Methods and compositions for the generation of retinal neurons in carrier-free 3D spheroid suspension culture

Abstract

In one aspect, the present invention provides a retinal neuron population including photoreceptor precursor cells (PRPCs) generated in vitro from human pluripotent stem cells (hPSCs), which can be used as a cell source for regenerative therapy, drug discovery, and disease modeling. Also provided are methods and compositions for making and using the same.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 62 / 728,088, filed on September 7, 2018, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure generally relates to methods and compositions for generating retinal neurons from, for example, human pluripotent stem cells.

[0004] background

[0005] Age-related macular degeneration and inherited retinal degeneration are the leading causes of untreatable blindness in developed countries [1]. They share a common ultimate pathophysiology, namely the loss of light-sensitive photoreceptors, which are composed of rods and cones. Replacement of lost photoreceptors may offer a potential therapeutic strategy for both patient populations that lack effective treatment options [1-3]. Most macular diseases involve the loss of photoreceptors and retinal pigment epithelial cells (RPE), which support and maintain the health of photoreceptors in the outer retina. Transplantation of human embryonic stem cell (hESC)-derived RPE has been published in clinical trials for the treatment of patients with dry age-related macular degeneration (AMD) and Stargardt's disease and has shown evidence of safety and efficacy, including evaluation after 4 years [4-6], but such studies have not been conducted in human clinical trials for photoreceptors derived from human pluripotent stem cells (hPSC) [7].

[0006] The main requirement for developing effective photoreceptor transplantation therapy is to establish a robust protocol that allows the generation of large numbers of homogeneous photoreceptor precursor cells (PRPCs) from renewable sources such as hESCs and human induced pluripotent stem cells, due to the limited supply of PRPCs that form human tissue and ethical barriers [8]. Over the past few decades, significant progress has been made in the ability to generate retinal cell types, including PRPCs and photoreceptors, in vitro from a variety of murine and human PSC sources with varying degrees of efficiency, and the potential of hPSC-derived PRPCs has been established in preclinical transplantation studies using different animal models [2, 7, 9-13].

[0007] Currently, most differentiation protocols use traditional adherent two-dimensional (2D) planar cultures, which typically initiate differentiation directly in a 2D monolayer by exposure to a mixture of protein factors [9-11, 14, 15], or form cell aggregates called embryoid bodies (EBs) in static culture, which are then attached to coated or uncoated surfaces for further differentiation [1, 2, 8]. Recently, Sasai and colleagues described an EB-based self-organizing 3D differentiation system called retinal organoids, which recapitulates many aspects of normal retinal development

[16] . Based on this technology, several laboratories have generated PRPCs and photoreceptors from human PSC-laminated 3D retinal organoids [12, 17-21]. However, long-term cultured organoids contain a large number of cell types at different developmental stages, and these cell types form tight junctions, which makes them susceptible to enzymatic or physical dissociation and generates unhealthy and heterogeneous cell populations. Furthermore, while all of these approaches generate photoreceptors or their progenitors that can be used for research purposes, they are not suitable for generating transplantable cells for clinical applications due to the use of undefined proteins of non-human origin, which raises major safety concerns.

[0008] Therefore, there is a need for a scalable platform that can generate homogeneous populations of specific cells from hPSCs, including PRPCs and photoreceptors, under defined conditions. Summary of the invention

[0009] The present disclosure provides, inter alia, a method for generating photoreceptor precursor cells in vitro, comprising:

[0010] (a) culturing a plurality of pluripotent stem cells in a three-dimensional (3D) spheroid to generate a plurality of first spheroids, wherein the first spheroids comprise early and late committed retinal neural progenitor cells (CRNPs);

[0011] (b) monitoring the spheroid size until the diameter of the first spheroid reaches an average size of about 300-500 μm;

[0012] (c) disassociating the first spheroid into a first plurality of substantially single cells;

[0013] (d) 3D spheroid culturing the first plurality of elementary single cells to generate a plurality of second spheroids, the plurality of second spheroids comprising photoreceptor precursor cells (PRPCs);

[0014] (e) monitoring the spheroid size until the second spheroids reach an average size of about 300-500 μm in diameter;

[0015] (f) dissociating the second sphere into a second plurality of elementary single cells;

[0016] (g) 3D spheroid culturing the second plurality of elementary single cells to generate a plurality of third spheroids, the plurality of third spheroids comprising post-mitotic pRPCs; and

[0017] (h) Optionally, further differentiating the post-mitotic PRPCs into photoreceptor-like cells.

[0018] In some embodiments, the pluripotent stem cells may be embryonic stem cells or induced pluripotent stem cells, preferably from human.

[0019] In some embodiments, steps (a), (d) and (g) comprise culturing in a spinner flask or stirred tank bioreactor, preferably under continuous stirring.

[0020] In some embodiments, step (a) further comprises gradually adapting to and culturing in a neural induction medium, preferably NIM-3D (Neural Induction Medium-3D) basal medium comprising DMEM / F12 with HEPES, N2 and B27 serum-free supplements, penicillin / streptomycin, MEM non-essential amino acids and glucose, supplemented with one or more of the following substances: sonic hedgehog, heparin, IWR-1e, SB431542, LDN193189 and IGF1.

[0021] In some embodiments, the method may further include providing SB431542, LDN193189, and IGF1 in the neural induction medium for a first time period, providing IWR-1e in the neural induction medium for a second time period, the second time period being shorter than the first time period, and providing sonic hedgehog or heparin in the neural induction medium for a third time period, the third time period being shorter than the second time period. In some embodiments, the first time period is 10-20 days, preferably 12-18 days, and more preferably 16 days. In some embodiments, the second time period is 5-15 days, preferably 8-14 days, and more preferably 11 days. In some embodiments, the third time period is 3-12 days, preferably 5-10 days, and more preferably 7 days.

[0022] In some embodiments, sonic hedgehog or heparin can be withdrawn during neural commitment, e.g., about 1-5 days, 2-4 days, or 2 days before IWR-1e is withdrawn. In neural induction medium, IWR-1e can be withdrawn at full adaptation, about 1-5 days (or 2-4 days or 1 day) before full adaptation. As used herein, full adaptation in a certain medium refers to culturing in 100% of such medium. SB431542, LDN193189, and IGF1 can be withdrawn at the beginning of adaptation to photoreceptor differentiation medium or 1-5 days (or 2-4 days or 3 days) before adaptation.

[0023] In some embodiments, in step (b), the diameter of the first spheres reaches an average size of about 350-450 μm. In some embodiments, in step (b), the diameter of the first spheres may reach an average size of less than about 400 μm.

[0024] In some embodiments, steps (c) and (f) comprise contacting the first spheroid and the second spheroid, respectively, with a cell dissociation enzyme.

[0025] In some embodiments, step (d) further comprises gradually adapting and culturing the cells in a photoreceptor differentiation medium, preferably a PRPC-3D medium comprising Neurobasal TM Media, N2 and B27 without serum supplement, penicillin / streptomycin, MEM non-essential amino acids and glucose.

[0026] In some embodiments, steps (g) and / or (h) further comprise switching to a maturation medium and culturing therein, preferably Neurobasal TM Culture medium containing L-glutamine (e.g., GlutaMAX TM ), penicillin / streptomycin, human brain-derived neurotrophic factor (BDNF), ascorbic acid and DAPT (N-[N-(3,5-difluorophenylacetyl)-l-alanyl]-S-phenylglycine tert-butyl ester). In certain embodiments, the switching may include gradual adaptation to a maturation medium.

[0027] In some embodiments, steps (g) and / or (h) further comprise monitoring the spheroid size until the diameter is about 300-500 μm; dissociating the third spheroid into a third plurality of elementary single cells, preferably using a cell dissociation enzyme; and reaggregating the third plurality of elementary single cells.

[0028] Another aspect relates to a neural induction medium comprising DMEM / F12 with HEPES, N2 and B27 serum-free supplements, penicillin / streptomycin, MEM non-essential amino acids, glucose, and one or more of sonic hedgehog, heparin, IWR-1e, SB431542, LDN193189 and / or IGF1.

[0029] Another aspect relates to a maturation medium comprising Neurobasal TM Culture medium, L-glutamine (e.g., GlutaMAX TM ), penicillin / streptomycin, human brain-derived neurotrophic factor (BDNF), ascorbic acid, and DAPT (N-[N-(3,5-difluorophenylacetyl)-l-alanyl]-S-phenylglycine tert-butyl ester).

[0030] On the other hand, it relates to a method for photoreceptor replacement therapy, which comprises administering to a patient in need thereof a plurality of post-mitotic PRPCs and / or photoreceptor-like cells prepared using the methods disclosed herein. Also provided is the use of post-mitotic PRPCs and / or photoreceptor-like cells prepared using the methods disclosed herein, for example, for photoreceptor replacement therapy. In some embodiments, photoreceptor replacement therapy is used to treat retinal diseases such as dry and wet age-related macular degeneration, rod or cone dystrophy, retinal degeneration, retinitis pigmentosa, diabetic retinopathy, Leber's congenital amaurosis and Stargardt disease.

[0031] Another aspect relates to a method for in vitro screening comprising testing a substance in a plurality of post-mitotic PRPCs and / or photoreceptor-like cells prepared using the methods disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 : Characterization of 2D monolayer and 3D spheroid hiPSCs. (A) From left to right, 10 cm 2D cell culture dish, hiPSC monolayer colonies, flow cytometry histogram of OCT4-positive hiPSCs (97.9%) and karyotype analysis of hiPSCs cultured in 2D. (B) (Top, from left to right) Spinner flask, 3D hiPSC spheroids cultured in a 30 ml spinner (bioreactor), flow cytometry histogram of OCT4-positive hiPSCs (98.8%) and karyotype analysis of hiPSCs from 3D spheroids. (Bottom) Typical 3D spheroid passaging cycle from one hiPSC line during 4-day passaging intervals, medium changes, morphology and spheroid size.

[0033] Figure 2 : 3D spheroid differentiation protocol and timeline. (Figure A) Schematic diagram showing 3D spheroid PRPC differentiation over a period of approximately 100 days. The number of days of differentiation, the specific culture medium and small molecules and growth factors used at each time point are indicated. (Figure B) Schematic diagram showing the step-by-step strategy for inducing and differentiating 3D-hiPSCs into early and late ocular directed retinal neural progenitor cells (CRNPs) and photoreceptor precursor cells (PRPCs). The time points for freezing cells are indicated in the figure.

[0034] Figures 3A-3C : Induction of committed retinal neuronal progenitor cells from hiPSCs in 3D spheroids. Figure 3A : Phase contrast images show the complete morphology and expansion of 3D neurospheres from D0-D19 in 30 ml spinner flasks. The cell numbers at D0 (30 million), D9 (approximately 250 million), and D19 (approximately 450 million) are indicated. Figure 3B: Table summarizes the results from 4 hiPSC lines used for retinal neuronal differentiation. Neural differentiation efficiency was assessed by flow cytometry using PAX6 antibody as a marker for neural cells. Figure 3C : Approximate yields of early and late committed retinal neural progenitors (CRNPs) at day 19 generated from one spinner flask starting with 30 million hiPSCs.

[0035] Figures 4A-4C : Expression dynamics of pluripotent stem cell and neural markers during induced neuronal differentiation in 3D-hiPSC spheres. Figure 4A : Graphs show the percentage of OCT4, PAX6, PAX6 / SOX2, and SOX2-positive cells from four hiPSC lines during differentiation from D0-D19 analyzed by flow cytometry. Figure 4B : Flow cytometric analysis of OCT4, PAX6, and PAX6 / SOX2 positive cells at different days of neural differentiation. Figure 4C : Table summarizing the results of flow cytometric analysis.

[0036] Figure 5A-5B : Characterization of neural markers in 3D spheroid cells. Figure 5A : Phase contrast images and immunofluorescence staining show PAX6, RAX1, nestin and SOX2 on cells derived from 3D neurospheres. Differentiated cells at D5 were plated and cultured for 8 days and then stained with antibodies against the neural-specific marker PAX6 (green), the CRNP marker RAX1 (red), and the universal neural markers nestin (green) and SOX2 (red). A high percentage of cells were double positive for PAX6 / RAX1 and NES / SOX2, indicating the acquisition of neural and early and late CRNP properties. Nuclei were counterstained with DAPI. Scale bar, 100 μm. Figure 5B : Gene expression was quantitatively analyzed by qRT-PCR at different days of differentiation induction. OCT4 expression completely disappeared at D5; PAX6 expression increased at D5 and continued until D19, then gradually decreased until D40; RAX1 expression showed a similar pattern to PAX6; CHX10 expression showed a peak at D13 and then decreased at D40, indicating that early and late CRNP phenotypes were acquired during this differentiation period and PRPC specification was initiated after D40.

[0037] Figure 6: Retinal neuron spheroids generated by a sequential dissociation / reaggregation and spheroid reconstitution protocol. (A) Phase contrast images show the morphology and expansion pattern of 3D retinal neuron spheres at different stages of hiPSC differentiation in 30 ml spinner flasks. Scale bar = 100 μm. (B) Schematic summary of the yield of early and late CRNPs and post-mitotic PRPCs from 30 million starting 3D-hiPSCs.

[0038] Figures 7A-7C ; Characterization of hiPSC-generated 3D-PRPCs. Fig. 7A : Phase contrast images show the morphology of 3D hiPSC-derived 3D neurospheres before isolation at D32 (upper panel) and D82 (lower panel), and dissociated single cells cultured on the surface for 5 days (D37) and 18 days (D100), respectively. Figure 7B : Immunofluorescence staining shows the expression of CRX (green), ThRB2 (red) and NRL (red), MAP2 (green) and GFAP (red) on cells derived from 3D-PRPC spheres cultured on the surface for another 18 days at D82. Ki67 staining (red) shows a very low percentage of mitotic cells at D100. DAPI was used for nuclear staining. Figure 7C : Immunofluorescence staining shows the expression of photoreceptor-specific proteins rhodopsin (RHOD, green) and recoverin (REC, red) on cells derived from 3D-PRPC spheroids at D82 cultured on the surface for another 18 days. DAPI was used for nuclear staining.

