Methods for the production of Müller cells and cell products

Abstract

The present invention relates to a novel process for producing therapeutic GMP-grade Müller cells derived from stem cells using animal-derived component-free products, and the Müller cells obtainable therefrom. The Müller cells are suitable for treating ocular diseases, including glaucoma. A cell culture medium is also provided.

Description

[Technical Field]

[0001] This invention relates to a novel process for producing therapeutic GMP-grade Müller cells derived from stem cells using a product free of animal-derived components, and to Müller cells obtained therefrom. The Müller cells are suitable for the treatment of eye diseases, including glaucoma. Cell culture media are also provided. [Background technology]

[0002] Glaucoma is the most frequent cause of irreversible blindness worldwide, and is estimated to affect approximately 111,000,000 people by 2040. Retinal ganglion cell (RGC) loss is a characteristic feature of ophthalmic neuropathy, including glaucoma, where damage to RGC axons occurs at the level of the optic nerve head (ONH). Under normal conditions, RGCs receive visual signals from photoreceptors through two anterior layers of neuronal cells (bipolar and non-axonal cells) and transmit this information to the brain through axons exiting the eyeball via the ONH and optic nerve. Damage to RGCs results from physical and molecular mechanisms, including mechanical compression, reduced paracrine neurotrophic factor support, glial activation, oxidative stress / reduction in the antioxidant defense system, immune system dysregulation, and mitochondrial dysfunction / metabolic deficiencies. These established mechanisms of RGC dysfunction, along with evidence emerging from the literature, indicate underlying multifactorial, metabolic deficiencies that lead to loss of RGC function and subsequent RGC degeneration and death in glaucoma. Currently, approved treatments for glaucoma aim to slow the progression of the disease, but ultimately these treatments do not stop the ongoing damage to the RGC, and therefore the disease still progresses, with many patients losing sight in one or both eyes.

[0003] Several studies highlight the importance and crucial role of Müller cells in providing functional and metabolic support to RGCs. Under homeostatic conditions, Müller cells provide numerous beneficial functions, including: supplying nutrients and protection against the neurotoxic glutamate; ion and water homeostasis; buffering mechanical stimuli; structural stabilization of the retina; modulation of immune and inflammatory responses; antioxidant production; glucose metabolism; exhibiting considerable metabolic activity / support, containing a significant number and large number of mitochondria; and promoting considerable neuroprotective levels of adenosine triphosphate (ATP), all of which are disrupted in glaucoma.

[0004] Based on the known role of Müller cells, it is expected that Müller cell therapy will promote the repair, survival, and function of RGCs, and therefore improve visual function for patients suffering from optic neuropathy.

[0005] To evaluate this hypothesis, several studies were conducted in relevant glaucoma-like animal models using Müller cells generated from different sources, including human adult cadaveric donor retina (Singhal et al, Stem Cells Translational Medicine, 2012); cat donor sources (Becker et al, Stem Cells Translational Medicine, 2016); and human iPSC lineage (Eastlake et al, Stem Cells Translational Medicine, 2019). Each of these pharmacological studies showed that single intravitreal administration of Müller cells could significantly improve RGC function.

[0006] The generation of embryoid bodies and the formation of retinal organoids were initially based on Nakano's protocol. Nakano et al (Cell Stem Cell 10, 771-785, June 14, 2012) found that fetal bovine serum (FBS) is an effective enhancer for retinal differentiation of stem cells, but fetal bovine serum and other animal-based products are not acceptable for cell-based therapeutics administered to humans.

[0007] Therefore, there is a global challenge to develop methods for culturing and differentiating Müller cells in accordance with Good Manufacturing Practice (GMP) standards for clinical application without negatively impacting the scalability, yield, morphology, or biological properties of the cells. This invention provides an improved method for culturing GMP-compliant stem cell-derived Müller cells suitable for cell therapy, and Müller cells derived therefrom. [Brief explanation of the drawing]

[0008] [Figure 1] Müller cells. Schematic diagram showing Müller cells with other retinal cell types. GCL = Ganglion cell layer. INL = Inner granular layer. ONL = Outer granular layer. [Figure 2] Stage 2 of the cell culture process - neuroretinal differentiation. A schematic diagram illustrating the initial process for stem cell differentiation to generate mature retinal organoids. The diagram shows embryoid body formation, neural differentiation, and induction of maturation from stem cells. [Figure 3] Stage 3 of the cell culture process - Dissociation and proliferation of Müller cells from retinal organoids. Schematic diagram illustrating the cell dissociation and proliferation process. Müller cells are isolated from retinal organoids. Single-cell suspensions can be prepared using dissociation with a weak cell dissociation reagent (GCDR), followed by centrifugation and plate culture on fibronectin-coated flasks. Next, proliferation is followed by nutrient supplementation and FGF (fibroblast growth factor) and EGF (epidermal growth factor). [Figure 4]Müller cells differentiated from RC-9 cells do not express the stem marker Tra-1-60. The expression of the stem cell marker Tra-1-60 was measured on the surface of undifferentiated RC-9 cells using flow cytometry (Figure 4A) and compared to the expression on the surface of Müller cells differentiated from RC-9 cells (Figure 4B) (generated using a GMP-compliant protocol). Undifferentiated RC-9 cells were highly positive for Tra-1-60 (99.44% of the population were positive), indicating the stem cell state of the cells. After differentiation of RC-9 cells into Müller cells, Tra-1-60 expression is lost. [Figure 5-1] Mueller cells differentiated from RC-9 cells express markers associated with Mueller cells. Mueller cells differentiated from RC-9 cells using a GMP-compliant protocol were characterized for Mueller marker expression using flow cytometry. Vimentin (Figure 5A and 5B), CD29 (Figure 5C and 5D), CD44 (Figure 5E and 5F), and nestin (Figure 5G and 5H) were highly expressed by the derived Mueller cells compared to negative isotype controls. [Figure 5-2] This is a continuation of Figure 5-1. [Figure 6] Mueller cells generated using the GMP-compliant protocol of the present invention secrete neuroprotective factors and antioxidants known to support RGC function. Mueller cells differentiated from RC-9 cells using the GMP-compliant protocol were characterized for neuroprotective factor and antioxidant secretion using ELISA. The concentrations of BDNF, PEDF, and PRDX6 in the supernatant from Mueller cells were measured by ELISA and compared to hESCs (starting material) of undifferentiated RC-9; BLQ = <quantification level. [Figure 7]Mueller cells generated using the GMP-compliant protocol of the present invention express and secrete more BDNF than published cells. Mueller cells differentiated from RC-9 cells using the GMP-compliant protocol were characterized for BDNF gene expression using transcriptomics and for BDNF secretion in the cell supernatant using ELISA. TPM = transcripts per million, B4 = Mueller cells of Eastlake et al. 2019, Eng1 = Mueller cells of the present invention. [Figure 8] Mueller cells generated using the GMP-compliant protocol of the present invention exhibit higher PEDF gene expression than published cells. Mueller cells differentiated from RC-9 cells using the GMP-compliant protocol were characterized for PEDF gene expression using transcriptomics and for PEDF secretion in the cell supernatant using ELISA. TPM = transcripts per million, B4 = Mueller cells of Eastlake et al. 2019, Eng1 = Mueller cells of the present invention. [Figure 9] Mueller cells generated using the GMP-compliant protocol of the present invention do not express pluripotency markers. Mueller cells differentiated from RC-9 cells using the GMP-compliant protocol were characterized for pluripotency marker expression using transcriptomics. Differential gene expression by DESeq of Bioconductor; the numbers are normalized transcripts per million readouts (TPM). TPM <10 is below the quantifiable level (BLQ). Undiff.RC-9 = undifferentiated RC-9 (starting material). [Figure 10] Mueller cells generated using the GMP-compliant protocol of the present invention have lower POU5F1(OCT3) gene expression than published cells. Mueller cells differentiated from RC-9 cells using the GMP-compliant protocol were characterized for the expression of pluripotency markers using transcriptomics. TPM = transcripts per million, B4 = Mueller cells of Eastlake et al. 2019, Eng1 = Mueller cells of the present invention. [Figure 11] Müller cells generated using the protocol compliant with GMP of the present invention improve RGC survival in vitro after treatment with excessive glutamate, as demonstrated by the increasing neurite length. Rat primary RGCs were treated with 25 μM glutamate for 24 hours and then treated with serum-free basal medium (BM) or Müller cell (MC) supernatant (SN) for 72 hours. A, RGCs were immunostained with anti-β-tubulin III antibody (TUJ, Alexa488, indicating cell shape), a marker of RGCs, and DAPI (indicating nuclei). Scale bar = 20 μm. B, The histogram plot shows the average length of primary neurites per RGC; error bars ± SEM; **p < 0.01; statistical analysis Mann-Whitney test. Data were generated with Müller cells differentiated from RC-9 cells using the protocol compliant with GMP of the present invention. [Figure 12] Müller cells differentiated from RC-9 improve RGC survival in vivo. The Müller cells of the present invention are effective in the RGC loss NMDA rodent model, improve RGC function in the NMDA model measured by ERG (measured by the scotopic negative threshold response of electroretinogram - nSTR), and are consistent with previous published data. A, 1×105 Müller cells were injected intravitreally 1 week after NMDA treatment. B, Complete ERG profile (box) by enhanced nSTR at a light intensity of -3.5, inset shows only the nSTR region. The upper line in the nSTR box = NMDA control, the middle line in the nSTR box = NMDA + Müller cells, the lower line in the nSTR box = control. C, The lowest point of nSTR is represented as %RGC function at a light intensity of -3.5; p = 0.04. The left column = control, the middle column = NMDA control, the right column = NMDA + Müller cells. Control = PBS-treated eyes; NMDA control = NMDA-treated eyes. Data were generated using Müller cells derived from RC-9 hESCs.

