium (RD1) containing a BMP inhibitor, a TGFβ inhibitor, a Wnt inhibitor, and a MEK inhibitor to further differentiate the iPSCs into anterior neuroectoderm cells. (c) Inducing retinal differentiation of the anterior neuroectoderm cells by culturing them in a second retinal differentiation medium (RD2) that is essentially free of BMP inhibitors but contains a TGFβ inhibitor, a Wnt inhibitor, and a MEK inhibitor, thereby forming retinal progenitor cells (RPCs). (d) Culturing the RPC in a retinal maturation (RM) medium containing nicotinamide and ascorbic acid to produce neuroretinal progenitor cells (NRP), and (e) Further culturing the NRP as a suspension aggregate in RM medium containing nicotinamide and ascorbic acid or in photoreceptor maturation (PM) medium containing nicotinamide and a γ-secretase inhibitor for a period of time sufficient to produce a population of PRPs. A method that includes this.

2. The method according to claim 1, wherein step (e) comprises culturing the NRP as a suspension aggregate in RM medium for a period of time sufficient to produce a population of PRP.

3. The method according to claim 1, wherein step (e) comprises culturing the NRP in PM medium containing nicotinamide and a γ-secretase inhibitor for a period of time sufficient to produce a population of PRPs.

4. The method according to claim 1, wherein the culturing in steps (a) to (d) is further defined as adhesive two-dimensional culture.

5. (ii) After culturing in RD2, at least 30% of the cells express CHX10 (VSX2); or (iii) The RPC expresses PAX6, MITF, and / or PMEL17, in particular the RPC does not express CRALBP and / or BEST1, or does not express them in any meaningful way; or (iv) RPC does not express CRALBP and / or BEST1, or does not express them in any meaningful way; or (v) After culturing in RM, at least 70% of the cells express PAX6 and CHX10 (VSX2); or (vi) The NRP expresses PAX6 and CHX10 (VSX2); or (vii) The NRP expresses one or more markers selected from the group consisting of PAX6, CHX10 (VSX2), Ki67, and PMEL17. The method according to claim 1.

6. The method according to claim 1, further comprising maturing the group of PRPs as a suspension aggregate in RM medium containing nicotinamide or photoreceptor maturation (PM) medium, thereby producing a group of mature PRP aggregates.

7. The method according to claim 6, wherein the PM medium further comprises a cyclin-dependent kinase inhibitor.

8. The method according to claim 7, wherein the cyclin-dependent kinase inhibitor is a CDK4 / 6 inhibitor.

9. The method according to claim 3, wherein the γ-secretase inhibitor is DAPT.

10. The method according to claim 6, further comprising cryopreserving the mature PRP aggregates.

11. The method according to claim 6, further comprising dissociating the mature PRP aggregates into essentially single cells in PM medium.

12. The method according to claim 11, further comprising cryopreserving the mature PRP as a single cell.

13. The method according to claim 1, further comprising purifying the PRP.

14. The method according to claim 13, comprising selecting cells that are positive for CD171 and / or SUSD2, thereby providing a purified PRP cell population.

15. The method according to claim 13, comprising selecting cells that are positive for CD171, SUSD2, CD56 (NCAM), CD57 (LAMP-3), CD81, CD111 (Nectin 1), CD133, CD147, CD184 (CXCR4), CD200, CD230, CD276, CD298, CD344 (Frizzled), PSA-NCAM, and / or PTK7, thereby obtaining a purified PRP cell population.

16. The method according to claim 13, wherein purification comprises selecting cells that are positive for CD133, thereby providing a purified PRP cell population.

17. The method according to any one of claims 13 to 17, wherein the purification comprises depletion of cells positive for two or more markers selected from the group consisting of CD9, CD49f, CD340, podoplanin, CD29, and / or CD63.

18. The method according to any one of claims 13 to 17, wherein at least 75% of the cells express recabinetin (RCVRN).

19. The method according to any one of claims 13 to 17, wherein at least 80% of the cells express recabinetin (RCVRN).

20. The method according to any one of claims 13 to 17, wherein at least 85% of the cells express recabinetin (RCVRN).

21. The method according to any one of claims 13 to 17, wherein at least 90% of the cells express recabinetin (RCVRN).

22. The method according to claim 1, wherein the PRP expresses one or more markers selected from the group consisting of OTX2, IRBP, SUSD2, CRX, BLIMP1, NEUROD1, RCVRN, TUBB3, and CD171 / L1CAM, and / or the PRP does not express, or essentially does not express, TRYP1, CRALBP, BEST1, Ki67, MITF, PMEL17, PAX6, CHX10, CHX10 (VSX2), and / or Onecut1.

23. The method according to any one of claims 13 to 16, wherein 15%, 10%, or less than 5% of the cells in the purified PRP population express PAX6.

24. A method for producing a pharmaceutical composition, comprising adding (a) to (e) of claim 1 and a pharmaceutically acceptable carrier.

25. The method according to claim 1, further comprising culturing the NRP population as a suspension aggregate in a medium containing a γ-secretase inhibitor and a ROCK inhibitor or brevistatin.

26. The method according to claim 1, further comprising culturing iPS cells in retinal induction medium (RIM) before culturing them in the RD1 medium to initiate differentiation into anterior neuroectoderm cells.

27. ​​The method according to claim 26, wherein the RIM includes IGF-1.

28. The method according to claim 26, wherein the RIM essentially does not contain CKI-7.

29. The method according to claim 26, wherein the RIM essentially does not contain activin A.

30. The method according to claim 1, wherein the RD2 medium does not essentially contain LDN193189.

31. The method according to claim 1, wherein at least 90% of the cells express PMEL17 after culturing in the RD2 medium.

32. The method according to claim 1, wherein at least 30% of the cells express VSX2 after culturing in the RD2 medium.

33. The method according to claim 1, wherein the RPC expresses PAX6, MITF, and / or PMEL.

34. The method according to claim 1, wherein the NRP expresses one or more cell markers selected from the group consisting of PAX6, CHX10 (VSX2), Ki67, and PMEL.

Images