[0039] Figures 8A-8B : Molecular characterization of hiPSC-derived 3D-PRPCs. Fig. 8A : Quantification of intracellular staining of OCT4 (0%), CRX (95.2%), NRL (96.6%), NR2E3 (91.3%), REC (96.8) and M-opsin (91.2%) in 3D spheroid cells after 80 days of differentiation by flow cytometry analysis. Secondary antibody alone served as negative control. Figure 8B : Quantitative RT-PCR analysis of PRPC and photoreceptor markers on 3D spheroid cells obtained from retinal neural differentiation on different days, showing the gradual acquisition of PRPC phenotype (NRL, NR2E3, ThRb2) and photoreceptor characteristics (REC, RHOD, M-opsin).

[0040] Fig. 9: Characterization of cells within 3D spheroids after 120 days of differentiation. (Panel A) Schematic diagram indicating how 3D spheroids at D120 are sliced ​​(broken lines). Phase contrast images show the intact morphology and integrity of the central core of the spheroid. Scale bar, 100 μm. (Panel B) Hematoxylin staining of sections indicates the presence of live cells on the sections. (Panel C) Immunohistochemical staining of sections of D120 differentiated 3D-PRPC spheres shows expression of the neuronal marker MAP2. Cell nuclei are counterstained with DAPI. (Panel D) Immunohistochemical staining of sections of D120 differentiated 3D spheroids shows expression of PRPC markers CRX (green) and NRL (red); while staining for the cell proliferation marker Ki67 (red) shows a very low percentage of proliferating cells in these spheres.

[0041] Fig.10 : 3D spheroids express photoreceptor markers after 120 days of differentiation. Immunohistochemical staining on sections of D120 differentiated 3D spheroids shows the expression of photoreceptor markers rhodopsin (RHOD, green) and recoverin (REC, red). Nuclei were counterstained with DAPI.

[0042] Fig.11 : Overview of an exemplary neural induction protocol for deriving retinal neural progenitor cells from human induced pluripotent stem cells. Efficient generation of retinal progenitor cells using small molecules in combination with sonic hedgehog (SHH).

[0043] Fig.12 : Comparative RT-PCR quantification of PAX6 mRNA gene expression in differentiating cells treated with heparin or SHH. Total RNA was collected from cells in both conditions during the photoreceptor differentiation timeline and analyzed for PAX6 RNA transcript levels.

[0044] Fig.13 : Characterization of differentiated day 122 photoreceptor cells derived from human induced pluripotent stem cells in a modified maturation medium. DETAILED DESCRIPTION

[0045] In one aspect, the present invention provides a retinal neuron population including photoreceptor precursor cells (PRPCs) generated in vitro from human pluripotent stem cells (hPSCs), which can be used as a cell source for regenerative therapy, drug discovery, and disease modeling. Also provided are methods and compositions for making and using the same.

[0046] A fundamental requirement for developing cell therapies is to establish robust manufacturing processes that allow large quantities of highly pure transplantable cells to be derived from renewable resources that reproduce the characteristics of the endogenous cell type to be replaced. However, many methods for differentiating hPSCs into retinal neurons and PRPCs for cell replacement therapies have produced unsatisfactory results in terms of efficiency, purity, homogeneity, and scalability. In one aspect, a robust, defined, and scalable three-dimensional (3D) spheroid culture system is disclosed herein for producing highly enriched retinal neurons, including early and late directed retinal neuronal progenitors (CRNPs), PRPCs, and photoreceptor-like cells, from hPSCs at different developmental stages through a synchronized differentiation process, which can be easily adapted to current general manufacturing practices (cGMP) protocols.

[0047] In some embodiments, the protocol or method can start with 3D spheroids of hPSCs, which are directly induced to differentiate into early CRNPs, late CRNPs, PRPCs, and photoreceptor-like cells in a defined serum-free medium. In some embodiments, the culture medium can include a combination of small molecules that can be introduced into the culture medium at specified time points during culture to induce differentiation, accompanied by a continuous spheroid dissociation / reaggregation and spheroid reconstitution process performed in a rotating bioreactor under matrix-free and carrier-free conditions. This well-controlled 3D spheroid system overcomes many limitations, particularly scalability, for conventional surface-adherent 2D cultures and traditional embryoid bodies and organoid systems. Surprisingly, the method is typically 3x10 6 HiPSCs start to generate 3-4.5x10 9 PRPCs were generated with a purity of approximately 95%. Multi-level analyses, including immunofluorescence staining, flow cytometry, and quantitative gene expression by RT-qPCR, confirmed the identity of early and late CRNPs, PRPCs, and photoreceptor-like cells generated using this system. Therefore, this 3D spheroid platform is suitable for the development of in vitro GMP-compliant retinal cell manufacturing protocols from a variety of renewable hPSC sources for future preclinical studies and human cell replacement therapies.

[0048] The 3D expandable spheroid culture system under the limited minimum culture conditions can provide multiple benefits, such as scalability, reproducibility and homogeneity microenvironment and cost-effectiveness. In addition, the development of a scheme for generating enriched retinal neuron groups (such as PRPC) at the right stage is the key to the success of cell replacement therapy (such as cell replacement therapy for patients with photoreceptor loss). In some embodiments, the method disclosed herein simulates the chemical and cytokine microenvironment of the signal known to guide retinal tissue to occur during normal development. We have developed a limited, continuous matrix-free and carrier-free 3D spheroid culture system, and supplemented with small molecules to promote retinal neuron differentiation directly by hPSC adapted by 3D spheroids. In some embodiments, the stepwise induction differentiation scheme disclosed herein includes regular spheroid dissociation / re-aggregation in each generation, which is used to reorganize the spheroid in a cycle of about 50-100 days, which leads to the formation of uniform spheroids with controlled and ideal size. It is not desired to be bound by theory, and it is believed that the size of the spheroid is very important, which allows oxygen, nutrients and other factors to fully penetrate the entire spheroid, enriching neuronal populations and synchronizing neuronal differentiation. In addition, hPSC spheres differentiated under this protocol sequentially acquired markers specific for neural cells (PAX6), early and late committed retinal neuronal progenitors (RAX1 and CHX10), PRPCs (CRX, NRL, NR2E3, ThRB2), and photoreceptors (REC, RHOD, and M-opsin) with a purity of approximately 95%.

[0049] Significantly, this article provides methods to integrate undifferentiated hPSC expansion and streamlined small molecule-induced retinal neuronal differentiation into a scalable 3D spheroid culture system under matrix-free and carrier-free conditions by, for example, using spinner flasks, which paves the way for current common manufacturing practice (cGMP)-compliant methods to scale up the generation of retinal neurons from hPSCs for cell replacement therapy.

[0050] definition

[0051] For convenience, specific terms used in the specification, examples, and appended claims are collected here. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this document belongs.

[0052] The articles "a" and "an" are used herein to refer to one or to more than one (ie, to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0053] As used herein, the term "about" means within 20%, more preferably within 10% and most preferably within 5%. The term "substantially" means greater than 50%, more preferably greater than 80% and most preferably greater than 90% or 95%.

[0054] As used herein, the term "plurality" means more than 1, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more, for example, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 or more, or any integer therebetween.

[0055] As used herein, the terms "comprising" or "including" are used with respect to the compositions, methods, and their respective components present in a given embodiment and are open ended and may include unspecified elements.

[0056] As used herein, the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristics of that embodiment of the invention.

[0057] The term "consisting of" refers to the compositions, methods, and respective components thereof described herein, to the exclusion of any elements not recited in the description of that embodiment.

[0058] The term "embryonic stem cell" (ES cell) refers to a pluripotent stem cell derived from the inner cell mass of the embryo of a blastocyst or morula that has been serially passaged as a cell line. ES can be derived from fertilization of an egg cell with a sperm or DNA, nuclear transplantation, parthenogenesis, etc. The term "human embryonic stem cell" (hES cell) refers to a human ES cell. The generation of ESC is disclosed in U.S. Pat. Nos. 5843780; 62000806, and ESC obtained from the inner cell mass of a blastocyst derived from somatic cell nuclear transplantation is described in U.S. Pat. Nos. 5945577; 5,994,619; 6235970, all of which are incorporated herein by reference. The significant features of embryonic stem cells determine the embryonic stem cell phenotype. Therefore, if a cell has one or more unique features of an embryonic stem cell, then the cell will have the phenotype of an embryonic stem cell, so that the cell can be distinguished from other cells. Exemplary different embryonic stem cell characteristics include, but are not limited to, gene expression profiles, proliferation capacity, differentiation capacity, karyotype, responsiveness to specific culture conditions, etc.

[0059] The term "pluripotent" as used herein refers to cells with the ability to differentiate into more than one differentiated cell type under different conditions, and preferably differentiates into cell types with all three germ cell layer characteristics. The main feature of pluripotent stem cells is that they are differentiated into multiple cell types, preferably the ability to differentiate into all three germ layers using, for example, nude mouse teratoma formation tests. Such cells include hES cells, human embryo-derived cells (hEDC) and adult-derived stem cells. Pluripotent stem cells can be genetically modified or not genetically modified. Genetically modified cells can include markers, such as fluorescent proteins, to facilitate their identification. Pluripotency is also demonstrated by the expression of embryonic stem cell (ES) markers, but the preferred test of pluripotency is to demonstrate the ability to differentiate into cells of each layer in the three germ layers. It should be noted that simply cultivating these cells cannot make them pluripotent themselves. Relative to primary cell parents that usually only have a limited number of division abilities in culture, reprogramming pluripotent stem cells (e.g., the term iPS cells defined herein) also has the characteristics of extended passage without losing the ability of growth potential.

[0060] As used herein, the terms "iPS cells" and "induced pluripotent stem cells" are used interchangeably and refer to pluripotent stem cells that are artificially derived (e.g., induced or by complete reversal) from non-pluripotent stem cells (usually adult somatic cells), for example, by inducing the forced expression of one or more genes.

[0061] The term "reprogramming" as used herein refers to the process of changing or reversing the differentiation state of somatic cells, thereby resetting the developmental clock of the nucleus; for example, resetting the developmental state of the adult differentiated nucleus so that it can execute the genetic program of the early embryonic nucleus, so that all proteins required for embryonic development can be synthesized. Reprogramming disclosed herein covers the complete reversal of the differentiation state of somatic cells to pluripotent or totipotent cells. Reprogramming generally involves changes in genetic patterns such as at least some nucleic acid modifications (e.g., methylation), chromatin condensation, epigenetic changes, genomic imprinting, etc. that occur during the cell differentiation process from zygote to adult, such as reversal.

[0062] The terms "renewal" or "self-renewal" or "proliferation" are used interchangeably herein to refer to the ability of stem cells to self-renew by dividing into the same non-specialized cell type over long periods of time and / or months to years. In some cases, proliferation refers to the expansion of cells by repeated division of a single cell into two identical daughter cells.

[0063] As used herein, the term "culturing" or "feeding" refers to in vitro laboratory procedures used to maintain cell viability and / or proliferation.

[0064] The term "carrier-free three-dimensional spheroid" culture or feeding refers to the technique of culturing cells under non-adherent conditions so that the cells can form spheroids on their own without any carrier. Conventional methods for culturing cells with adhesion are characterized by culturing cells on a flat surface of a container such as a culture dish (two-dimensional culture). In contrast, in three-dimensional culture methods, no attachment support is provided to the cells, and the culture relies heavily on cell-cell contact.

[0065] As used herein, "carrier" or "microcarrier" refers to a solid spherical bead made of plastic, ceramic or other material such as gelatin or hydrogel, designed to provide an adherent surface for suspended cell culture. Other forms or shapes of carriers have also been reported, such as fiber structures.

[0066] The term "sphere" or "spheroid" refers to a three-dimensional spherical or substantially spherical mass or aggregate of cells. In some embodiments, an extracellular matrix can be used to help cells move within their spheres, similar to the way cells move in living tissues. The most common types of ECM are basement membrane extracts or collagen. In some embodiments, matrix-free or scaffold-free spheroid cultures can also be used, in which cells are grown in suspension in culture medium. This can be achieved by continuous rotation or using low attachment plates. In embodiments, spheroids can be created by single culture or co-culture techniques such as hanging drop method, rotation culture method, forced floating method, stirring method or concave plate method (see, e.g., Breslin et al., Drug Discovery Today 2013, 18, 240-249; Pampaloni et al., Nat. Rev. Mol. Cell Biol. 2007, 8, 839-845; Hsiao et al., Biotechnol. Bioeng. 2012, 109, 1293-1304; and Castaneda et al., J. Cancer Res. Clin. Oncol. 2000, 126, 305-310; all incorporated by reference). In some embodiments, the size of the spheroid can be increased during 3D culture.

[0067] The term "culture medium" is used interchangeably with "culture medium" and refers to any culture medium that allows cell proliferation. Suitable culture media do not need to promote maximum proliferation, only measurable cell proliferation. In some embodiments, the culture medium maintains the cells in a pluripotent state. In some embodiments, the culture medium encourages cells (e.g., pluripotent stem cells) to differentiate into, for example, early and late eye-directed retinal neural progenitor cells (CRNP) and photoreceptor precursor cells (PRPC). Some exemplary basal culture media used herein include DMEM / F-12 (Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12; available from Thermo Fisher Scientific), without growth factors. It does not contain bFGF or TGFb (no With Pluriton TM (“PL”) are used interchangeably and are available from Biological Industries) and Neurobasal TM Culture medium (available from Thermo Fisher Scientific Inc.). Each may be supplemented with one or more of the following: a suitable buffer (e.g., HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)), chemically defined supplements such as N2 (0.1-10%, e.g., 1%) and B27 (0.1-10%, e.g., 1%) serum-free supplement (available from Thermo Fisher Scientific Inc.), antibiotics such as penicillin / streptomycin (0.1-10%, e.g., 1%), MEM non-essential amino acids (Eagle's Minimal Essential Medium (MEM), which consists of a balanced salt solution, amino acids, and vitamins that are essential for the growth of cultured cells and, when supplemented with non-essential amino acids, produces a MEM non-essential amino acid solution), glucose (0.1-10%, e.g., 0.30%), L-glutamine (e.g., GlutaMAX TM ), human brain-derived neurotrophic factor (BDNF), ascorbic acid and / or DAPT (N-[N-(3,5-difluorophenylacetyl)-1-alanyl]-S-phenylglycine tert-butyl ester). Factors for inducing differentiation as disclosed herein, such as sonic hedgehog, heparin, IWR-1e, SB431542, LDN193189 and IGF1, may also be added to the culture medium.