Summary of the Invention

[0009] The present invention is based on a novel process for producing Müller cells from stem cells using a culture process that does not contain animal-derived components. Surprisingly, during the conversion to a GMP-compliant method, there was no decrease in the yield of Müller cells or deterioration in quality. The cells cultured from pluripotent stem cells by the method of the present invention provide Müller cells that are morphologically similar to Müller cells derived from the iPSC BJ cell line described in Eastlake et al 2019 (Stem Cells Translational Medicine). Furthermore, the novel process for producing Müller cells from pluripotent stem cells using products that do not contain animal-derived components is less labor-intensive, cost-effective, time-efficient, reduces the risk of infectious diseases, and is easier to scale up for industrial and clinical applications.

[0010] The present invention also relates to Müller cells derived from human embryonic stem cells (hESC). In a preferred embodiment, the hESC is the RC-9 cell.

[0011] In the present disclosure, in the context of Müller cells derived from the methods described herein, the terms "Müller" and "Müller-like" are understood to be used interchangeably to describe cells that have been artificially differentiated to share the characteristics of Müller cells normally present in the eye.

[0012] Thus, in one embodiment of the present invention, an isolated human Müller cell, a) expresses detectable levels of CD29, vimentin, CD44, and nestin and does not express detectable levels of Tra-1-60, b) is provided that can secrete the neurotrophins BDNF and PEDF.

[0013] In a further embodiment of the present invention, a purified substantially homogeneous population of two or more Müller cells according to the present invention is provided.

[0014] In a further embodiment of the present invention, a population of human Müller cells is provided, wherein at least 95% of the cells in the population express CD29, vimentin, CD44, and nestin to detectable levels, less than 5% of the cells express Tra-1-60 to detectable levels, and the cells are capable of secreting the neurotrophins BDNF and PEDF.

[0015] In a further embodiment of the present invention, Mueller cells derived from human embryonic stem cells, a) Expressing detectable levels of CD29, vimentin, CD44, and nestin, but not expressing detectable levels of Tra-1-60, b) Mueller cells capable of secreting neurotrophins BDNF and PEDF are provided.

[0016] A further embodiment of the present invention is a method for producing therapeutic-grade human Müller cells, a) A step of culturing RC-9 human embryonic stem cells in a plate-based suspension system in xeno-free and serum-free medium for at least 15 days in the presence of a ROCK signaling pathway inhibitor and a Wnt inhibitor. b) Adding a synthetic cell adhesion promoter to the xeno-free and serum-free media of step (a) and culturing the cells for at least 8 days, c) Add synthetic enrichment growth factor and a smoothed protein agonist of the Hedgehog signaling pathway to the xeno-free and serum-free medium of step (b), and culture the cells for at least 3 days. d) Add retinoic acid to the xeno-free and serum-free medium from step (c) and culture the cells from step (c) for a further 2-300 days until the retinal organoids are visible. e) The step of dissociating the retinal organoids and isolating the Müller cells. A method including this is provided.

[0017] Further embodiments of the present invention provide a pharmaceutical composition comprising Müller cells of the present invention or a population of Müller cells according to the present invention, or Müller cells that can be derived from the method of the present invention, and a pharmaceutically acceptable carrier.

[0018] In further embodiments of the present invention: a) A step of culturing stem cells in a plate-based suspension in xeno-free and serum-free medium for at least 15 days in the presence of a ROCK signaling pathway inhibitor and a Wnt inhibitor. b) Adding a synthetic cell adhesion promoter to the xeno-free and serum-free media of step (a) and culturing the cells for at least 8 days, c) Add synthetic enriched growth factor and a smoothed protein agonist of the Hedgehog signaling pathway to the xeno-free and serum-free media of step (b), and culture the cells for at least 3 days. d) Add retinoic acid to the xeno-free and serum-free medium from step (c) and culture the cells from step (c) for a further 2-300 days until the retinal organoids are visible. e) The step of dissociating the retinal organoids and isolating the Müller cells. A population of Müller cells obtainable by a method including the present invention is provided, and a pharmaceutical composition comprising a pharmaceutically acceptable carrier is also provided.

[0019] In further embodiments of the present invention, a method for treating a retinal disease or condition is provided, comprising administering a pharmaceutical composition according to the present invention, Müller cells according to the present invention, a population of Müller cells according to the present invention, or Müller cells that can be derived from a method of the present invention, to a patient in need thereof.

[0020] In a further embodiment of the present invention, a method for treating a retinal disease or condition comprises administering a pharmaceutical composition to a patient in need thereof, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier and a population of Müller cells, the Müller cells being: a) A step of culturing stem cells in a plate-based suspension in xeno-free and serum-free medium for at least 15 days in the presence of a ROCK signaling pathway inhibitor and a Wnt inhibitor. b) Adding a synthetic cell adhesion promoter to the xeno-free and serum-free media of step (a) and culturing the cells for at least 8 days, c) Add synthetic enriched growth factor and a smoothed protein agonist of the Hedgehog signaling pathway to the xeno-free and serum-free media of step (b), and culture the cells for at least 3 days. d) Add retinoic acid to the xeno-free and serum-free medium from step (c) and culture the cells from step (c) for a further 2-300 days until the retinal organoids are visible. e) The step of dissociating the retinal organoids and isolating the Müller cells. A method is provided which is obtained from a method including the following.