[0068] As used herein, the term "differentiated cell" refers to any cell that is differentiating into a somatic cell lineage or has terminally differentiated. For example, an embryonic cell can differentiate into an epithelial cell lining the intestine. For example, a differentiated cell can be isolated from a fetus or a live born animal.

[0069] In the context of cellular ontogeny, the adjective "differentiated" or "differentiated" is a relative term, meaning that a "differentiated cell" is a cell that is further along in the developmental process than the cell to which it is being compared. Thus, a stem cell can differentiate into a lineage-restricted precursor cell (such as a mesodermal stem cell), which can further differentiate into other types of precursor cells (such as a photoreceptor precursor cell), and then differentiate into a terminally differentiated cell that will play a characteristic role in certain tissue types and may or may not retain the ability to expand further.

[0070] The terms "enrich" or "enriched" are used interchangeably herein and mean that the yield (fraction) of one type of cells is increased by at least 10% over the fraction of that type of cells in the starting culture or preparation.

[0071] As used herein, the term "agent" or "substance" refers to any compound or substance, such as but not limited to small molecules, nucleic acids, polypeptides, peptides, drugs, ions, etc. "Agent" can be any chemical substance, entity or part, including but not limited to synthetic and naturally occurring proteinaceous and non-proteinaceous entities. In some embodiments, the agent is a nucleic acid, a nucleic acid analog, a protein, an antibody, a peptide, an aptamer, a nucleic acid oligomer, an amino acid or a carbohydrate, including but not limited to proteins, oligonucleotides, ribozymes, DNA enzymes, glycoproteins, siRNA, lipoproteins, aptamers, and in certain embodiments, the agent is a small molecule having a chemical moiety. For example, the chemical moiety includes unsubstituted or substituted alkyl, aromatic or heterocyclic moieties, including macrolides, leptomycin and related natural products or their analogs. The compound can be known to have the desired activity and / or properties, or can be selected from a library of multiple compounds.

[0072] The term "small molecule" refers to an organic compound having multiple carbon-carbon bonds and a molecular weight of less than 1500 Daltons. Typically such compounds contain one or more functional groups that mediate structural interactions with the protein's structure, such as hydrogen bonds, and typically include at least one amine, carbonyl, hydroxyl or carboxyl group, and in some embodiments, at least two chemical functional groups. Small molecule agents may contain cyclic carbon or heterocyclic structures, and / or aromatic or polycyclic aromatic hydrocarbon structures substituted with one or more chemical functional groups.

[0073] The term "marker" as used herein is used to describe the characteristics and / or phenotype of a cell. Markers can be used to select cells containing features of interest. Markers vary depending on the specific cell. Markers are characteristics that can be either cell morphology, function, or biochemical (enzymatic) characteristics of a specific cell type, or molecules expressed by the cell type. Preferably, such markers are proteins, and more preferably, have epitopes for antibodies or other binding molecules accessible in the art. However, markers can be composed of any molecule present in the cell, including but not limited to proteins (peptides and polypeptides), lipids, polysaccharides, nucleic acids, and steroids. Examples of morphological characteristics or characteristics include but are not limited to shape, size, and nuclear-cytoplasmic ratio. Examples of functional characteristics or characteristics include but are not limited to the ability to adhere to a specific substrate, the ability to include or exclude specific dyes, the ability to migrate under specific conditions, and the ability to differentiate along a specific lineage. Markers can be detected by any method accessible to those skilled in the art. Markers can also be lacking morphological characteristics or lacking proteins, lipids, etc. Markers can be a combination of a set of unique characteristics with or without polypeptides and other morphological characteristics.

[0074] The term "isolated population" used herein with respect to an isolated cell population refers to a cell population that has been removed and separated from a mixed or heterogeneous cell population. In some embodiments, an isolated population is a substantially pure cell population compared to the heterogeneous population from which the cells were isolated or enriched.

[0075] With respect to a particular cell population, the term "substantially pure" refers to a cell population that is at least about 75%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% pure relative to the cells that make up the total cell population. With respect to a defined endoderm cell population, the term "substantially pure" or "substantially pure" refers to a cell population that contains less than about 20%, more preferably less than about 15%, 10%, 8%, 7%, and most preferably less than about 5%, 4%, 3%, 2%, 1%, or less than 1% of cells that are not defined endoderm cells or their progeny, as such terms are defined herein. In some embodiments, the present disclosure includes a method of expanding a defined endoderm cell population, wherein the expanded defined endoderm cell population is a substantially pure defined endoderm cell population. Similarly, reference to a "substantially pure" or "substantially pure" population of SCNT-derived stem cells or pluripotent stem cells refers to a population containing less than about 20%, more preferably less than about 15%, 10%, 8%, 7%, and most preferably less than about 5%, 4%, 3%, 2%, 1% cells.

[0076] The term "retina" refers to the ocular neuron, which is stratified into three nuclear layers and consists of photoreceptors, horizontal cells, bipolar cells, amacrine neurons, Müller glial cells, and ganglion cells.

[0077] "Progenitor cell" refers to a cell that maintains mitosis and can give rise to more progenitor cells or precursor cells from which cells can differentiate into terminal cell lineages.

[0078] "Precursor cell" refers to a cell that is capable of differentiating into terminal cell lineages.

[0079] In an embodiment of the present disclosure, "retinal neural progenitor cells" refer to cells differentiated from embryonic stem cells or induced pluripotent stem cells, which express cell markers PAX6 and CHX10. This may include early and late committed retinal neuronal progenitor cells, which may express markers PAX6, RAX1 and CHX10.

[0080] In embodiments of the present disclosure, "photoreceptor precursor cells" (PRPCs) refer to cells differentiated from embryonic stem cells or induced pluripotent stem cells, and which express the marker PAX6, but do not express the marker CHX10 (i.e., CHX10-). These cells transiently express CHX10 at the retinal neural progenitor cell stage, but when the cells differentiate into the photoreceptor progenitor cell stage, CHX10 expression is turned off. PRPCs may also express the markers CRX, NRL, NR2E3, and ThRB2.

[0081] Likewise, a "photoreceptor" may refer to a postmitotic cell differentiated from an embryonic stem cell or an induced pluripotent stem cell and expressing the cell marker rhodopsin (RHOD) or any of the three cone opsins (e.g., M-opsin), and optionally, a rod or cone cGMP phosphodiesterase. A photoreceptor may also express the marker recoverin (REC) found in photoreceptors. A photoreceptor may be a rod and / or cone photoreceptor.

[0082] "Photoreceptor-like cells" are cells that express most or all of the photoreceptor-specific markers but whose function has not been tested.

[0083] The terms "treat" or "treatment" are intended to refer to the administration of a substance for the purpose of: (i) preventing a disease or condition, i.e., causing clinical symptoms of the disease or condition not to develop; (ii) inhibiting a disease or condition, i.e., arresting the development of clinical symptoms; and / or (iii) relieving a disease or condition, i.e., causing regression of clinical symptoms.

[0084] Various aspects of the disclosure are described in further detail below. Additional definitions are provided throughout the specification.

[0085] Pluripotent stem cells

[0086] In various embodiments, PRPCs and photoreceptor cells can be generated from human pluripotent stem cells (hPSCs), including but not limited to human embryonic stem cells (hESCs), human parthenogenetic stem cells (hPSCs), nuclear transfer-derived stem cells, and induced pluripotent stem cells (iPSCs). Methods for obtaining such hPSCs are well known in the art.

[0087] Pluripotent stem cells are functionally defined as stem cells that: (a) are capable of inducing teratomas when transplanted into immunodeficient (SCID) mice; (b) are capable of differentiating into cell types of all three germ layers (e.g., can differentiate into ectoderm, mesoderm, and endoderm cell types); and (c) express one or more embryonic stem cell markers (e.g., express Oct4, alkaline phosphatase, SSEA-3 surface antigen, SSEA-4 surface antigen, NANOG, TRA-1-60, TRA-1-81, SOX2, REX1, etc.). In certain embodiments, pluripotent stem cells express one or more markers selected from the group consisting of OCT4, alkaline phosphatase, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. For example, exemplary pluripotent stem cells can be generated using methods known in the art. Exemplary pluripotent stem cells include embryonic stem cells derived from the ICM of blastocyst stage embryos, and embryonic stem cells derived from one or more blastomeres of cleavage stage or morula stage embryos (optionally without destroying the rest of the embryo). Such embryonic stem cells can be generated by embryonic material produced by fertilization or asexual means, including somatic cell nuclear transfer (SCNT), parthenogenesis and androgenesis. Other exemplary pluripotent stem cells include induced pluripotent stem cells (iPSCs) generated by reprogramming somatic cells by expression factors (referred to herein as reprogramming factors) combinations. iPSCs can be generated using fetal, postnatal, neonatal, youth or adult somatic cells.

[0088] In some embodiments, factors that can be used to reprogram somatic cells into pluripotent stem cells include, for example, OCT4 (sometimes referred to as OCT3 / 4), SOX2, c-Myc, and a combination of Klf4. In other embodiments, factors that can be used to reprogram somatic cells into pluripotent stem cells include, for example, a combination of OCT4, SOX2, NANOG, and LIN28. In some embodiments, at least two reprogramming factors are expressed in somatic cells to successfully reprogram somatic cells. In other embodiments, at least three reprogramming factors are expressed in somatic cells to successfully reprogram somatic cells. In other embodiments, at least four reprogramming factors are expressed in somatic cells to successfully reprogram somatic cells. In other embodiments, identify other reprogramming factors and use them alone or in combination with one or more known reprogramming factors to reprogram somatic cells into pluripotent stem cells. Induced pluripotent stem cells are functionally defined and include cells that are reprogrammed using any one of a variety of methods (integrated vectors, non-integrated vectors, chemical means, etc.). Pluripotent stem cells can be genetically modified or otherwise modified to increase lifespan, potency, homing, to prevent or reduce allogeneic immune responses, or to deliver desired factors in cells differentiated from such pluripotent stem cells (e.g., photoreceptor cells, photoreceptor progenitor cells, rod cells, cone cells, etc., and other cells such as described in the Examples herein).

[0089] Induced pluripotent stem cells (iPS cells or iPSCs) can be produced by protein transduction of reprogramming factors in somatic cells. In some embodiments, at least two reprogramming proteins are transduced into somatic cells to successfully reprogram somatic cells. In some embodiments, at least three reprogramming proteins are transduced into somatic cells to successfully reprogram somatic cells. In some embodiments, at least four reprogramming proteins are transduced into somatic cells to successfully reprogram somatic cells.

[0090] Pluripotent stem cells can be from any species. Embryonic stem cells have been successfully derived from, for example, mice, various species of non-human primates and people, and embryonic stem-like cells have been generated in various other species. Therefore, those skilled in the art can generate embryonic stem cells and embryo-derived stem cells by any species, including but not limited to people, non-human primates, rodents (mice, rats), ungulates (cows, sheep, etc.), dogs (domestic and wild dogs), cats (domestic and wild cats, such as lions, tigers, cheetahs), rabbits, hamsters, gerbils, squirrels, guinea pigs, goats, elephants, pandas (including giant pandas), pigs, raccoons, horses, zebras, marine mammals (dolphins, whales, etc.), etc. In some embodiments, the species is an endangered species. In some embodiments, the species is a currently extinct species.

[0091] Similarly, iPS cells can be from any species. These iPS cells have been generated using mice and human cells. In addition, iPS cells have been successfully generated using embryos, fetuses, newborns and adult tissues. Therefore, iPS cells can be easily generated using donor cells from any species. Therefore, iPS cells can be produced by any species, including but not limited to people, non-human primates, rodents (mice, rats), ungulates (cows, sheep, etc.), dogs (domestic and wild dogs), cats (domestic and wild cats, such as lions, tigers, cheetahs), rabbits, hamsters, goats, elephants, pandas (including giant pandas), pigs, raccoons, horses, zebras, marine mammals (dolphins, whales, etc.), etc. In some embodiments, the species is an endangered species. In some embodiments, the species is a currently extinct species.

[0092] Induced pluripotent stem cells can be generated using almost any somatic cell at any stage of development as a starting point. For example, the cell can be from an embryo, fetus, newborn, adolescent or adult donor. Exemplary somatic cells that can be used include fibroblasts, such as dermal fibroblasts obtained by skin samples or biopsy tissue, synoviocytes from synovial tissue, foreskin cells, cheek cells or lung fibroblasts. Although skin and cheeks provide a ready-made and easily accessible source of suitable cells, almost any cell can be used. In some embodiments, the somatic cell is not a fibroblast.

[0093] Induced pluripotent stem cells can be produced by expressing or inducing the expression of one or more reprogramming factors in somatic cells. Somatic cells can be fibroblasts, such as dermal fibroblasts, synovial fibroblasts or lung fibroblasts, or non-fibroblast somatic cells. Somatic cells can be reprogrammed by causing at least 1, 2, 3, 4, 5 kinds of reprogramming factors to be expressed (such as by viral transduction, integration or non-integration vectors, etc.) and / or contacting with them (for example, using protein transduction domains, electroporation, microinjection, cationic amphiphiles, fusions with lipid bilayers, detergent permeabilization, etc.). Reprogramming factors can be selected from: OCT3 / 4, SOX2, NANOG, LIN28, C-MYC and KLF4. The expression of reprogramming factors can be induced by contacting somatic cells with at least one reagent (such as an organic small molecule reagent) that induces the expression of reprogramming factors.

[0094] Other exemplary pluripotent stem cells include induced pluripotent stem cells produced by reprogramming somatic cells by expression or a combination of inducing factors ("reprogramming factors"). iPS cells can be obtained from a cell bank. Preparing iPS may be the first step in producing differentiated cells. iPS cells can be specifically generated using materials from a specific patient or a matched donor, with the goal of generating tissue-matched PHRPS or photoreceptor cells. iPSCs can be generated from cells that are substantially non-immunogenic in the intended recipient, for example, from autologous cells or from cells that are compatible with the intended recipient tissue.