[0021] Therefore, in a further embodiment of the present invention, a method for producing therapeutic-grade Müller cells, a) A step of culturing stem cells in a plate-based suspension in xeno-free and serum-free medium for at least 15 days in the presence of a ROCK signaling pathway inhibitor and a Wnt inhibitor. b) Adding a synthetic cell adhesion promoter to the xeno-free and serum-free media of step (a) and culturing the cells for at least 8 days, c) Add synthetic enriched growth factor and a smoothed protein agonist of the Hedgehog signaling pathway to the xeno-free and serum-free media of step (b), and culture the cells for at least 3 days. d) Add retinoic acid to the xeno-free and serum-free medium from step (c) and culture the cells from step (c) for a further 2-300 days until the retinal organoids are visible. e) The step of dissociating the retinal organoids and isolating the Müller cells. A method including this is provided.

[0022] In further embodiments, the process includes a preliminary pre-differentiation step in which stem cells are cultured to a concentration density of 50-99%, frozen at -80 degrees Celsius in cryogenic storage medium, then transferred to liquid nitrogen for long-term storage and thawed before step (a) above. Thus, the present invention provides a method for producing therapeutic-grade Müller cells, comprising culturing stem cells on the surface of xeno-free and serum-free medium to a concentration density of 50-99%, preferably 50-80%, freezing the cells in cryogenic storage medium, thawing the cells, and then culturing them by the process described above.

[0023] In preferred embodiments, the stem cells are human embryonic stem (hES) cells or induced pluripotent stem (iPS) cells, preferably human embryonic stem cells. The stem cells used in the present invention are WA09(hESC) from WiCell, Shef 1.3(hESC) from University College London, Man-15hES cell lineage from University of Manchester, BJ cell (iPSC) lineage from University College London, preferably RC-9 hES cell lineage provided by Roslin Cell Therapies.

[0024] In this process, stem cells are on a coated surface, and the preferred coating is a glycoprotein, a combination of laminin-111 and laminin-521, Matrigel®, or more preferably a GMP-compliant synthetic vitronectin or a synthetic vitronectin-based substrate.

[0025] The basal medium for this process includes minimally essential media, such as GMEM containing glutamine, and synthetic media such as KOSR. The medium is added at each stage. For example, during the pre-differentiation stage of stem cell proliferation (before steps a) to d), the medium may be TeSR-E8™ (an animal component-free medium available from STEMCELL Technologies Inc.), but preferably iPS-Brew or TeSR2, which are xeno-free cell media. iPS-Brew may have TGF-beta (transforming growth factor beta) added. During the differentiation stage (a) to d), the basal medium may contain a carbon source such as sodium pyruvate, essential and non-essential amino acids, and one or more antibiotics such as gentamicin, penicillin, and / or streptomycin.

[0026] The adhesion promoter may be a synthetic matrix protein to avoid animal products, and is preferably a GMP-compliant synthetic vitronectin or a synthetic vitronectin-based substrate.

[0027] The enriched growth factor is preferably a synthetic compound or a product such as GMP-grade human platelet lysate (HPL). At the stage of adding the enriched growth factor, it is also preferable to add a neuronal differentiation enhancer, such as an agonist of a Hedgehog signaling pathway protein, such as smoothed protein (SAG).

[0028] Mammalian cells require iron for cell proliferation, DNA replication, cellular respiration, and metabolism. Transferrin is a natural physiological method by which iron is transported to cells. Therefore, at the final differentiation stage, when retinoic acid is added to the culture medium, it is also preferable to add human transferrin, such as a B27 or N2 adjuvant, which further contains human insulin, selenite, putrescine, and progesterone, which are important for cell survival.

[0029] In this process, stem cells are differentiated in a plate-based system suspension, preferably in a V-bottom well plate, ideally in a 96-well plate.

[0030] Cell culture may be performed for different durations at each stage, but a preferred culture protocol involves seeding on day 0, supplying Rock signaling pathway inhibitors and Wnt inhibitors initially, preferably from day 0 to at least 15 days, on days 2, 5, and 9, adding cell adhesion promoters ideally from day 2 and / or for at least 8 days, again supplying enrichment growth factor and SAG initially and / or for at least 2, 3, or 4 days on days 12 and 15, and supplying retinoic acid initially and / or for at least 2 days from day 17. The ideal time for retinoic organoids to become visible from embryoid bodies is 15–90 days, preferably 15–70 days, or at least 15–40 days. From this time, cells are ideally supplied with medium containing retinoic acid twice a week.

[0031] Retinal organoids arising from embryoid bodies can be carefully dissected using a microblade such as a microscalpel or diamond-tipped cutter between 25 and 40 days to avoid damaging cells under a dissection microscope, and then transferred to a new, low-adhesion plate prepared for dissection to collect enriched Müller cell suspension. The retinal organoids are maintained on the low-adhesion plate in a medium containing retinoic acid. These retinal organoids are ideally harvested between 30 and 300 days.

[0032] Between approximately 15 and 90 days, embryoid bodies containing retinal organoids can be dissociated without incision in cell dissociation reagents such as papain or Gentle Cell Dissociation Reagent (GCDR) from STEMCELL Technologies Inc. (allowing for considerably greater scalability) to isolate enriched Müller cell suspension. This appears possible, as cells seem to proliferate much more densely in human platelet lysates, without being constrained by theory.

[0033] Therefore, in a further embodiment of the present invention, a method for producing Müller cells, a) A step of culturing retinal organoids in human platelet lysate, b) Steps to dissociate the retinal organoids without incision and isolate the Müller cells. A method including this is provided.

[0034] Müller cells are the only cells of the neuroretina that express CD29, a ligand that binds to fibronectin. Therefore, it is advantageous to pre-coat the surface of culture plates or flasks with fibronectin to allow isolation of a pure population of Müller cells (meaning that they are pure and free from contamination by non-Müller cells by removing undesirable cells suspended in the medium), and, if possible, to produce a pure population of Müller cells by growing them in a medium supplemented with fibroblast growth factor (FGF) and epidermal growth factor (EGF) (the last step in Figure 3), before forming a bank of frozen cells in xeno-free medium in vials.

[0035] A further embodiment of the present invention is a method for producing a pure population of Müller cells, a) A step of culturing retinal organoids, b) A step of isolating a Müller cell suspension obtained by dissociating the retinal organoids and enriching them, c) A step of culturing the Müller cells on a surface coated with fibronectin, d) The step of isolating the Müller cells on fibronectin to form a pure population thereof, e) Depending on the case, growing the pure population of cells in a medium supplemented with fibroblast growth factor (FGF), or epidermal growth factor (EGF), or FGF and EGF. A method including this is provided.

[0036] A further aspect of the present invention is a method for producing therapeutic-grade pure human Müller cells, a) A step of culturing stem cells in a plate-based suspension in xeno-free and serum-free medium for at least 15 days in the presence of a ROCK signaling pathway inhibitor and a Wnt inhibitor. b) Adding a synthetic cell adhesion promoter to the xeno-free and serum-free media of step (a) and culturing the cells for at least 8 days, c) Add synthetic enriched growth factor and a smoothed protein agonist of the Hedgehog signaling pathway to the xeno-free and serum-free media of step (b), and culture the cells for at least 3 days. d) Add retinoic acid to the xeno-free and serum-free medium from step (c) and culture the cells from step (c) for a further 2-300 days until the retinal organoids are visible. e) A step of isolating a Müller cell suspension obtained by dissociating the retinal organoids and enriching them, f) A step of culturing the Müller cells on a surface coated with fibronectin, g) Depending on the case, a step of growing the pure population of cells in a medium supplemented with fibroblast growth factor (FGF), or epidermal growth factor (EGF), or FGF and EGF, h) The step of isolating the Müller cells on fibronectin to form a pure population thereof. A method including this is provided.

[0037] A further aspect of the present invention provides Müller cells that can be obtained by any of the methods outlined above.

[0038] A further aspect of the present invention involves the use of Müller cells, obtainable by any of the methods outlined above, in the manufacture of pharmaceuticals for the treatment of eye diseases, including, but not limited to, age-related macular degeneration, proliferative diabetic retinopathy, proliferative vitreoretinopathy, retinal detachment, pigmentary retinitis, glaucoma, and damage and degeneration of the optic nerve.

[0039] A further aspect of the present invention provides a cell medium for the differentiation of Müller cells (up to day 15) essentially comprising a minimal essential synthetic basal medium, a carbon source, non-essential amino acids, a ROCK inhibitor, human platelet lysate, a Wnt inhibitor, and GMP-compliant synthetic vitronectin, a synthetic vitronectin-based glycoprotein, or a hydrogel skeleton. A preferred medium for use (generally from day 12 to 18) contains an agonist of a smoothed protein of the Hedgehog signaling pathway.

[0040] A further aspect of the present invention provides a cell medium for the differentiation of Müller cells (from day 15), which essentially consists of a minimal essential synthetic basal medium, an N2 auxiliary, retinoic acid, and human platelet lysate.

[0041] A further aspect of the present invention provides a cell culture kit for culturing Müller cells, comprising the xeno-free and serum-free media described above. [Modes for carrying out the invention]

[0042] It should be understood that different applications of the disclosed products and methods can be adapted to the specific needs of the art. It should also be understood that the terms used herein are for the sole purpose of describing and not limiting specific embodiments of the invention.

[0043] Furthermore, as used herein and in the appended claims, unless the content clearly indicates otherwise, the singular forms "a," "an," and "the" are included in the plural form. Thus, for example, a reference to "cell" includes "multiple cells," a reference to "tissue" includes two or more such tissues, a reference to "subject" includes two or more such subjects, and so on.

[0044] All publications, patents, and patent applications cited herein, whether above or below, are incorporated herein in their entirety by reference.

[0045] Stem cells have the ability to differentiate into various cell types in response to appropriate signals. These properties give stem cells the ability to repair, replace, and regenerate tissue. Therefore, human stem cells, more specifically human embryonic stem cells (hESCs), are of particular interest in medical research. Embryonic stem cells have the ability to differentiate into more cell types than adult stem cells and therefore have great potential in therapy. Differentiation is induced in vivo by various factors, some of which can be replicated in in vitro stem cell culture. Induced pluripotent stem cells (iPSCs) are a form of stem cell often used in autologous procedures because they can be produced from the tissue of the same patient receiving the transplant and thus avoid immune rejection. iPSCs obtained in this way do not have the ethical considerations of stem cells derived from embryos.

[0046] The nature of stem cells necessitates the use of special stem cell media and reagents. Culture is ideally carried out in media that are xeno-free and serum-free. Being xeno-free (non-human) and serum-free means that there are absolutely no uncertain animal products in the culture process, which is crucial for GMP compliance. For example, FBS (fetal bovine serum) can be replaced with GMP-quality HPL (human platelet lysate). Furthermore, Matrigel® can be replaced with human-compatible Matrigel® substitutes such as Synthemax. Media beneficial in this invention include minimal essential media such as Glasgow's Minimum Essential Medium (GMEM) with optionally added L-glutamine, and / or synthetic media such as knockout serum supplementation medium (KOSR). The culture medium may also contain non-essential amino acids, growth promoters, carbon sources such as sodium pyruvate, antibiotics such as penicillin and / or streptomycin, biological antioxidants such as 2-mercaptoethanol, Wnt signaling pathway inhibitors, and more specifically, the Wnt antagonist IWR-1-endo, as well as SAG (smoothened agonists) that enhance neuronal differentiation in human stem cells.

[0047] Regarding cell culture in general, most cells require a surface or artificial substrate (adherent or monolayer culture), while others can be grown floating in a culture medium (suspension culture). In the present invention, stem cells are preferably cultured on a coated surface, which is preferably coated using a protein-based material, ideally a glycoprotein, because it improves cell adhesion and performance. The glycoprotein to be selected is synthetic vivonectin because it acts as a substrate that promotes cell adhesion through its binding domain RGD sequence (arginine, glycine, and aspartic acid). Human recombinant vivonectin is preferred for GMP compliance and reduced batch-to-batch variability. Matrigel® is another cell adhesion promoter, but is not a preferred coating material. The cell medium may be TeSR-E8®, but is preferably iPS-Brew, TeSR2, or StemPro containing TGF beta. TeSR-E8™ is a feeder and animal component-free culture medium for human embryonic stem cells and human induced pluripotent stem (iPS) cells, available from STEMCELL Technologies Inc. iPS-Brew is a xeno-free cell medium commercially available from Miltenyi Biotec. TGF beta can be added to iPS-Brew.

[0048] Stem cell colonies divide when they reach a certain level of concentration. "Concentration" or "density" refers to the percentage of the culture vessel or well surface covered by adherent cells. It is preferable to culture and expand stem cells so that there are enough cells to seed for the generation of retinal organoids.

[0049] The ability to freeze and thaw is crucial for quality control in the large-scale preparation of clinical-grade cells. Stem cells supplied by suppliers are typically provided frozen. Cryogenic storage media are used as part of the freezing process. Common cryogenic storage media support cells and prevent ice crystal formation. The freeze and thaw process can be applied to stem cells before and / or after Stage 1 maintenance. At the end of the complete process, when vials of Müller cells are produced, these can also be frozen for storage and for subsequent thawing at a later time.

[0050] After thawing, stem cells can be further cultured. This culture may include subculturing in several different media, such as those described in the subsequent Examples section (Stage 2 differentiation).

[0051] Stem cells form embryoid bodies; three-dimensional aggregates of pluripotent stem cells. Embryoid bodies form the "mantle," an indicator of retinal organoids. "Organoids" are self-organizing three-dimensional tissue cultures derived from stem cells. Such cultures can replicate many of the complexities of an organ or express a selected aspect thereof. The culture is performed on low-adhesion plates, and therefore the organoids are in a suspended state.

[0052] Retinal organoids are cultured for at least 15 days, and may be up to 300 days, but preferably 15 to 90 days, within a time frame of 15 to 80 days, 15 to 70 days, 15 to 60 days, 15 to 50 days, 15 to 40 days, or 15 to 30 days. The culture medium supporting retinal organoid production is critical to the present invention, and appropriate specific therapeutic-grade reagents are added to the production stage as shown above. Key reagents for production up to approximately day 15 are ROCK signaling pathway inhibitors, such as p160 ROCK inhibitors, which may be GMP-grade ROCK inhibitors in preference to non-GMP ROCK inhibitors or research-grade ROCK inhibitors. Further reagents included up to day 15 are Wnt inhibitors, which may also be GMP-grade or research-grade.

[0053] The key reagents for production, generally starting from day two, are synthetic cell adhesion promoters, such as human recombinant vivonectin, which can be used in preference to Matrigel™.

[0054] Further important reagents, starting around day 12, may include synthetic enrichment growth factors, which may be GMP-quality human platelet lysates (HPL), and preferably agonists of smoothed proteins of the Hedgehog signaling pathway, starting around day 15-18.

[0055] Ideally, additional reagents added to the culture medium from day 15 include human transferrin, such as N2 adjuvants and retinoic acid.

[0056] Retinal organoids can be isolated from embryoid bodies using a microblade. Other cutting tools such as blades, scalpels, micro-scalpels, and / or diamond-tipped cutters can also be used. From days 15 to 90, retinal organoids can be dissected from embryoid bodies under sterile microscopic conditions using a microblade to purify the optic cup structures from the embryoid bodies. A variation of this method is to dissect all organoids on a single day between days 50 and 57. The organoids can then be transferred to low-adhesion plates and kept in culture medium for long-term culture with two medium changes per week.