[0095] Somatic cells can also be reprogrammed using a combination of methods, in which reprogramming factors are expressed (e.g., using viral vectors, plasmids, etc.) and the expression of reprogramming factors is induced (e.g., using organic small molecules). For example, reprogramming factors can be expressed in somatic cells by infection using viral vectors such as retroviral vectors or lentiviral vectors. Similarly, non-integrated vectors (such as episomal plasmids) can be used to express reprogramming factors in somatic cells. See, for example, Yu et al., Science. May 8, 2009; 324(5928):797-801, incorporated by reference in its entirety. When reprogramming factors are expressed using non-integrated vectors, electroporation, transfection or transformation of somatic cells (using vectors) can be used to express factors in cells. For example, in mouse cells, expression of four factors (OCT3 / 4, SOX2, C-MYC and KLF4) using integrative viral vectors is sufficient to reprogram somatic cells. In human cells, expression of four factors (OCT3 / 4, SOX2, NANOG and LIN28) using integrative viral vectors is sufficient to reprogram somatic cells.

[0096] Once the reprogramming factor is expressed in the cell, the cell can be cultured. Over time, cells with ES characteristics appear in the culture dish. Cells can be selected and subcultured based on, for example, ES morphology or based on the expression of selectable or detectable markers. These cells can be cultured to produce cell cultures similar to ES cells (they are assumed iPS cells).

[0097] To confirm the pluripotency of iPS cells, the cells can be tested in one or more pluripotency assays. For example, the cells can be tested for expression of ES cell markers; the ability of the cells to produce teratomas when transplanted into SCID mice can be assessed; the ability of the cells to differentiate to produce cell types of all three germ layers can be assessed. Once pluripotent iPSCs are obtained, they can be used to generate cell types disclosed herein, e.g., photoreceptor progenitors, photoreceptor cells, rods, cones, etc., as well as other cell types described herein.

[0098] Another method of obtaining hPSC is by parthenogenesis." parthenogenesis " (" parthenogenesis activated " and " activated by parthenogenesis " are used interchangeably herein) refers to the process of oocyte activation occurring in the absence of sperm penetration, and refers to the development of early embryos, which includes trophectoderm and inner cell mass obtained by activating oocytes or embryonic cells (such as blastomeres) containing all female source DNA. In related aspects, " parthenogenetic body " refers to the cell obtained by this type of activation. In another related aspect, " blastocyst " refers to the cleavage stage of fertilized activated oocytes, including hollow cell balls composed of external trophoblast cells and internal cell mass (ICM). In another related aspect, " blastocyst formation " refers to such a process: after oocyte fertilization or activation, oocytes are subsequently cultured in culture medium for a period of time to develop into hollow cell balls (e.g., 5-6 days) composed of external trophoblast cells and ICM.

[0099] Another method of obtaining hPSC is by nuclear transplantation. As used herein, "nuclear transplantation" refers to the fusion or transplantation of donor cells or DNA from donor cells to suitable recipient cells, typically oocytes of the same or different species, which are treated to remove or inactivate endogenous nuclear DNA before, during or after transplantation or fusion. Donor cells for nuclear transplantation include embryonic cells and differentiated cells, such as somatic cells and germ cells. Donor cells may be in a proliferative cell cycle (G1, G2, S or M) or a non-proliferative cell cycle (GO or static). Preferably, donor cells or DNA from donor cells are derived from proliferating mammalian cell cultures, such as fibroblast cultures. Optionally, donor cells may be transgenic, that is, they may include one or more gene additions, substitutions or deletions for modification.

[0100] Another method for obtaining hPSC is to obtain induced pluripotent stem cells by cell reprogramming. Takahashi et al. (Cell 131861-872 (2007)) have disclosed such a method for reprogramming differentiated cells without using any embryonic or ES (embryonic stem) cells, and establishing inducible pluripotent stem cells with pluripotency and growth capacity similar to ES cells. The nuclear reprogramming factors for differentiating fibroblasts include the products of the following four genes: Oct family genes; Sox family genes; Klf family genes; and Myc family genes.

[0101] Preferably, the pluripotency state of the cells is maintained by culturing the cells under appropriate conditions, for example, by culturing on a fibroblast feeder layer or another feeder layer or culture medium that includes leukemia inhibitory factor (LIF). The pluripotency state of such cultured cells can be confirmed by a variety of methods, for example: (i) confirming the expression of markers characteristic of pluripotent cells; (ii) generating chimeric animals containing cells expressing a pluripotent stem cell genotype; (iii) injecting cells into animals (e.g., SCID mice) to generate different differentiated cell types in vivo; and (iv) observing the differentiation of cells in vitro (e.g., cultured in the absence of a feeder layer or LIF) into embryoid bodies and other differentiated cell types.

[0102] The pluripotency state of the cells used in the present disclosure can be confirmed by a variety of methods. For example, the cells can be tested for the presence of characteristic ES cell markers. In the case of human ES cells, the above markers are identified (as examples), and include SSEA-4, SSEA-3, TRA-1-60, TRA-1-81, and OCT 4, and are known in the art.

[0103] Additionally, pluripotency can be confirmed by injecting the cells into a suitable animal (eg, SCID mouse) and observing the production of differentiated cells and tissues.Another method to confirm pluripotency is to generate chimeric animals using the subject's pluripotent cells and observe the contribution of the introduced cells to different cell types.

[0104] Another method to confirm pluripotency is to observe that ES cells differentiate into embryoid bodies and other differentiated cell types when cultured under conditions favorable for differentiation (e.g., removing fibroblast feeder layers). This method has been applied and has confirmed that subject pluripotent stem cells produce embryoid bodies and different differentiated cell types in tissue culture.

[0105] By routine passaging, hPSCs can be maintained in a pluripotent state in culture until neural stem cell derivation is desired.

[0106] 3D matrix and carrier-free spheroid culture to generate photoreceptors

[0107] In order to practically apply hPSCs in cell therapy, it is necessary to improve their large-scale and 3D culture. A variety of 3D spheroid culture operations can be used, including, for example, forced-floating methods, which modify the cell culture surface and thus promote 3D culture formation by preventing cells from attaching to its surface; hanging drop methods, which support cell suspension growth; stirring / rotation systems, which encourage cells to attach to each other to form 3D spheroids.

[0108] One method for generating 3D spheroids is to prevent the 3D spheroids from attaching to the surface by modifying the vessel surface, thereby causing the cells to be forced to float. This promotes cell-cell contact, which in turn promotes multicellular spheroid formation. Exemplary surface modifications include poly-2-hydroxyethyl methacrylate (polyHEMA) and agarose.

[0109] The hanging drop method of 3D spheroid generation uses a small aliquot (usually 20 ml) of a single cell suspension that is dropped into the wells of a tray. Similar to forced flotation, the cell density of the inoculated suspension (e.g., 50, 100, 500 cells / well, etc.) can be changed accordingly depending on the desired spheroid size. After cell inoculation, the tray is then inverted and the aliquot of the cell suspension is allowed to become a hanging drop that remains in place due to surface tension. The cells accumulate at the droplet tip, the liquid-air interface, and are allowed to diffuse.

[0110] The stirring-based methods for producing 3D spheroids can be roughly divided into two categories: (i) spinner flask bioreactors and (ii) rotating culture systems. The general principle behind these methods is to place a cell suspension in a container and keep the suspension in motion, i.e., gently stir or rotate the container. The continuous movement of the suspended cells means that the cells do not adhere to the walls of the container, but instead form cell-cell interactions. Spinner flask bioreactors (commonly referred to as "rotators") include a container for holding the cell suspension and a stirring element for ensuring continuous mixing of the cell suspension. Rotating cell culture bioreactors work in a similar manner to spinner flask bioreactors, but instead of using a stirring bar / rod to keep the cell suspension moving, the culture container itself is rotated.

[0111] In some embodiments, the present invention provides a spinner bottle-based 3D spheroid culture protocol. In the absence of feeder cells and matrix, multiple hPSCs can be continuously cultured as substantially identical spheroids in a spinner bottle with a defined medium. The medium can be any defined, xeno-free, serum-free cell culture medium designed to support the growth and expansion of hPSCs (such as hiPSCs and hES cells). In one example, the medium is Culture medium. In some embodiments, the culture medium can be mTeSR1, mTeSR2 or E8 culture medium or other stem cell culture medium. The culture medium can be supplemented with small molecule inhibitors, which are Rho-related, containing coiled-coil protein kinase (ROCK) such as Y27632 or other ROCK inhibitors such as Thiazovivin, ROCK II inhibitors (such as SR3677) and GSK429286A. Using this suspension culture system, hPSC cultures can be continuously passaged and continuously expanded for at least 10 generations. A typical passage downgrade of 3D-hiPSC spheres can be about 3-6 days, at which time the spheres can grow to a size of about 230-260 μm in diameter. The size of the spheres can be monitored by taking an aliquot of the culture and observing it under a microscope. The spheres can then be dissociated into single (or substantially single) cells using enzymes with proteolytic and collagenase activities, such as those used to separate primary and stem cell lines and tissues. In one example, the enzyme is Or TrypLE, or trypsin / EDTA. After this, under continuous stirring of, for example, 60-70RPM, the dissociated cells can be reassembled to reconstitute the sphere in a rotating bottle. After at least 3-5 repeated passages, the sphere gradually increases, while maintaining a consistent structure and high pluripotency marker expression (OCT4) and a normal karyotype. As used herein, "passage" should be understood as meaning a cell sphere culture of a sphere grown from a single cell to an ideal size, at which point the sphere is dissociated into a single cell and inoculated again for another passage. For 3D-hiPSC spheres, passage may require 3-6 days, or longer or shorter, depending on the type and culture conditions of hPSC. Once a sufficient amount of 3D-hPSC spheres are obtained, they can perform 3D sphere retinal neuron differentiation, as described in more detail below.

[0112] To generate retinal neuronal progenitors at different developmental stages from hPSCs, a stepwise approach using mainly small molecules can be used to directly induce 3D-hPSC spheroids in suspension ( Figure 2 ). In some embodiments, this can be done in 3D spinner bottles or other 3D spheroid culture methods. In various embodiments, continuous 3D spheroid culture can be integrated with multiple dissociation / reaggregation steps, and small molecules can be added at different developmental stages to induce retinal neuronal differentiation. Unlike previously reported use of protein factors to induce hPSC differentiation into retinal lineages, small molecules whose quality is easily controlled are used when possible to sequentially differentiate hPSC spheres into retinal cells at different developmental stages.

[0113] like Figure 2 As shown, one can firstly sample single cells (e.g., 1x10 6hPSCs (e.g., hiPSCs) are plated in a defined medium supplemented with a ROCK inhibitor (e.g., Y27632, "Y") in a spinner flask at 400 cells / mL. 24 hours later (designated as D0 of induction), hiPSC spheres can be first treated with dual SMAD inhibitors SB431542 ("SB") and LDN193189 ("LDN") to block signal transduction of activin / transforming growth factor b (TGF-β) and bone morphogenetic protein (BMP) and promote neural patterning, and then treated with Wnt inhibitor IWR-1e, IGF1 (an inducer of eye field cell development) and heparin to further induce retinal neural lineage commitment.

[0114] For pRPC differentiation, the differentiation can be performed by serially diluting Pluriton with the above induction factors. TM / No GF and NIM-3D to achieve gradual adaptation of NIM-3D medium. For example, 100% Pluriton TM / No GF The gradual adaptation to 100% NIM-3D can include sequentially 75%:25%, 50%:50% and 25%:75%, using Pluriton TM / No GF Intermediate culture with NIM-3D, where cells are cultured for 2-6 days in various media compositions. Other dilution series may also be used. NIM-3D (Neural Induction Medium-3D) (interchangeably used with "NIM") basal medium comprises DMEM / F12 with HEPES, N2 (0.1-10%, e.g., 1%) and B27 (0.1-10%, e.g., 1%) serum-free supplements, penicillin / streptomycin (0.1-10%, e.g., 1%), MEM non-essential amino acids and glucose (0.1-10%, e.g., 0.30%). Upon full adaptation in NIM-3D medium, heparin may be withdrawn during neural orientation and IWR-1e.

[0115] Thereafter, the spheres can be adapted to pRPC-3D photoreceptor differentiation medium by adapting to a 50 / 50 mixture of NIM-3D / pRPC-3D medium. Fig.11 Also known as “PRPM” in Neurobasal TMCulture medium, N2 (0.1-10%, e.g., 1%) and B27 (0.1-10%, e.g., 1%) serum-free supplements, penicillin / streptomycin (0.1-10%, e.g., 1%), MEM non-essential amino acids and glucose (0.1-10%, e.g., 0.30%). SB431542, LDN193189 and IGF1 can be withdrawn at the beginning of adaptation to NIM-3D / PRPC-3D culture medium.

[0116] Another exemplary differentiation protocol is Fig.11 As shown. Figure 2 The key difference in the protocol shown is the use of sonic hedgehog (SHH) instead of heparin. Surprisingly, SHH is more effective than other mitogen-activated proteins such as heparin in achieving neuronal induction and proliferation of retinal progenitor cells and maintaining neurogenesis in PAX6-positive cells.

[0117] During the differentiation process, cell dissociation enzymes such as trypsin (Thermo Fisher Scientific), Accutase enzymes or trypsin / EDTA can be used to dissociate the spheres into single cells at different time points. When the diameter of the spheres usually or on average reaches about 300-500 μm or about 350-450 μm, PRPCs can be generated every 2-5 weeks by continuously dissociating the spheres into single cells and reaggregating them into a spinner bottle to avoid the generation of hypoxic cells in the center of the spheres. It should be noted that sphere sizes exceeding about 450 or 500 μm in diameter may be undesirable because this may prevent oxygen, nutrients, and differentiation-inducing factors / molecules from penetrating into the central core of the spheres, resulting in necrosis and other causes of nuclear cell death. Therefore, once the average size of the spheres grows to about 300-400 μm or about 350-450 μm in diameter, various enzymes known in the art for cell dissociation can be used to dissociate the spheres into single cells (or substantially single cells).

[0118] Without wishing to be bound by theory, it is believed that if the spheroids become too large (e.g., greater than 500 or greater than 400 μm in diameter) then cells near the center of the spheroids may be at risk of malnutrition. Therefore, in some embodiments, it may be desirable to control the size of the spheroids. In certain embodiments, the spheroids may be dissociated when they reach a diameter of about 300-500 μm, about 350-450 μm, or about 230-260 μm.