[0057] Retinal organoids can be dissociated and released from Müller cells 25–300 days after the start of the differentiation protocol. Dissociation can be achieved using a papain-based protocol. Papain is a cysteine ​​protease enzyme. A commercially available papain kit (Worthington Biochemical) can be used to dissociate organoids. A variation of this protocol is to completely replace the papain method with the use of Gentle Cell Dissociation reagent. Gentle Cell Dissociation reagent (GCDR) is a suitable enzyme-free reagent for dissociation of human embryonic stem cells or human induced pluripotent stem cells into small cell aggregates or single-cell suspensions for routine passage. GCDR is available from STEMCELL Technologies Inc.

[0058] When using GMP-compliant reagents, such as xeno-free and serum-free media, this process generates therapeutic-grade Müller cells. The resulting Müller cells are suitable for the treatment of eye diseases associated with cell loss or cytotoxicity, particularly age-related macular degeneration, proliferative diabetic retinopathy, proliferative vitreoretinopathy, retinal detachment, pigmentary retinitis, glaucoma, and optic nerve damage and degeneration.

[0059] The process described provides a cell culture medium, which may be part of a cell culture kit.

[0060] This improved cell culture-based process generates therapeutic-grade Müller cells without any reduction in yield or cell quality.

[0061] Therefore, a further aspect of the present invention provides a cell medium for differentiation of retinal organoids containing Müller cells (from day 15) or an enriched population of Müller cells (from day 15), essentially comprising a minimal essential synthetic basal medium, an N2 auxiliary, retinoic acid, and human platelet lysate.

[0062] Therefore, in a further aspect of the present invention, a cell culture kit for inducing retinal organoids containing Müller cells or a population rich in Müller cells is provided, comprising xeno-free and serum-free media as shown above.

[0063] Müller cells of the present invention As shown above, the present invention also provides Müller cells. Preferably, the Müller cells are human cells. In one embodiment of the present invention, isolated or purified human Müller cells are provided. The terms “purified” or “isolated” mean that cells, tissues, polynucleotides, peptides, polypeptides, proteins, antibodies or fragments thereof are separated from cellular or other components to which they are naturally and conventionally associated. Cells are isolated or purified if they substantially do not contain any other components, such as culture media and other cells. Purified or isolated cells are separated from tissues to which they are naturally and conventionally associated. Isolated or purified cells are cells separated from tissues or cells of different phenotypic or genotypes. Müller cells derived from the methods of the present invention have the Müller cell marker identity described below. Müller cells derived from the methods of the present invention can be screened for Müller cell markers as described below to confirm their identity.

[0064] The Müller cells of the present invention can be advantageously used to treat diseases in subjects. The Müller cells of the present invention may be autologous or allogeneic to the subject being treated. The Müller cells of the present invention are generated from stem cells, preferably from human stem cells, preferably from hESCs, and preferably from RC-9 cells, by the method described herein. Müller cells derived from the method described herein can be characterized with respect to the marker profiles shown below.

[0065] The Müller cells of the present invention can be identified as Müller cells using standard methods known in the art, including the expression of lineage restriction markers and analysis of structural and functional characteristics. The Müller cells of the present invention express detectable levels of cell surface markers known to be characteristic of Müller cells. Compared to undifferentiated stem cells, the Müller cells of the present invention express detectable levels of cell surface markers known to be characteristic of Müller cells. The Müller cells of the present invention express detectable levels of CD29, vimentin, CD44, and nestin.

[0066] The Müller cells of the present invention do not express detectable levels of cell surface markers known to be characteristic of undifferentiated stem cells. The Müller cells of the present invention do not express detectable levels of Tra-1-60. In a preferred embodiment, the Müller cells of the present invention do not express detectable levels of Tra-1-60 and further do not express one or more detectable levels of LIN28, SOX2, OCT3 / OCT4, NANOG, and ESRG. In a preferred embodiment, the Müller cells of the present invention do not express detectable levels of Tra-1-60 and further do not express one or more detectable levels of LIN28, SOX2, OCT3 / OCT4, NANOG, ESRG, and DPPA4.

[0067] CD29, also known as integrin beta-1, VLA-β chain, or gpIIa, acts as a fibronectin receptor and is involved in various cell matrix interactions. Members of the integrin family are membrane receptors involved in cell adhesion and recognition in a variety of processes, including embryogenesis, hemostasis, tissue repair, immune responses, and metastatic spread of tumor cells.

[0068] Vimentin is a type III intermediate filament (IF) protein that acts as a structural protein. It is expressed by many cells, including mesenchymal and glial cells. Vimentin plays a role in maintaining cell shape, cytoplasmic integrity, and stabilizing cytoskeletal interactions.

[0069] CD44 is a cell surface glycoprotein involved in cell-cell interactions, cell adhesion, and cell migration.

[0070] Nestin (a neuroectodermal stem cell marker) is a type VI intermediate filament (IF) protein. Nestin is expressed in dividing cells during the early stages of neuronal development. It is downregulated after neuronal maturation and is also expressed in many other tissues. However, it is mostly expressed in neurons, where it is linked to the radial growth of axons. Nestin acts in conjunction with vimentin.

[0071] Tra-1-60 is a cell surface antigen expressed in undifferentiated human embryonic stem cells.

[0072] LIN28 is an RNA-binding protein that is highly expressed in human embryonic stem cells.

[0073] SOX2 is a transcription factor essential for maintaining the pluripotency of undifferentiated embryonic stem cells.

[0074] OCT3 / OCT4 (known as OCT3, OCT4, or POU5F1) is a homeodomain transcription factor of the POU family. It is critically involved in the autorenewal of undifferentiated embryonic stem cells and is a marker of pluripotency.

[0075] NANOG is a transcription factor of the homeobox family that helps maintain the pluripotency of embryonic stem cells.

[0076] ESRGs (embryonic stem cell-associated genes, also known as HESRGs) are specifically expressed in undifferentiated human ESCs.

[0077] DPPA4 (developmental pluripotency-related 4) is a highly specific marker for pluripotent cells.

[0078] The Müller cells of the present invention are distinguished from known cells, including human embryonic stem cells, through their marker expression patterns. The Müller cells of the present invention express detectable levels of CD29, vimentin, CD44, and nestin. The Müller cells of the present invention preferably express increased amounts of these markers compared to human embryonic stem cells. This can be determined by comparing the expression levels / amounts of the markers in the Müller cells of the present invention with the expression levels / amounts in human embryonic stem cells using the same technique under the same conditions. Suitable hESCs are commercially available.

[0079] The Müller cells of the present invention do not express a detectable level of Tra-1-60. In a preferred embodiment, the Müller cells of the present invention do not express a detectable level of Tra-1-60 and further do not express one or more detectable levels of LIN28, SOX2, OCT3 / OCT4, NANOG, and ESRG. In a preferred embodiment, the Müller cells of the present invention do not express a detectable level of Tra-1-60 and further do not express one or more detectable levels of LIN28, SOX2, OCT3 / OCT4, NANOG, ESRG, and DPPA4.

[0080] Standard methods known in the art can be used to determine the detectable or increased expression of the various markers discussed above (and below). Preferred methods include, but are not limited to, immunocytochemistry, immunoassays, flow cytometry, e.g., fluorescence-activated cell sorting (FACS), polymerase chain reaction (PCR), e.g., reverse transcription PCR (RT-PCR), and transcriptomics methods, e.g., RNA sequencing (RNA-Seq). Preferred immunoassays include, but are not limited to, Western blotting, enzyme-linked immunoassays (ELISA), enzyme-linked immunosorbent spot assays (ELISPOT assays), enzyme proliferation immunoassay techniques, radioallergosolvent (RAST) tests, radioimmunoassays, radiobinding assays, and immunofluorescence methods. Western blotting, ELISA, and RT-PCR are all quantitative and can therefore be used to measure the expression levels of various markers, if present. The use of transcriptomics is disclosed in the examples, where gene expression levels are shown as normalized transcripts per million (TPM) reads. A level of less than 10 TPM is defined as below the quantification level. Expression or increased expression of any of the markers disclosed herein is preferably performed using flow cytometry. Antibodies and fluorescently labeled antibodies for all of the various markers discussed herein are commercially available.