[0119] Once isolated, the single cell suspension can be filtered through a filter (e.g., with a mesh size of about 10-200 or about 20-100 or about 40 μm). The single cells are then inoculated into appropriate culture media in spinner flasks. The morphology (size, appearance, and ability to incorporate spheres) of the resulting spheres can be monitored 2-3 days after each reaggregation and every week thereafter until the next dissociation / reaggregation step. Monitoring is monitored by taking an aliquot of the culture and observing it under a microscope.

[0120] Therefore, this article discloses a stepwise hPSC sphere differentiation process for different developmental stages of retinal neurons, including early and late committed retinal neuronal progenitor cells (CRNPs), photoreceptor precursor cells (PRPCs), and photoreceptor-like cells. Compared with previously reported differentiation protocols, the 3D sphere platform has the following advantages:

[0121] 1) Defined medium with small molecules but without undefined matrix. In the 3D spheroid culture system disclosed herein, serum and undefined matrix or carrier are not added to the culture medium from hPSC maintenance and expansion to 3D spheroid retinal differentiation. In addition, small molecules are used to replace protein factors, which makes it easy to control quality and consistency, making the 3D spheroid system more consistent and reproducible than previously reported systems;

[0122] 2) A robust platform for cell processing and manufacturing. The process of directly switching from 3D-hPSC sphere expansion culture to 3D sphere differentiation does not require cell manipulation, such as cell attachment to the surface or matrix embedding. Simply replacing the culture medium can make the transition smooth. In addition, retinal neurons at different developmental stages are generated during the process, and these cells can be cryopreserved for further differentiation, making the quality control process easier.

[0123] 3) The consistency and integrity of the 3D spheroids leads to synchronized retinal neuron differentiation. The diameter and integrity of the 3D spheroids during hPSC expansion and differentiation induction can be well controlled for a long time (usually 3-4 months) by controlling the agitation / agitation speed and initial cell density. This allows oxygen, nutrients, and differentiation-inducing factors / molecules to penetrate into the central core of the spheroid, avoiding necrosis and other causes of cell death, which are common in uncontrolled embryoid and organoid systems. Maintaining the consistency and integrity of the 3D spheroids synchronizes the differentiation process with the generation of pure retinal neuron populations;

[0124] 4) A scalable process for large-scale production of desired cells. Although the examples used a 30-50 ml bioreactor for proof-of-concept studies, this is easily scalable to multiple and larger bioreactors. Each 30-50 ml bioreactor typically produces approximately 3×10 8The system can produce retinal neuronal cells, equivalent to the capacity of 20-25 T-75 tissue culture flasks, making it a cost-effective and manageable process for cell production.

[0125] 5) Generate a more homogeneous cell population. During the induction of differentiation, a dissociation / reaggregation step is integrated for spheroid reconstitution when separating these cells, which serves as a purification step to remove non-neuronal cells and enrich neuronal cells. This step results in the generation of retinal cells at different developmental stages, including early and late CRNPs, PRPCs, and photoreceptor-like cells, at different steps, with high purity (about 95%) and without contamination of undifferentiated pluripotent stem cells; and

[0126] 6) An adaptable system that can be expanded to generate other types of neurons. Because the 3D spheroid differentiation process is a multi-step process, and each step generates a unique neuronal process, the process can be easily expanded to generate other neuronal cell types by changing the differentiation induction conditions, including but not limited to other retinal neuronal cells, such as retinal ganglion cells and progenitor cells, bipolar cells, Müller cells, horizontal cells and amacrine neurons and other universal neuronal cells.

[0127] In various embodiments, a novel, high-resolution and defined 3D spheroid platform is provided herein to generate desired cells from hPSCs, specifically, pRPCs and photoreceptor-like cells that can be used for photoreceptor replacement therapy for blind patients. This system is not only suitable for large-scale production work, but also eliminates the dependence on animal serum and matrix, plus supplementation with small molecules rather than protein factors, making it amenable to cGMP-compliant cell manufacturing protocols and making the process more suitable for clinical translation.

[0128] Photoreceptor replacement therapy

[0129] Retinal diseases often lead to blindness due to loss of postmitotic neuronal cells. Retinal diseases include rod or cone dystrophy, retinal degeneration, retinitis pigmentosa, diabetic retinopathy, macular degeneration, Leber congenital amaurosis, and Stargardt disease. In most retinal degenerations, cell loss occurs primarily in the outer nuclear layer, which includes rod and cone photoreceptors. Due to the loss of postmitotic neuronal cell populations, an exogenous source of new cells is required to replace the photoreceptor cells.

[0130] Retinal degeneration is an irreversible process that ultimately leads to blindness. Rod and cone photoreceptors are the primary photoreceptors in the retina, but these cells lack the ability to regenerate. Currently, no treatment exists to regenerate lost photoreceptors, and cell replacement is the only therapeutic strategy for treating patients with photoreceptor loss. MacLaren et al.

[46] were the first group to demonstrate that transplantation of mouse postmitotic photoreceptor precursor cells into completely blind mice restored some visual function. These transplanted cells integrated into the ONL layer, differentiated into rod photoreceptors, formed synaptic connections, and improved visual function in these animals. More recently, several groups have reported improved visual function in animal models with a diverse range of retinal dysfunction following transplantation of postmitotic photoreceptor precursor cells derived from mouse and human PSCs [9, 42, 43]. These results provide proof of concept in animal models that cell replacement therapy may be effective for patients with photoreceptor degeneration if the appropriate cells are transplanted into the appropriate location with an appropriate microenvironment. Clinical trials using human embryonic stem cells (hESC) and human embryonic stem cells (hiPSC)-derived retinal pigment epithelial (RPE) cells to prevent vision loss in patients with AMD and Stargardt disease are currently ongoing [3-5], but cell transplantation to replace lost photoreceptors has not yet begun. Therefore, there is an urgent need to obtain human photoreceptor precursor cells at the appropriate developmental stage from renewable sources for cell replacement therapy. Obviously, postmitotic human photoreceptor precursors derived from human donors do not represent a suitable source of cell replacement. Differentiating human PSCs in vitro to generate retinal neurons, especially postmitotic photoreceptor precursor cells, will achieve this goal.

[0131] Therefore, the PRPCs or photoreceptor cells disclosed herein can be formulated with a pharmaceutically acceptable carrier and used in photoreceptor replacement therapy. For example, the PRPCs or photoreceptor cells can be administered alone or as a component of a pharmaceutical preparation. The test compound can be formulated for administration in any convenient manner for use in medicine. Pharmaceutical preparations suitable for administration may include the PRPCs or photoreceptor cells, in combination with one or more pharmaceutically acceptable sterile isotonic aqueous solutions or non-aqueous solutions (e.g., balanced salt solutions (BSS)), dispersions, suspensions or emulsions, or sterile powders that can be reconstituted into sterile injectable solutions or dispersions immediately before use, which may contain antioxidants, buffers, bacteriostats, solutes, suspending agents or thickening agents. Exemplary pharmaceutical preparations include the PRPCs or photoreceptor cells, in combination with BSS (Balanced salt solution, each mL contains 7.14 mg of sodium chloride, 0.38 mg of potassium chloride, 0.154 mg of calcium chloride dihydrate, 0.2 mg of magnesium chloride hexahydrate, 0.42 mg of sodium dihydrogen phosphate, 2.1 mg of sodium bicarbonate, 0.92 mg of levulose, 0.184 mg of glutathione disulfide (oxidized glutathione), and aqueous solution of hydrochloric acid and / or sodium hydroxide (the pH value is adjusted to about 7.4)).

[0132] When administered, the pharmaceutical preparations used in the present disclosure may be in a pyrogen-free, physiologically acceptable form.

[0133] The preparation containing the pRPCs or photoreceptor cells used in the methods described herein can be transplanted in a suspension, gel, colloid, slurry or mixture. In addition, it is ideal to encapsulate the preparation in or inject it into the vitreous in a viscous form for delivery to the site of retinal or choroidal damage. In addition, at the time of injection, the cryopreserved pRPCs or photoreceptors can be resuspended using a commercially available balanced salt solution to obtain the desired osmotic pressure and concentration for administration by subretinal injection. The preparation can be administered to areas of the central peripheral macula that have not been completely lost due to disease, which can promote the attachment and / or survival of the administered cells.

[0134] The pRPCs or photoreceptor cells of the present disclosure can be delivered by intraocular injection in a pharmaceutically acceptable ophthalmic formulation. For example, when the formulation is administered by intravitreal injection, the solution can be concentrated so that the minimum volume can be delivered. The injection concentration can be any amount that is effective and non-toxic, depending on the factors described herein. The pharmaceutical preparation of pRPCs or photoreceptor cells for treating a patient can be formulated to be at least about 10 4 The dosage of pRPC or photoreceptor cell preparations used to treat annular lesions is at least about 10 3 , 10 4 , 10 5 , 10 6 , 107, 10 8 , 10 9 or 10 10 PRPCs or photoreceptor cells / mL. For example, PRPCs or photoreceptor cells can be formulated in a pharmaceutically acceptable carrier or excipient.

[0135] The pharmaceutical preparations of pRPCs or photoreceptor cells described herein may include at least about 1000; 2,000; 3,000; 4,000; 5,000; 6,000; 7,000; 8,000; or 9,000 pRPCs or photoreceptor cells. The pharmaceutical preparations of pRPCs or photoreceptor cells may include at least about 1×10 4 , 2×10 4 , 3×10 4 , 4×104 、 5×10 4 、6×10 4 、7×10 4 、8×10 4 、9×10 4 、1×10 5 、2×10 5 、3×10 5 、4×10 5 、5×10 5 、6×10 5 、 7×10 5 、8×10 5 、9×10 5 、1×10 6 、2×10 6 、3×10 6 、4×10 6 、5×10 6 、6×10 6 、7×10 6 、8×10 6 、 9×10 6 、1×10 7 、2×10 7 、3×10 7 、4×10 7 、5×10 7 、6×10 7 、7×10 7 、8×10 7 、9×10 7 、1×10 8 、 2×10 8 、3×10 8 、4×10 8 、5×108、6×10 8 、7×10 8 、8×10 8 、9×10 8 、1×10 9 、2×10 9 、 3×10 9 、4×10 9 、5×10 9 、6×10 9 、7×10 9 、8×10 9 、9×10 9 、1×10 10 、2×10 10 、3×10 10 、 4×10 10 、5×10 10 、6×1010 ,7×10 10 , 8×10 10 or 9×10 10 PRPCs or photoreceptor cells, or more or less. A pharmaceutical preparation of PRPCs or photoreceptor cells may contain at least about 1×10 2 -1×10 3 , 1×10 2 -1×10 4 , 1×10 4 -1×10 5 or 1×10 3 -1×10 6 PRPCs or Photoreceptor Cells For example, a pharmaceutical preparation of PRPCs or photoreceptor cells can comprise at least about 20,000-200,000 PRPCs or photoreceptor cells in a volume of at least about 50-200 μL.

[0136] In the above-mentioned pharmaceutical preparations and compositions, the number of PRPCs or photoreceptor cells or the concentration of PRPCs or photoreceptor cells can be determined by counting live cells and excluding non-live cells. For example, non-live PRPCs or photoreceptor cells can be detected by failing to exclude active dyes (such as trypan blue) or using functional tests (such as the ability to adhere to the culture matrix, phagocytosis, etc.). In addition, the number of PRPCs or photoreceptor cells or the concentration of PRPCs or photoreceptor cells can be determined by counting cells expressing one or more PRPCs or photoreceptor cell markers and / or excluding cells expressing one or more markers indicating cell types other than PRPCs or photoreceptor cells.

[0137] The PRPCs or photoreceptor cells can be formulated for delivery in a pharmaceutically acceptable ophthalmic carrier such that the preparation remains in contact with the ocular surface for a sufficient period of time to allow the cells to penetrate the affected ocular region, such as the anterior chamber, posterior chamber, vitreous, aqueous humor, vitreous, cornea, iris / ciliary body, lens, choroid, retina, sclera, suprachoroidal space, conjunctiva, subconjunctival space, suprascleral space, intracorneal space, corneal surface space, pars plana, surgically induced avascular zone, or macula.

[0138] PRPCs or photoreceptor cells may be included in the cell sheet. For example, a cell sheet comprising pRPCs or photoreceptor cells can be prepared by culturing pRPCs or photoreceptor cells on a substrate that releases the intact cell sheet, e.g., a thermoresponsive polymer, such as a thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) grafted surface, to which the cells will adhere and proliferate at the culture temperature, and then upon a change in temperature, the surface characteristics change, resulting in release of the cultured cell sheet (e.g., by cooling to below the lower critical solution temperature (LCST) (see da Silva et al., Trends Biotechnol. 2007 Dec;25(12):577-83; Hsiue et al., Transplantation. 2006 Feb 15;81(3):473-6; Ide, T. et al. (2006); Biomaterials 27,607-614, Sumide, T. et al. (2005), FASEB J. 20,392-394; Nishida, K. et al. (2004), Transplantation 77, 379-385; and Nishida, K. et al. (2004), N. Engl. J. Med. 351, 1187-1196, each of which is incorporated herein by reference in its entirety). The cell sheet can be attached to a substrate suitable for transplantation, such as a substrate that can dissolve in vivo when the cell sheet is transplanted into a host organism, for example, prepared by culturing the cells on a substrate suitable for transplantation, or releasing the cells from another substrate (such as a thermoresponsive polymer) onto a substrate suitable for transplantation. An exemplary substrate that may be suitable for transplantation may include gelatin (see Hsiue et al., supra). Other substrates that may be suitable for transplantation include fibrin-based matrices and other matrices. The cell sheet can be used to manufacture a drug for preventing or treating retinal degenerative diseases. The PRPC or photoreceptor cell sheet can be formulated for introduction into the eye of a subject in need thereof. For example, the cell sheet can be introduced into an eye in need thereof by subfoveal membrane resection accompanied by transplantation of the PRPC or photoreceptor cell sheet, or it can be used to manufacture a drug for transplantation after subfoveal membrane resection.

[0139] The volume of the preparation administered according to the methods described herein can depend on factors such as the mode of administration, the number of pRPCs or photoreceptor cells, the age and weight of the patient, and the type and severity of the disease being treated. If administered by injection, the volume of the pharmaceutical preparation of pRPCs or photoreceptor cells of the present disclosure can be at least about 1, 1.5, 2, 2.5, 3, 4, or 5 mL or more or less. The volume can be at least about 1-2 mL.