[0081] The Müller cells of the present invention possess the functional characteristics of Müller cells and can express genes and secrete neurotrophin proteins BDNF and PEDF. The Müller cells of the present invention can secrete neurotrophin BDNF and PEDF and the antioxidant PRDX6. The ability of Müller cells to secrete neurotrophin and antioxidant can be measured using standard assays known in the art. Preferred methods include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunostaining. The Müller cells of the present invention preferably secrete neurotrophin BDNF and PEDF at detectable levels. The Müller cells of the present invention preferably secrete increased amounts of neurotrophin BDNF and PEDF compared to hESCs.

[0082] The Müller cells of the present invention are preferably able to migrate to the retina and intercept cells such as rod and cone receptors, bipolar cells, and ganglion cells.

[0083] A preferred embodiment of the present invention is a method for producing therapeutic-grade human Müller cells, a) A step of culturing human embryonic stem cells in a plate-based suspension system in xeno-free and serum-free medium for at least 15 days in the presence of a ROCK signaling pathway inhibitor and a Wnt inhibitor. b) Adding a synthetic cell adhesion promoter to the xeno-free and serum-free media of step (a) and culturing the cells for at least 8 days, c) Add synthetic enriched growth factor and a smoothed protein agonist of the Hedgehog signaling pathway to the xeno-free and serum-free media of step (b), and culture the cells for at least 3 days. d) Culturing the cells from step (c) for a further 2-300 days, and adding retinoic acid to the xeno-free and serum-free medium from step (c) until retinal organoids are visible. e) The step of dissociating the retinal organoids and isolating the Müller cells. A method is provided that includes Müller cells, where the Müller cells express detectable levels of CD29, vimentin, CD44, and nestin, do not express detectable levels of Tra-1-60, and can secrete the neurotrophins BDNF and PEDF.

[0084] Preferably, the human embryonic stem cells are RC-9 cells.

[0085] The population of the present invention The present invention also provides a population of two or more Müller cells of the present invention. Any number of cells may be present in the population. The population of the present invention preferably contains at least about 5×10 5 Müller cells of the present invention. The population more preferably contains at least about 1×10 6 cells, at least about 2×10 6 cells, at least about 2.5×10 6 cells, at least about 5×10 6 cells, at least about 1×10 7 cells, at least about 2×10 7 cells, at least about 5×10 7 cells, at least about 1×10 8 cells, or at least about 2×10 8 cells of the present invention. In some cases, the population can contain at least about 1.0×10 7 cells, at least about 1.0×10 8 cells, at least about 1.0×10 9 cells, at least about 1.0×10 10 cells, at least about 1.0×10 11 , or at least about 1.0×10 12 cells of the present invention, or more.

[0086] A population comprising two or more Müller cells of the present invention may include other cells in addition to the Müller cells of the present invention. However, at least 70% of the cells in the population are preferably Müller cells of the present invention. More preferably, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 97%, at least about 98%, or at least about 99% of the cells in the population are Müller cells of the present invention.

[0087] The present invention also provides a specific population of Müller cells.

[0088] The present invention also provides substantially homogeneous, purified populations of Müller cells. A “substantially homogeneous” cell population describes a population of cells in which more than 50%, or instead more than 60%, or instead more than 70%, or instead more than 75%, or instead more than 80%, or instead more than 85%, or instead more than 90%, or instead more than 95% of the cells have the same or similar phenotype. The phenotype is determined by markers of Müller cell identity, which are described in further detail herein.

[0089] The present invention provides a population of Müller cells in which at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or preferably at least 99%, of the cells express CD29 to a detectable level, and less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or preferably less than 1%, of the cells express Tra-1-60 to a detectable level.

[0090] The present invention provides a population of Müller cells in which at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or preferably at least 99%, of the cells in the population express vimentin to a detectable level, and less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or preferably less than 1%, of the cells express Tra-1-60 to a detectable level.

[0091] The present invention provides a population of Müller cells in which at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or preferably at least 99%, of the cells in the population express CD44 at a detectable level, and less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or preferably less than 1%, of the cells express Tra-1-60 at a detectable level.

[0092] The present invention provides a population of Müller cells in which at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or preferably at least 99%, percent of the cells express nestin to a detectable level, and less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or preferably less than 1%, of the cells express Tra-1-60 to a detectable level.

[0093] The present invention provides a population of Müller cells in which at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or preferably at least 99% of the cells in the population express at least two of CD29, vimentin, CD44, and nestin to a detectable level, and less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or preferably less than 1%, of the cells express Tra-1-60 to a detectable level.

[0094] The present invention provides a population of Müller cells in which at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or preferably at least 99% of the cells in the population express at least three of CD29, vimentin, CD44, and nestin to a detectable level, and less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or preferably less than 1%, of the cells express Tra-1-60 to a detectable level.

[0095] In a preferred embodiment, the present invention provides a population of Müller cells, wherein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or preferably at least 99% of the cells in the population express CD29, vimentin, CD44, and nestin to a detectable level, and less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or preferably less than 1%, of the cells express Tra-1-60 to a detectable level.

[0096] In a preferred embodiment, the present invention provides a population of Müller cells in which at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or preferably at least 99% of the cells in the population express CD29, vimentin, CD44, and nestin to a detectable level; less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or preferably less than 1%, of the cells express Tra-1-60 to a detectable level; and further less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or preferably less than 1%, of the cells express one or more of LIN28, SOX2, OCT3 / OCT4, NANOG, and ESRG to a detectable level.

[0097] In a preferred embodiment, the present invention provides a population of Müller cells in which at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or preferably at least 99% of the cells in the population express CD29, vimentin, CD44, and nestin to a detectable level; less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or preferably less than 1%, of the cells express Tra-1-60 to a detectable level; and further less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or preferably less than 1%, of the cells express one or more of LIN28, SOX2, OCT3 / OCT4, NANOG, ESRG, and DPPA4 to a detectable level.

[0098] Cells in these preferred populations may further express detectable levels of other markers with respect to Müller cells. Cells in these preferred populations may possess any of the advantageous properties of Müller cells discussed above.

[0099] In any of the above embodiments in which a population is defined with respect to a percentage of cells expressing a particular marker, the population preferably includes at least 5,000 cells, for example, at least 6,000, at least 7,000, at least 8,000, at least 9,000, at least 10,000, at least 20,000, at least 30,000, at least 40,000 cells, at least 50,000 cells, at least 100,000 cells, at least 200,000 cells, at least 250,000 cells, or at least 500,000 cells. The population more preferably includes at least 5,000 cells, at least 50,000 cells, or at least 250,000 cells. These populations may include any of the cell counts discussed above.

[0100] The cells and populations of the present invention are advantageous for the therapies discussed below. The cells or populations of the present invention can be isolated, substantially isolated, purified, or substantially purified. The cells or populations are isolated or purified if they contain no other components whatsoever, such as culture medium and other cells. The cells or populations are substantially isolated if they are mixed with a carrier or diluent that does not interfere with the intended use, such as culture medium. Other carriers and diluents are discussed in more detail below. The cells or populations that are substantially isolated or substantially purified do not contain any cells other than the Müller cells of the present invention.

[0101] Mueller cells can be administered to a subject in a single opportunity. Alternatively, Mueller cells can be administered to a subject in at least two opportunities, for example, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten opportunities. The intervals between opportunities can be determined by a qualified expert.

[0102] Pharmaceutical composition and administration The present invention further provides pharmaceutical compositions comprising Müller cells or populations of the present invention in combination with a pharmaceutically acceptable carrier or diluent.

[0103] The term “pharmaceutically acceptable carrier” (or “culture medium or diluent”), which can be used interchangeably with the term “biocompatible carrier” or “culture medium,” refers to reagents, cells, compounds, materials, compositions and / or dosage forms that are not only compatible with cells and other agents administered therapeutically, but also, within the bounds of sound medical judgment, free from excessive toxicity, inflammation, allergic responses or other complications in proportion to a reasonable benefit / risk ratio, and suitable for use in contact with human and animal tissues. Suitable pharmaceutically acceptable carriers for use in the present invention include liquids, semi-solids (e.g., gels) and solid materials (e.g., cell scaffolds and matrices, tubular sheets, and other such materials known in the art and described in more detail herein). These semi-solids and solid materials may be designed to withstand degradation in the body (non-biodegradable) or they may be designed to degrade in the body (biodegradable, biocorrosive). Biodegradable materials may also be bioreabsorbable or bioabsorbable, meaning they can be dissolved and absorbed into bodily fluids (water-soluble implants being one example), or they can be broken down and ultimately eliminated from the body by conversion into other materials or by decomposition and elimination through natural pathways.

[0104] Various compositions of the present invention can be formulated using any preferred method. Formulation of cells with standard pharmaceutically acceptable carriers and / or excipients can be carried out using routine methods in the pharmaceutical field. The precise nature of the formulation depends on several factors, including the cells to be administered and the desired route of administration. Preferred types of formulations are described in Remington's Pharmaceutical Sciences, 19 th This is fully documented in Edition, Mack Publishing Company, Eastern Pennsylvania, USA.

[0105] The composition can be prepared with a physiologically acceptable carrier or diluent. Generally, such compositions are prepared as a liquid suspension of cells. The cells can be mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients include, for example, water, saline solution, dextrose, glycerol, etc., and combinations thereof.

[0106] Furthermore, if desired, the pharmaceutical composition of the present invention may contain small amounts of auxiliary substances, such as humectants or emulsifiers, pH buffers, and / or adjuvants that enhance efficacy.

[0107] In a preferred embodiment: (a) A step of culturing human embryonic stem cells in a plate-based suspension in xeno-free and serum-free medium for at least 15 days in the presence of a ROCK signaling pathway inhibitor and a Wnt inhibitor. (b) Adding a synthetic cell adhesion promoter to the xeno-free and serum-free media of step (a) and culturing the cells for at least 8 days, (c) Add synthetic enriched growth factor and a smoothed protein agonist of the Hedgehog signaling pathway to the xeno-free and serum-free medium of step (b), and culture the cells for at least 3 days. (d) Add retinoic acid to the xeno-free and serum-free medium from step (c) and culture the cells from step (c) for a further 2-300 days until the retinal organoids are visible. (e) Step of dissociating the retinal organoids and isolating the Müller cells. A pharmaceutical composition is provided comprising a population of Müller cells obtainable by a method including and a pharmaceutically acceptable carrier. In a preferred embodiment, the human embryonic stem cells are RC-9 cells.

[0108] In a preferred embodiment, Müller cells of the pharmaceutical composition express detectable levels of CD29, vimentin, CD44, and nestin, but do not express detectable levels of Tra-1-60, and can secrete neurotrophins BDNF and PEDF. In a preferred embodiment, Müller cells of the pharmaceutical composition express detectable levels of CD29, vimentin, CD44, and nestin, but do not express detectable levels of Tra-1-60, and further do not express one or more detectable levels of LIN28, SOX2, OCT3 / OCT4, NANOG, and ESRG, and can secrete neurotrophins BDNF and PEDF. In a preferred embodiment, Müller cells of the pharmaceutical composition express detectable levels of CD29, vimentin, CD44, and nestin, but do not express detectable levels of Tra-1-60, and further do not express one or more detectable levels of LIN28, SOX2, OCT3 / OCT4, NANOG, ESRG, and DPPA4, and can secrete neurotrophins BDNF and PEDF.

[0109] The Müller cells or populations of the present invention, or the pharmaceutical compositions of the present invention, are administered in a manner suitable for a drug formulation and are therapeutically effective in such amounts. The amount administered depends on the subject being treated. The exact amount of Müller cells to be administered may be determined by the practitioner and may be specific to each subject.

[0110] It can be administered to any suitable number of cells. For example, at least, or about 0.2 × 10⁶ cells per kg of the target. 6 , 0.25 × 10 6 , 0.5 × 10 6 , 1.5×10 6 , 4.0×10 6 or 5.0 × 10 6 A number of cells can be administered. For example, at least, or about 10 5 , 10 6 , 10 7 , 10 8 , 10 9A number of cells can be administered. As a guideline, the number of cells of the present invention administered is 10 5 ~10 9 Preferably 10 6 ~10 8 That is acceptable. Generally, the maximum is 2 × 10 8 Individual Müller cells are administered to each subject. Any of the specific numbers discussed above with respect to the population of the present invention may be administered. In such cases where cells are administered or present, a culture medium may be present to facilitate cell survival. In some cases, the cells of the present invention may be provided in frozen aliquots, and a substance such as DMSO may be present to facilitate survival during freezing. Such frozen cells are generally thawed and then placed in a buffer or culture medium for maintenance or administration.

[0111] The cells can be administered through any appropriate route, such as intraocular, intravitreous, or subretinal.

[0112] Pharmaceuticals, methods, and therapeutic uses The Müller cells, populations, or pharmaceutical compositions of the present invention can be used in therapeutic treatments of the human body. Therefore, the present invention provides the Müller cells, populations, or pharmaceutical compositions of the present invention for use in therapeutic treatments of the human body. More specifically, the present invention relates to the use of the Müller cells, populations, or pharmaceutical compositions of the present invention for treating diseases such as retinal diseases or conditions.

[0113] A preferred embodiment is a method for treating a retinal disease or condition, comprising administering a pharmaceutical composition to a patient in need thereof, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier and a population of Müller cells, the Müller cells being: (a) A step of culturing human embryonic stem cells in a plate-based suspension in xeno-free and serum-free medium for at least 15 days in the presence of a ROCK signaling pathway inhibitor and a Wnt inhibitor. (b) Adding a synthetic cell adhesion promoter to the xeno-free and serum-free media of step (a) and culturing the cells for at least 8 days, (c) Add synthetic enriched growth factor and a smoothed protein agonist of the Hedgehog signaling pathway to the xeno-free and serum-free medium of step (b), and culture the cells for at least 3 days. (d) Add retinoic acid to the xeno-free and serum-free medium from step (c) and culture the cells from step (c) for a further 2-300 days until the retinal organoids are visible. (e) Step of dissociating the retinal organoids and isolating the Müller cells. A method is provided which includes obtaining the following. In a preferred embodiment, the human embryonic stem cells are RC-9 cells.

[0114] In a preferred embodiment, Müller cells of the pharmaceutical composition express detectable levels of CD29, vimentin, CD44, and nestin, but do not express detectable levels of Tra-1-60, and can secrete neurotrophins BDNF and PEDF. In a preferred embodiment, Müller cells of the pharmaceutical composition express detectable levels of CD29, vimentin, CD44, and nestin, but do not express detectable levels of Tra-1-60, and further do not express one or more detectable levels of LIN28, SOX2, OCT3 / OCT4, NANOG, and ESRG, and can secrete neurotrophins BDNF and PEDF. In a preferred embodiment, Müller cells of the pharmaceutical composition express detectable levels of CD29, vimentin, CD44, and nestin, but do not express detectable levels of Tra-1-60, and further do not express one or more detectable levels of LIN28, SOX2, OCT3 / OCT4, NANOG, ESRG, and DPPA4, and can secrete neurotrophins BDNF and PEDF.