[0140] The method for treating retinal degeneration may further include administering an immunosuppressant. The immunosuppressants that may be used include, but are not limited to, anti-lymphocyte globulin (ALG) polyclonal antibodies, anti-thymocyte globulin (ATG) polyclonal antibodies, azathioprine, (anti-IL-2Rα receptor antibody), cyclosporine (cyclosporine A), (anti-IL-2Rα receptor antibody), everolimus, mycophenolic acid, (anti-CD20 antibodies), sirolimus and tacrolimus. The immunosuppressant may be administered at least about 1, 2, 4, 5, 6, 7, 8, 9 or 10 mg / kg. When immunosuppressants are used, they may be administered systemically or locally and may be administered before, simultaneously with or after administration of the pRPCs or photoreceptor cells. Immunosuppressive therapy may continue for weeks, months, years, or indefinitely after administration of the pRPCs or photoreceptor cells. For example, 5 mg / kg of cyclosporine may be administered to a patient for 6 weeks after administration of the pRPCs or photoreceptor cells.

[0141] Methods for treating retinal degeneration may include administering a single dose of PRPCs or photoreceptor cells. In addition, the methods of treatment described herein may include a course of treatment in which PRPCs or photoreceptor cells are administered multiple times over a period of time. Exemplary treatment courses may include weekly, biweekly, monthly, quarterly, semi-annual, or annual treatments. Alternatively, treatment may be staged, whereby multiple doses are initially administered (e.g., daily doses for the first week), with fewer and fewer doses required subsequently.

[0142] If administered by intraocular injection, PRPCs or photoreceptor cells can be delivered periodically once or more throughout the patient's life cycle. For example, PRPCs or photoreceptor cells can be delivered once a year, once every 6-12 months, once every 3-6 months, once every 1-3 months, or once every 1-4 weeks. Alternatively, more frequent administration may be required for certain situations or disorders. If administered by implant or device, PRPCs or photoreceptor cells can be administered periodically once or more throughout the patient's life cycle, depending on the needs of the specific patient and the disorder or condition being treated. Similarly envisioned are therapeutic regimens that change over time. For example, more frequent treatments (e.g., daily or weekly treatments) may be required at the beginning. Over time, as the patient's condition improves, less frequent treatments may be required, and no further treatment may even be required.

[0143] The methods described herein may further include the step of monitoring the efficacy of treatment or prevention by measuring the electroretinogram response, visual motion acuity threshold or brightness threshold in the subject. The method may also include monitoring the efficacy of treatment or prevention by monitoring the immunogenicity of the cells or the migration of the cells in the eye.

[0144] PRPC or PR can be used to manufacture a drug for treating retinal degeneration. The present disclosure also encompasses the use of a preparation comprising PRPC or PR in treating blindness. For example, a preparation comprising human PRPC or PR can be used to treat retinal degeneration associated with many vision-changing diseases that cause photoreceptor damage and blindness, such as diabetic retinopathy, macular degeneration (including age-associated macular degeneration, for example, wet age-associated macular degeneration and dry age-associated macular degeneration), retinitis pigmentosa and Stargardt disease (fundus flavimaculatus), night blindness and color blindness. The preparation can contain at least about 5,000-500,000 PRPC or PR (e.g., 100,00 PRPC or PR), which can be administered to retinal degeneration associated with many vision-changing diseases that cause photoreceptor damage and blindness, such as diabetic retinopathy, macular degeneration (including age-associated macular degeneration, retinitis pigmentosa and Stargardt disease (fundus flavimaculatus).

[0145] The PRPCs or PRs provided herein can be PRPCs or PRs. However, it should be noted that human cells can be used in human patients, as well as animal models or animal patients. For example, human cells can be tested in mouse, rat, cat, dog or non-human primate retinal degeneration models. In addition, human cells can be used therapeutically to treat animals in need thereof, such as in veterinary medicine.

[0146] The following examples illustrate the present disclosure and should not be construed as limiting the scope of the present disclosure.

[0147] Example

[0148] Example 1: Materials and Methods

[0149] Human induced pluripotent stem cell suspension culture

[0150] The human induced pluripotent stem cells (hiPSCs) used in this study were generated by using StemRNA TM -NM Reprogramming Kit (Stemgent, catalog number 00-0076) was used to generate hiPSCs from normal human dermal fibroblasts. 2 Routine growth as colonies on iMatrix-511 stem cell culture substrate (recombinant laminin-511) (ReproCell) and in NutriStem XF / FF TMhiPSCs were cultured in 1% paraformaldehyde (Bio-Industry) medium (Bio-Industry). hiPSCs were dissociated using Accutase (Innovative Cell Technologies) and transferred from traditional 2D monolayer cell culture to 3D spheroid culture in disposable spinner bottles (ReproCell) on nine-position stirring plates (Dura-Mag, ChemCell). Spheroid-adapted hPSCs were cultured at 0.5-1x10 6 Single cells were inoculated at a density of 10 cells / ml into a culture medium containing 10 μM Y27632 (ReproCell) 30 ml spinner flasks (ReproCell) were used. The stirring speed was adjusted to 50-80 RPM, depending on the hiPSC line. The medium without Y27632 was replaced once a day except for the first day after seeding when only half of the medium was replaced. When the sphere size was approximately 230-260 μm, the spheres were dissociated into single cells using Accutase and / or TrypLE and passaged every 4-5 days. The cell sphere culture was maintained at 5% CO 2 , 95% humidity, 37°C.

[0151] Neural Commitment and Photoreceptor Progenitor Differentiation of 3D-hiPSC Spheroids

[0152] After 3-5 generations of 3D culture expansion in spinner flasks, dissociated 3D-hiPSC spheres were plated at 1x10 6 After 24 hours, undifferentiated hiPSC spheroids in spinner flasks were directly used for differentiation, with an agitation speed of 50-80 RPM throughout the differentiation protocol. Figure 2 Figure A of lists all the culture medium compositions and factors. Briefly, cell spheroids were first patterned at D0 using the dual SMAD inhibitors SB431542 (1.5-15, Reagents Direct) and LDN193189 (0.25-2.5 μM, ReproCell) and IFG1 (2.5- 50 μg / ml, Peprotech). At D1, the Wnt inhibitor IWR-1e (0.25-10 μM, Sigma) and heparin (0.25 -15 μg / ml, Sigma) were added to the differentiation induction medium. Heparin was withdrawn at D9 of the neural commitment period, and IWR-1e was withdrawn at D11. All other factors were withdrawn at D15 of differentiation. For PRPC differentiation, from D2-D13, Pluriton was serially diluted with the above-mentioned induction factors. TM / No GF Gradually adapt spheroids to NIM-3D medium by adapting to NIM-3D and NIM-3D. At D18-D27, spheroids were adapted to PRPC-3D photoreceptor differentiation medium by 50 / 50 adaptation containing NIM-3D / PRPC-3D medium. From D27 onwards, spheroids were maintained in PRPC-3D medium. Medium was changed as follows: D0-D1: Pluriton TM / No GF D2-D5: 75% Pluriton TM / No GF NIM-3D; D6-D9: 50% Pluriton TM / No GF NIM-3D;D10-D13: Pluriton TM / No GF 25% - NIM-3D 75%; D13-D17: NIM-3D 100%; D18-D27: NIM-3D 50% - PRPC-3D 50%; starting from D27, PRPC-3D 100%.

[0153] NIM-3D (Neural Induction Medium-3D) basal medium consisted of DMEM / F12 with HEPES, 1% N2 and 1% B27 serum-free supplements (Thermo Fisher Scientific), 1% penicillin / streptomycin, MEM non-essential amino acids (Thermo Fisher Scientific), 0.30% glucose (Sigma), and Figure 2 All factors described in . PRPC-3D culture medium contains Neurobasal TM Culture medium, 1% N2 and 1% B27 serum-free supplement (Thermo Fisher Scientific), 1% penicillin / streptomycin, MEM non-essential amino acids (Thermo Fisher Scientific), 0.30% glucose (Sigma). Cells were cultured at 37°C and 5% CO. 2 Approximately 85% of the medium was replaced daily from D0 to D19 of neural differentiation and every 2-4 days after D19.

[0154] During the differentiation process, spheres were dissociated into single cells using TrypLE (Thermo Fisher Scientific) at different time points, and RNA was extracted for qRT-PCR analysis. Other cells were processed for flow cytometry analysis and immunofluorescence staining. When spheres typically reached 350-450 μm in diameter, pRPCs were generated at D19, D30-D32, D50-D52, and D80-82 by serially dissociating spheres into single cells and reaggregating them into 30 ml spinner flasks to avoid generating hypoxic cells in the center of the spheres. Briefly, the dissociation / reaggregation process involved collecting all spheres into a 50 ml conical tube, followed by washing once with PBS and incubating with TrypLE in a 37 °C water bath for 30-60 min. Cells were gently triturated into single cells to ensure a homogenous single-cell suspension, which was then filtered through a 40 μm filter. Single cells were centrifuged at 0.5–1x10 6 The spheroids were seeded at a density of 10 μM Y27632 / ml in 30 ml spinner flasks in the appropriate medium. The morphology (size, appearance and ability to incorporate spheroids) of the resulting spheroids was monitored 2-3 days after each reaggregation and every week thereafter until the next dissociation / resaggregation step.

[0155] For neural rosettes selection, additional sphere attachment / manual scraping steps were introduced at D5 / D12 and D11 / D18 of neural differentiation. In brief, 3 ml sphere suspension cultures at D5 and D11 were attached to 3 Matrigel (Corning) coated wells (in 6-well plates) and kept in the same culture medium for continuous differentiation for 7 days, when neural rosettes were formed in the center of the attached spheres. After one week of cultivation, cells were manually lifted with a scraper (Corning) on ​​the 12th and 18th days, respectively, and inoculated in ultra-low attachment wells at a ratio of 1: 1 to form suspended spheres under static conditions. At D30-32 of differentiation, static spheres were dissociated into single cells and reaggregated in 30 ml spinner bottles, and then cultured using a similar scheme to our continuous dissociation / reaggregation process.

[0156] Freezing and thawing of early and late CRNPs and PRPCs

[0157] hiPSC-derived retinal progenitor cells at different stages of in vitro differentiation were obtained by suspension culture dissociated in 30 ml spinner bottles (early CRNPs collected at D19, late CRNPs collected at D30-D34, and PRPCs collected at D50-D120 days). The cells were separated into single cells and cryopreserved in CryostorCS10 (Sigma) freezing medium supplemented with 10 μM Y-26632 using a rate-controlled freezer, wherein in 1 ml aliquots, 40-50 million cells / bottle for early and late CRNPs, and 5-10 million cells / bottle for PRPCs. The ability of cells to recover / reaggregate in culture and the viability after cryopreservation were detected. Vials from 2 different differentiations were thawed, and the total viable cells were counted to determine recovery and viability. Cell viability averaged 80-90% when thawed, and the recovery rate was 60-80%. Placing thawed single cells into spinner flasks retained the ability to reaggregate, with sphere sizes ranging from 100-150 μm within 2-4 days after thawing, which is comparable to serial dissociation / resaggregation. Flow cytometric analysis of spheres at different days post-thawing showed that the percentage of cells expressing PAX6 / SOX2 was similar to spheres formed after dissociation and reaggregation from the initial suspension culture. qPCR analysis also confirmed similar levels of marker expression, further demonstrating the feasibility of cryopreservation and storage of large numbers of cells for future applications.

[0158] Immunocytochemistry

[0159] PRPC spheres were dissociated into single cells using TrypLE and cultured at 5.0 × 10 4 Cells / wells were seeded in vitro on Matrigel-coated 24-well plates for 10-20 days in PRPC-3D culture medium. For differentiated D13, the entire attached sphere was used for staining. The culture medium was removed, and the cells were washed 3 times with DPBS (Thermo Fisher Scientific) with Ca / Mg, then fixed with 4% PFA (paraformaldehyde) (Electron Microscopy Sciences) at room temperature for 15 minutes, and then washed 3 times with DPBS. The cells were infiltrated and blocked with a DPBS solution of 5% normal donkey serum (NDS) (Jackson Immunolaboratory) and 0.1% Triton X-100 (Sigma) at room temperature for up to 1 hour, and then incubated overnight at 4°C with a primary antibody diluted in blocking buffer. Table 1 summarizes the primary antibodies and their dilutions and sources for staining. After overnight incubation with antibodies, cells were washed 3 times with DPBS and then incubated for 2 h at room temperature in the dark with the appropriate species-specific fluorescently conjugated secondary antibodies diluted in DPBS containing 2.5% NDS and 0.1% Triton X-100: donkey anti-mouse Donkey anti rabbit Conjugated secondary antibodies [1:100] (Thermo Fisher Scientific). Table 2 lists the secondary antibodies used for immunostaining. Wash cells 3 times with DPBS and counterstain nuclei with 1 μg / m 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) (Thermo Fisher) for 3 minutes at room temperature, followed by DPBS washing. Cells were examined using a computer-assisted Nikon inverted microscope (Eclipse Ti-S) with 4x, 10x and 20x objectives, and images were captured and analyzed using NIS-Elements-BR software (version 4.50, Nikon).

[0160] Immunohistochemistry

[0161] After implantation in OCT.compound (Scigen), D120 spheres were sliced ​​at 10-12 μm in a cryostat (Leica CM 1950). For staining, slides stored at -80°C were air-dried for 1 hour at room temperature, fixed in cold 4% PFA for 15 minutes, and then washed 3 times with DPBS for 3 minutes each. Primary and secondary antibody blocking and incubation were performed directly on the sections. Slides were fixed with DAPI (Southern Biotech) containing Fluorogold-G using coverslips, dried at room temperature, and images were acquired as described above.

[0162] Flow cytometric analysis

[0163] Spheroids were dissociated into single cells using TrypLE (Thermo Fisher Scientific), filtered through a 40 μm filter, and fixed with fixation / permeabilization buffer (BD Biosciences) for 12 minutes on ice. For flow cytometry using fluorescently conjugated antibodies to detect intracellular antigens, fixed 1.0-2.0×10 5The cells / tubes were permeabilized on ice for 30 minutes with ice-cold 1X BD Perm / Wash buffer containing FBS and saponin (BD Biosciences) and then incubated with appropriately conjugated conjugated antibodies (summarized in Table 1) for 30 minutes in the dark. The cells were then washed with 2 ml of Perm / Wash buffer and prepared for analysis. Control cells were incubated with mouse or rabbit IgG. For flow cytometry using unconjugated antibodies to detect intracellular antigens, fixed cells were blocked on ice for 30 minutes with a blocking buffer consisting of 0.05% Triton X-100 (Sigma) and 5% normal donkey serum (NDS) (Jackson Immuno Research) in DPBS (Life Technologies) (Life Technologies) and then incubated with primary antibodies diluted in blocking buffer for 1 hour at room temperature and then washed with blocking buffer. The cells were then incubated with appropriate donkey anti-rabbit diluted in blocking buffer [1:1000] (Invitrogen) or donkey anti-mouse The cells were incubated with conjugated secondary antibodies (Invitrogen) for 1 hour in the dark. After washing, the cells were resuspended in blocking buffer. Control cells were incubated with secondary antibodies only. Cell analysis was performed on an Accuri C6 flow cytometer (BD Life Sciences) according to standard procedures. Data were analyzed using BD Accuri C6 Plus software (BD).

[0164] Quantitative real-time polymerase chain reaction (qPCR)

[0165] Total RNA was isolated from cultured cells using the RNeasy Minikit (Qiagen) and the concentration was measured using NanoDropOne (Thermo Fisher Scientific). qPCR was performed in a two-step reaction. For reverse transcription (RT), 0.5 μg of total RNA was transcribed into cDNA using the SuperScript OSM-IV VILO Master Mix cDNA Synthesis Kit (Invitrogen) according to the manufacturer's instructions using a SympliAmp Thermal Cycler (Applied Biosystems). For qPCR reactions, a QuantStudio RT-PCR system with a 96-well plate was used. TM15 ng of cDNA was amplified in a 20 μl reaction mixture containing TaqMan Gene Expression Assays and TaqMan Fast Advanced Master Mix (Applied Biosystems) using the 6Flex Real-Time PCR System. Table 3 lists all TaqMan Gene Expression Assays (TaqMan probes) used in the experiments. In all experiments, the housekeeping gene GAPDH was used as an internal control for data normalization. (-ΔΔCT) Methods

[31] , QuantStudio real-time PCR v1.3 software was used to analyze the relative quantitative data of each target gene, and induced iPSCs were used as a reference control. Samples were set up in triplicate and collected from 3 independent differentiations.

[0166] Example 2: HiPSC spheroid culture

[0167] In order to practically apply hPSCs in cell culture, it is necessary to culture them on a large scale and in 3D. hiPSCs cultured in feeder-free 2D monolayers in culture medium ( Figure 1 , Figure A) Scheme for transition to 3D dynamic suspension culture, in which hiPSCs are grown as homogeneous spheres in spinner flasks supplemented with the small molecule Y27632 and without feeder cells and matrix. Continuous culture medium ( Figure 1 , Figure B) [25, 32-38]. Using this suspension culture system, hPSC cultures can be serially passaged and continuously expanded for at least 10 generations. The typical passage interval of 3D hiPSC spheres is as follows Figure 1 As shown in Figure B, spheres with a size of 230-260 μm were dissociated into single cells using Accutase and reaggregated in a spinner bottle under continuous stirring at 60-70 RPM to reconstitute the spheres. Flow cytometry analysis demonstrated that after 3-5 repeated passages, the spheres gradually increased in size while maintaining a uniform structure and high expression of pluripotency markers (OCT4: 98.8%) and a normal karyotype. Our 3D-hPSC passaging method is widely applicable as it has been successfully applied to all routinely tested hPSC lines in our laboratory.

[0168] Example 3: Effective induction of retinal neuronal differentiation of 3D-hiPSC spheres using small molecules

[0169] To generate retinal neuronal progenitors at different developmental stages from hiPSCs, we developed a novel method in which 3D-hiPSC spheroids in suspension were directly induced in a stepwise manner using mainly small molecules. Figure 2). This novel protocol in 3D spinner flasks integrates continuous 3D spheroid culture with multiple dissociation / reaggregation steps using small molecules to induce retinal neuronal differentiation. Unlike previously reported methods that used protein factors to induce hPSC differentiation toward the retinal lineage [2, 8, 10, 11, 16, 21, 22], we identified and primarily used small molecules whose quality was easily controlled to sequentially differentiate hPSC spheres into retinal cells at different developmental stages. Cells were first cultured as single cells (1x10 6 cells / ml) were inoculated in a spinner flask supplemented with Y27632 culture medium to form spheres. 24 h later (designated as D0 of induction), hiPSC spheres were first treated with the dual SMAD inhibitors SB431542 and LDN193189 to block activin / transforming growth factor b (TGF-β) and bone morphogenetic protein (BMP) signaling and promote neural patterning

[39] , and then treated with the Wnt inhibitor IWR-1e [10, 11, 24], IGF1 (an inducer of eye field cell development), and heparin to further induce retinal neural lineage commitment

[16] . It has been demonstrated that manipulation of Wnt signaling with the small molecule IWR-1e at the early stage of neural induction has been reported to effectively induce hPSC differentiation into early photoreceptor progenitors in adherent and retinal organoid suspension cultures

[23] . Based on the results of the four hiPSC lines, the size of the spheres formed within 24 h was 100 μm-150 μm, and the optimal size was 110 μm-125 μm ( Figure 3B As differentiation progressed from D0 to D19, hiPSC spheres maintained a uniform size while growing and expanding, and gradually acquired neural characteristics without dissociation. By D3-D5 of differentiation, the spheres began to change their appearance, with more irregular edges; by D19, these irregular edges disappeared completely, and the spheres became translucent and smooth, which is typical for neurospheres. During the differentiation process, we observed a gradual increase in cell number, with cell numbers of 1.5-2.5X 10 at D9 when the sphere size was 250-300μm in diameter. 8 cells (5-8 times), and continued to increase to about 3.0–4.5X 10 8 Cells (10-15 times, Figure 3C ) The diameter of the sphere reaches 400 μm ( Figure 3A ).

[0170] Example 4: Kinetic analysis of retinal neuronal differentiation of 3D-hPSC spheroids

[0171] The retinal differentiation kinetics and neural induction efficiency were examined by flow cytometry analysis to monitor the appearance of paired box 6 gene (PAX6) and sex determining region Y-box 2 (SOX2) positive cells and PAX6 / SOX2 double positive cells. The pluripotency octamer binding transcription factor 4 gene (OCT4), which is known to be highly expressed in hPSCs, was detected at a high level (98%) at D0 before neural induction, while during differentiation, it was gradually downregulated to about 50% at D2, about 5% at D3, and completely shut down to 0% at D4 ( Figures 4A-4C SOX2 (a marker of pluripotent and neural stem cells) was highly expressed in hPSCs at D0 and in cells after neural induction (until D19). PAX6-positive cells and PAX6 / SOX2 double-positive cells, representing early committed retinal progenitor cells (CRNPs), appeared at D3 (about 10%) and gradually upregulated from D3 to D4 (about 77%). In the next 1-3 days, PAX6-positive and PAX6 / SOX2-positive cells increased dramatically from D5 to D7 (≥90%, Figures 4A-4C ). 3D spheroid cultures with at least about 90% of cells positive for PAX6 and PAX6 / SOX2 were considered successfully induced.

[0172] It has been reported that different hPSC lines may differ in lineage-specific differentiation and require optimization of differentiation conditions. Using the above protocol, we tested our neural differentiation conditions with 4 different hiPSC lines. In summary, each undifferentiated hPSC line was adapted to suspension culture in 30 ml spinner flasks and spheres of approximately 100-150 μm in size were used for retinal neural differentiation. Figure 2 The optimal concentrations for the time points indicated in Figure A were 10 μM SB431542, 1 μM LDN193189, 10 ng / ml IGF1, IWR-1e (2 μM) and heparin 2 μg / ml), and all hiPSC lines generated PAX6+ and PAX6+ / SOX2+ double positive cells ( Figure 4A ). We also noticed that hiPSC line #2 had slightly different kinetics compared to the other 3 lines, and while OCT4 expression was completely shut down in a similar manner to the other lines, the PAX6 population never reached >90% at D5-D7 by flow cytometry analysis, suggesting that there is variability in neural induction between different cell lines and that the quality of different hiPSC lines needs to be tested before starting large-scale cell production for future cell replacement therapies. Nonetheless, these results suggest that our small molecule cocktail and neural induction medium effectively and consistently induce 3D-hPSC spheroids toward the neural lineage without disrupting the spheroids.

[0173] To further characterize cells derived from the above step-wise differentiation protocol, we seeded D11 spheroids on Matrigel-coated wells, cultured for another 2 days, and examined the expression of PAX6, SOX2, and nestin (NES), as well as RAX1, a retinal-specific transcription factor expressed in early retinal progenitor cells. These attached retinal spheroids showed an arrangement similar to neural rosettes, and the cells were double positive for PAX6-RAX1 and NES-SOX2 ( Figure 5A ), which further confirmed their retinal lineage. Quantitative real-time PCR gene expression analysis of spheres performed at different time points during early differentiation showed that the OCT4 gene was completely turned off at D5 ( Figure 5B ), which confirmed our flow cytometry results and further demonstrated the absence of PSCs in our 3D spheroid cultures. In contrast, the expression of the neural marker PAX6 increased from D5 and continued during the early neural differentiation period at D19, and then gradually downregulated at D34-D40 ( Figure 5B ), which coincides with the onset of differentiation into more mature retinal cells. RAX1 expression at D5 indicated the emergence of early CRNPs, followed by high levels of Ceh-10 homology domain containing homeotic gene (CHX10), a marker for retinal progenitor cells, at D13, suggesting the sequential emergence of different developmental retinal cell types in our 3D spheroid cultures, recapitulating the temporal development of retinogenesis observed in vivo. Our results, together with previous reports [9, 13, 40-42], suggest that early retinal progenitor fate regulation occurs in our 3D spheroid culture system at approximately 5-13 days, as Figure 2 We named the cells at D5-D13 as early committed retinal neuron progenitor cells (early C ommitted R etinal N euron P rogenitor, CRNP), while cells at D13-D40 were named late CRNP.

[0174] Example 5: Generation of Photoreceptor Precursor Cells from 3D CRNP Spheroids

[0175] Although previous studies have reported that the generation of photoreceptor progenitors from hPSCs in 2D and 3D retinal organoids has varying degrees of efficiency [9, 11, 15, 16, 18, 21, 43, 44], we also investigated whether our differentiation protocol could efficiently generate postmitotic photoreceptor progenitors and photoreceptor-like cells. Figure 5A-5B), when the diameter of the continuously differentiated 3D spheroids reached approximately 400 μm, the spheroids were first dissociated into single cells and cultured in a 30 ml spinner flask with neural differentiation medium ( Figure 6 )0.5-1x10 6 The cells were reaggregated at a density of 10 cells / ml to reconstitute spheres. The cells were allowed to reaggregate immediately and completely into spheres with neurosphere morphology (bright in phase contrast, translucent and with small micro-protrusions around the sphere). As early as one day after reaggregation, the sphere size was approximately 110 μm ( Figure 6 , Figure A). Under differentiation conditions, spheroids were observed to gradually and continuously grow to about 400 μm from about D14 to about D30, at which time the spheroids were again dissociated into single cells and reaggregated in a spinner bottle. A similar dissociation / reaggregation process was repeated at D50-52, D80-82, and D100-102 (Figure 6, Figure A). Similar spheroid formation and growth patterns were observed in each dissociation / reaggregation cycle, and from the initial 3×10 7 hiPSCs to about 3.0-4.5X 10 at D100 9 Retinal neuronal progenitor cells, achieving an approximately 100-fold increase in cell number, which is far more efficient than previously reported ( Figure 6 , Figure B)

[0176] To examine the cellular composition of spheroids, we cultured spheroids at D32 and D82 ( Fig. 7A Dissociated cells were plated at the dissociation / reaggregation time point and cultured in vitro for approximately 1-3 weeks. Morphological characterization of attached single cells cultured under retinal differentiation conditions revealed neuronal connectivity similar to that of photoreceptor progenitors in vitro ( Fig. 7A). To further identify the true identity of these cells, we examined the expression of multiple photoreceptor progenitor-specific markers by immunofluorescence cytochemical analysis. Our results clearly showed that these cells expressed high levels of cone-rod homeobox (CRX), neural retinal leucine zipper (NRL), and thyroid hormone receptor-β2 (ThRB2), key transcription factors that are essential for D100 development and photoreceptor fate regulation, after multiple rounds of dissociation / reaggregation (Figure 7B). However, only a small number of Ki67+ proliferating cells were detected in these spheres, indicating that postmitotic retinal neurons were effectively generated in this 3D sphere protocol. In addition, in these cells, microtubule-associated protein 2 (MAP2, green), a general marker of relatively mature neuronal cells, was highly expressed, whereas glial fibrillary acidic protein (GFAP, red), a marker of astrocytes and / or Müller glia, was rarely detected, indicating a nearly homogeneous population of neurons expressing photoreceptor progenitor-specific markers, which we termed postmitotic retinal neurons (PRNs). P hoto R eceptor P recursor C We also examined the expression of D100 mature photoreceptor cell markers, and our results showed that the majority of these cells expressed the rod visual pigment protein rhodopsin and the neuronal calcium-binding protein recoverin (REC), both markers of rod rhodopsin ( Figure 7C ).

[0177] Efficient differentiation into PRPCs and photoreceptor cell-like cells was also confirmed by flow cytometry analysis at D80. The results showed that cells expressing the pluripotent gene OCT4 were not detected, and more than 90% of the cells expressed PRPCs and photoreceptor markers (CRX, 95.2%; NRL, 96.6%; NR2E3, 91.3%, REC, 96.8%, cone-specific opsin red / green M-opsin, 91.2%, Fig. 8A ). To further characterize the expression dynamics of these genes during differentiation, we performed RT-qPCR analysis. These analyses further revealed a gradual increase in PRPC marker genes such as NRL, nuclear receptor subfamily 2, group E member 3 (NR2E3), and ThRβ2 starting from D40 of differentiation ( Figure 8B ). Similarly, the expression kinetics of photoreceptor markers REC, RHOD, and M-opsin showed the same trend, but the expression of M-opsin was only detectable at D70 ( Figure 8B). However, PAX6, RAX1, and CHX10 genes (all markers of early and late CRNP cells) were significantly downregulated, such as Figure 5B As shown. The concomitant downregulation of early retinal neuronal genes and upregulation of late retinal neuronal markers in these cells indicated that these cells were at the developmental stage of PRPCs and photoreceptors. Therefore, we named cells that differentiated for more than 80 days (D80) as photoreceptor-like cells. The same analysis was performed on three other hiPSC lines (data not shown), demonstrating the reproducibility and consistency of this 3D sphere differentiation system. Karyotype analysis of hiPSC-derived PRPCs showed good genetic stability after long-term 3D sphere culture by repeated dissociation / reaggregation steps (data not shown). After cryopreservation in liquid nitrogen, the viability of spheres and single cells derived from these spheres was tested, and ≈80% of living cells were recovered from cryopreserved early and late CRNPs and PRPCs (data not shown).

[0178] To further characterize the cells in the differentiated spheroids, we embedded D120 spheroids in OCT and sectioned them to examine general neuronal and retinal neuron-specific markers ( Fig. 9 Qualitative assessment of spheroid morphology and hematoxylin staining revealed intact cells without necrotic cores across the entire cross-sectional surface of the spheroids, further demonstrating that the spheroids were properly supplied with oxygen and nutrients and that metabolic wastes were transported into the culture medium during extended suspension culture ( Fig. 9 MAP2 is expressed at high levels in a compact manner throughout the slice ( Fig. 9 , Figure C), as well as high percentages of cells expressing the PRPC-specific markers CRX and NRL, and very low numbers of Ki67+ proliferating cells further confirmed the efficient generation of PRPCs in these spheroids ( Fig. 9 , Figure D). More mature rod photoreceptors are abundant at D120, as indicated by widespread expression of RHOD and REC in spheres organized within recognizable structures of the neuroepithelial cells ( Fig.10 ). Taken together, these results demonstrate that our culture system supports the robust generation of large numbers of highly pure and homogeneous pRPCs from hiPSCs through the combined effects of small molecules, dissociation / reaggregation, and a stimulating microgravity-enhanced microenvironment under continuous agitation.

[0179] Table 1: List of unconjugated primary antibodies for flow cytometry and immunofluorescence

[0180]

[0181]

[0182] Table 2: List of conjugated antibodies for flow cytometry

[0183]

[0184] Table 3: List of TaqMan assays for qPCR

[0185] Gene name TaqMan Assay ID Number ARR3 Hs01020134_m1 ASCL1(MASH1) Hs00269932_m1 CRX Hs00230899_m1 GAPDH Hs02786624_g1 NR2E3 Hs00183915_m1 NRL Hs00172997_m1 OCT4 Hs04260367_gH OPN1MW (opsin-M) Hs04194752_g1 OPN1SW (opsin-S) Hs00181790_m1 PAX6 Hs01088114_m1 PDE6a Hs00166495_m1 RAX Hs00429459_m1 RCVRN (recovery protein) Hs00610056_m1 RHO (rhodopsin) Hs00892431_m1 THRB(ThRB2) Hs00230861_m1 VSX2(Chx10) Hs01584046_m1

[0186] Example 6: Use of Sonic Hedgehog

[0187] Fig.11 This is an overview of the neural induction protocol for deriving retinal neural progenitor cells from human induced pluripotent stem cells. Retinal progenitor cells were efficiently generated using a small molecule in combination with sonic hedgehog (SHH). Note that rh-SHH refers to recombinant human SHH.

[0188] After 3-5 generations of 3D culture expansion in spinner flasks, dissociated 3D-hiPSC spheres were plated at 1x10 6 After 24 hours, undifferentiated hiPSC spheroids in spinner flasks were directly used for differentiation, with an agitation speed of 50-80 RPM throughout the differentiation protocol. Fig.11 All media compositions and factors are listed. Briefly, at D0, spheroids were first patterned using dual SMAD inhibitors SB431542 ("SB", 1.5-15, Reagents Direct) and LDN193189 ("LDN", 0.25-2.5 μM, ReproCell) and IFG1 (2.5-50 μg / ml, Pipetech). At D1, differentiation induction medium Pluriton TM Wnt inhibitor IWR-1e (0.25-10 μM, Sigma) was added. At D3, rh-SHH (0.5-20 nM, Sigma) was added. rh-SHH was withdrawn at D9 for neural commitment, while IWR-1e was withdrawn at D11. All other factors were withdrawn at D15 of differentiation. For PRPC differentiation, from D2-D13, Pluriton was serially diluted with the above induction factors. TM / No GF Gradually adapt spheroids to NIM-3D medium by adapting to NIM-3D and NIM-3D. At D18-D27, spheroids were adapted to PRPC-3D photoreceptor differentiation medium by 50 / 50 adaptation containing NIM-3D / PRPC-3D medium. From D27 onwards, spheroids were maintained in PRPC-3D medium. Medium was changed as follows: D0-D1: Pluriton TM / No GF D2-D5: 75% Pluriton TM / No GF NIM-3D; D6-D9: 50% Pluriton TM / No GF NIM-3D; D10-D12: Pluriton TM / No GF 25% - NIM-3D 75%; D13-D18: NIM-3D 100%; D19-D21: NIM-3D 50% - PRPC-3D 50%; starting from D22, PRPM-3D 100%.

[0189] NIM-3D (Neural Induction Medium-3D) basal medium consisted of DMEM / F12 with HEPES, 1% N2 and 1% B27 serum-free supplements (Thermo Fisher Scientific), 1% penicillin / streptomycin, MEM non-essential amino acids (Thermo Fisher Scientific), 0.30% glucose (Sigma), and Fig.11 All factors described in PRPM-3D medium contain Neurobasal TM Culture medium, 1% N2 and 1% B27 serum-free supplement (Thermo Fisher Scientific), 1% penicillin / streptomycin, MEM non-essential amino acids (Thermo Fisher Scientific), 0.30% glucose (Sigma). Cells were cultured at 37°C and 5% CO 2 Approximately 85% of the culture medium was replaced daily from D0 to D19 of neural differentiation, and every 2-4 days after D19.

[0190] On the 7th day of differentiation, compared to the control treated with heparin, the culture treated with SHH showed a higher percentage of cells expressing PAX6 (transcription factor necessary for retinal neurogenesis). On the 19th day, relative to the control, the culture treated with SHH showed that the cells expressing PAX6 increased by more than 10 times. Contrary to the SHH conditions that showed a stable reduction in PAX6 expression from the 7th day to the 21st day, the control showed a sharp reduction in the cells expressing PAX6 to the 9th day, which may indicate that the cell population has adapted to the alternative cell fate mainly from the inner nuclear layer (INL) such as bipolar and amacrine cell types. In addition, the RT-PCR quantification of neurons and retina-specific markers in the heparin-treated control was not quite consistent in multiple independent retinal progenitor cell differentiation. These results show that, relative to other mitogen-activated proteins, such as heparin, SHH is a more effective alternative in achieving retinal progenitor cells for neuronal induction and proliferation.

[0191] Fig.12Comparative RT-PCR quantification of PAX6 mRNA gene expression in differentiated cells treated with heparin or SHH is shown. Total RNA was collected from cells in both conditions during the photoreceptor differentiation timeline and analyzed for PAX6 RNA transcript levels.

[0192] Heparin-treated controls showed a slow and gradual increase in PAX6 mRNA gene expression from D0 to D20, indicating that hiPSC neural induction efficiency was low. In contrast, the SHH-treated group showed a significant and robust increase in PAX6 RNA levels by day 5 relative to the control, and maintained high levels until day 19. By day 5, the PAX6 gene expression level of SHH-treated cells was more than 4 times higher than that of control cells. These results indicate that SHH is more effective than heparin in inducing hiPSCs toward the neural lineage and maintaining neurogenesis of PAX6-positive cells for the first 19 days of in vitro differentiation.

[0193] Example 7: Improved culture medium promotes maturation toward photoreceptor cell fate

[0194] To promote the survival and maturation of precursor cells and early photoreceptor-like cells, we optimized our maturation medium to robustly determine rod and cone photoreceptor fate. On differentiation day 99, cell culture was switched to Neurobasal medium containing 1% glutamine, 1% penicillin / streptomycin, human brain-derived neurotrophic factor (BDNF) (20 ng / mL), ascorbic acid (0.2 mM), and DAPT (1 μM). TM Culture medium in the culture medium. Fig.13 As shown, the above-defined maturation medium increased rod (RHO) and cone (ThRβ2) markers by 20% and 10%, respectively, compared to the control. The photoreceptor precursor marker recovery protein was about 9% higher than the control. Fewer nestin-positive cells were also observed in the modified maturation medium, indicating that most cells underwent differentiation and exited the neural stem cell state. These immunocytochemical data indicate that the modified medium promotes the maturation of photoreceptor cells compared to the control medium.

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[0242] Improvements and variations to the methods and compositions described in the present disclosure will be apparent to those skilled in the art without departing from the scope and spirit of the present disclosure. Although the present disclosure has been described in conjunction with specific preferred embodiments, it should be understood that the present disclosure should not be unduly limited to these specific embodiments as described in the claims. Indeed, various modifications to the described manner for carrying out the present disclosure are intended and understood by those skilled in the relevant art to which the present disclosure belongs, and these modifications fall within the scope of the present disclosure as represented by the appended claims.

[0243] Incorporation by Reference

[0244] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each individual patent or publication was specifically and individually indicated to be incorporated by reference.

Claims

1. A method for producing photoreceptor precursor cells in vitro, comprising: (a) culturing a plurality of pluripotent stem cells in a three-dimensional (3D) spheroid culture medium for differentiating committed retinal neural progenitor cells (CRNP) to generate a plurality of first spheroids, the plurality of first spheroids comprising early and late ocular committed retinal neural progenitor cells, wherein the neural induction medium in step (a) comprises DMEM / F12 with HEPES, N2 and B27 serum-free supplements, supplemented with one or more of the following: sonic hedgehog, heparin, IWR-1e, SB431542, LDN193189 and IGF1; (b) monitoring the spheroid size until the first spheroids reach an average size of 300-500 µm in diameter; (c) dissociating the first spheres into a first plurality of elementary single cells when the diameters of the first spheres reach an average size of 300-500 μm; (d) 3D spheroid culturing the first plurality of elementary single cells in a photoreceptor differentiation medium for differentiating CRNPs into photoreceptor precursor cells (PRPCs) to generate a plurality of second spheroids, wherein the plurality of second spheroids comprises photoreceptor precursor cells, wherein the photoreceptor differentiation medium in step (d) comprises Neurobasal medium, N2 and B27 serum-free supplements; (e) monitoring the spheroid size until the second spheroids reach an average size of 300-500 µm in diameter; (f) dissociating the second spheres into a second plurality of elementary single cells when the diameters of the second spheres reach an average size of 300-500 µm; (g) 3D spheroid culturing the second plurality of elementary single cells in a maturation medium for differentiating pRPCs into post-mitotic pRPCs to generate a plurality of third spheroids, wherein the plurality of third spheroids comprises post-mitotic pRPCs, wherein the maturation medium of step (g) comprises Neurobasal medium comprising L-glutamine, human brain-derived neurotrophic factor (BDNF), ascorbic acid and DAPT (N-[N-(3,5-difluorophenylacetyl)-l-alanyl]-S-phenylglycine tert-butyl ester).

2. The method of claim 1, wherein: The pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells.

3. The method of claim 1, wherein: Steps (a), (d) and (g) involve culturing in a spinner flask or stirred tank bioreactor.

4. The method of claim 1, wherein: The neural induction medium in step (a) is NIM-3D (Neural Induction Medium-3D) basal medium, which comprises DMEM / F12 with HEPES, N2 and B27 serum-free supplements, penicillin / streptomycin, MEM non-essential amino acids and glucose, supplemented with one or more of the following: Sonic Hedgehog, heparin, IWR-1e, SB431542, LDN193189 and IGF1.

5. The method of claim 4, further comprising providing SB431542, LDN193189, and IGF1 for a first time period, providing IWR-1e for a second time period, the second time period being shorter than the first time period, and providing Sonic Hedgehog or heparin for a third time period, the third time period being shorter than the second time period.

6. The method of claim 5, wherein: The first time period is 10-20 days.

7. The method of claim 5, wherein: The second time period is 5-15 days.

8. The method of claim 5, wherein: The third time period is 3-12 days.

9. The method of claim 1, wherein: In step (b), the diameter of the first spheres reaches an average size of 350-450 µm.

10. The method of claim 1, wherein: In step (b), the diameter of the first spheres reaches an average size of less than 400 μm.

11. The method of claim 1, wherein: Steps (c) and (f) include contacting the first spheroid and the second spheroid, respectively, with a cell dissociation enzyme.

12. The method of claim 1, wherein: The photoreceptor differentiation medium in step (d) is pRPC-3D medium, which contains Neurobasal medium, N2 and B27 serum-free supplements, penicillin / streptomycin, MEM non-essential amino acids, and glucose.

13. The method of claim 1, further comprising: step h) differentiating the post-mitotic PRPCs into photoreceptor-like cells, wherein the maturation medium in step (h) comprises Neurobasal medium comprising L-glutamine, human brain-derived neurotrophic factor (BDNF), ascorbic acid and DAPT (N-[N-(3,5-difluorophenylacetyl)-l-alanyl]-S-phenylglycine tert-butyl ester).

14. The method according to claim 1 or 13, wherein: The maturation medium in step (g) and / or (h) is Neurobasal medium containing L-glutamine, penicillin / streptomycin, human brain-derived neurotrophic factor (BDNF), ascorbic acid and DAPT (N-[N-(3,5-difluorophenylacetyl)-l-alanyl]-S-phenylglycine tert-butyl ester).

15. The method of claim 1 or 13, wherein: Steps (g) and / or (h) further include monitoring the spheroid size until the diameter is 300-500 µm; dissociating the third sphere into a third plurality of elementary single cells; and reaggregating the third plurality of elementary single cells.

Images