[0115] In some embodiments, retinal diseases or conditions include vision loss, blindness, glaucoma, optic nerve injury, optic nerve degeneration, diseases causing damage or degeneration of the optic nerve, dominant ocular atrophy, Leber hereditary optic neuropathy, congenital amaurosis, optic neuritis, mitochondrial disorders causing optic nerve injury, ganglion cell diseases, optic nerve cell diseases, or ischemic optic neuropathy.

[0116] In some embodiments, glaucoma is as follows: (a) Primary glaucoma, including primary open-angle glaucoma, acute primary closed-angle glaucoma, chronic primary angle glaucoma, normal-tension glaucoma, childhood glaucoma and juvenile glaucoma; or (b) Developmental glaucoma, such as Axenfeld anomaly, Rieger anomaly, Rieger syndrome, aniridia and Peters anomaly, traumatic glaucoma, steroid-induced glaucoma, pseudoexfoliative glaucoma, pigmentary glaucoma, uveitis glaucoma, neovascular glaucoma, mixed-mechanism glaucoma, iris-corneal endothelial syndrome, diseases causing optic nerve damage, Posner-Schlossman syndrome, juvenile chronic rheumatoid arthritis and secondary glaucoma, including ankylosing spondylitis with secondary uveitis.

[0117] In some embodiments, the Müller cells, populations, or pharmaceutical compositions of the present invention are used to treat conditions associated with cell loss or cytotoxicity. Conditions associated with cell loss or cytotoxicity may include, for example, age-related macular degeneration, proliferative diabetic retinopathy, proliferative vitreoretinopathy, retinal detachment, pigmentary retinitis, glaucoma, or optic nerve damage and degeneration.

[0118] The present invention also provides Müller cells, populations, or pharmaceutical compositions of the present invention for use in the manufacture of pharmaceuticals for the treatment of diseases or conditions described herein.

[0119] The present invention also provides Müller cells, populations, or pharmaceutical compositions of the present invention for use in treating the diseases or conditions described herein. Various embodiments of the present invention are shown below. 1. Isolated human Müller cells, (a) expressing detectable levels of CD29, vimentin, CD44, and nestin, and not expressing detectable levels of Tra-1-60, (b) Human Müller cells capable of secreting neurotrophins BDNF and PEDF. 2. A purified, substantially homogeneous population of two or more Müller cells as described in item 1 above. 3. A population of human Müller cells, wherein at least 95% of the cells in the population express CD29, vimentin, CD44, and nestin to a detectable level, less than 5% of the cells express Tra-1-60 to a detectable level, and the cells are capable of secreting neurotrophins BDNF and PEDF. 4. Müller cells derived from human embryonic stem cells, (a) expressing detectable levels of CD29, vimentin, CD44, and nestin, and not expressing detectable levels of Tra-1-60, (b) Müller cells capable of secreting neurotrophins BDNF and PEDF. 5. Müller cells as described in item 4 above, derived from RC-9 human embryonic stem cells. 6. A purified, substantially homogeneous population of two or more Müller cells as described in 4 or 5 above. 7. A method for producing therapeutic-grade human Müller cells, a) A step of culturing RC-9 human embryonic stem cells in a plate-based suspension system in xeno-free and serum-free medium for at least 15 days in the presence of a ROCK signaling pathway inhibitor and a Wnt inhibitor. b) Adding a synthetic cell adhesion promoter to the xeno-free and serum-free media of step (a) and culturing the cells for at least 8 days, c) Add synthetic enriched growth factor and a smoothed protein agonist of the Hedgehog signaling pathway to the xeno-free and serum-free media of step (b), and culture the cells for at least 3 days. d) Add retinoic acid to the xeno-free and serum-free medium from step (c) and culture the cells from step (c) for a further 2-300 days until the retinal organoids are visible. e) The step of dissociating the retinal organoids and isolating the Müller cells. A method that includes this. 8. A pharmaceutical composition comprising Müller cells as described in 1, 4, or 5 above, or a population of Müller cells as described in 2, 3, or 6 above, or Müller cells that can be derived from 7 above, and a pharmaceutically acceptable carrier. 9. (a) A step of culturing stem cells in a plate-based system suspension in xeno-free and serum-free medium for at least 15 days in the presence of a ROCK signaling pathway inhibitor and a Wnt inhibitor. (b) Adding a synthetic cell adhesion promoter to the xeno-free and serum-free media of step (a) and culturing the cells for at least 8 days, (c) Add synthetic enriched growth factor and a smoothed protein agonist of the Hedgehog signaling pathway to the xeno-free and serum-free media of step (b), and culture the cells for at least 3 days. (d) Add retinoic acid to the xeno-free and serum-free medium from step (c) and culture the cells from step (c) for a further 2-300 days until the retinal organoids are visible. (e) Step of dissociating the retinal organoids and isolating the Müller cells. A pharmaceutical composition comprising a population of Müller cells obtainable by a method including and a pharmaceutically acceptable carrier. 10. The pharmaceutical composition according to item 9 above, wherein the stem cells are human embryonic stem cells, and optionally RC-9 human embryonic stem cells. 11. The pharmaceutical composition according to any one of 8 to 10 above, wherein the Müller cells have the characteristics of the Müller cells described in 1 above. 12. A method for treating a disease or condition of the retina, comprising administering to a patient in need a pharmaceutical composition described in 8 to 10 above, Müller cells described in 1, 4 or 5 above, a population of Müller cells described in 2, 3 or 6 above, or Müller cells that can be derived from 7 above. 13. A method for treating a disease or condition of the retina, comprising administering a pharmaceutical composition to a patient in need thereof, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier and a population of Müller cells, the Müller cells being: (a) A step of culturing stem cells in a plate-based suspension in xeno-free and serum-free medium for at least 15 days in the presence of a ROCK signaling pathway inhibitor and a Wnt inhibitor. (b) Adding a synthetic cell adhesion promoter to the xeno-free and serum-free media of step (a) and culturing the cells for at least 8 days, (c) Add synthetic enriched growth factor and a smoothed protein agonist of the Hedgehog signaling pathway to the xeno-free and serum-free media of step (b), and culture the cells for at least 3 days. (d) Add retinoic acid to the xeno-free and serum-free medium from step (c) and culture the cells from step (c) for a further 2-300 days until the retinal organoids are visible. (e) Step of dissociating the retinal organoids and isolating the Müller cells. A method obtained from a method that includes a method. 14. The method according to 13 above, wherein the stem cells are human embryonic stem cells, and in some cases RC-9 human embryonic stem cells. 15. The method according to any one of items 12 to 14 above, wherein the disease or condition of the retina is visual impairment, blindness, glaucoma, optic nerve injury, optic nerve degeneration, a disease causing damage or degeneration of the optic nerve, dominant ocular atrophy, Leber hereditary optic neuropathy, congenital amaurosis, optic neuritis, mitochondrial disorder causing optic nerve injury, ganglion cell disease, optic nerve cell disease, or ischemic optic neuropathy. 16. The aforementioned glaucoma: (a) Primary glaucoma, including primary open-angle glaucoma, acute primary closed-angle glaucoma, chronic primary angle glaucoma, normal-tension glaucoma, childhood glaucoma and juvenile glaucoma; or (b) Developmental glaucoma, such as Axenfeld anomaly, Rieger anomaly, Rieger syndrome, aniridia and Peters anomaly, traumatic glaucoma, steroid-induced glaucoma, pseudoexfoliative glaucoma, pigmentary glaucoma, uveitis glaucoma, neovascular glaucoma, mixed-mechanism glaucoma, iris-corneal endothelial syndrome, diseases causing optic nerve damage, Posner-Schlossman syndrome, juvenile chronic rheumatoid arthritis and secondary glaucoma, including ankylosing spondylitis with secondary uveitis. The method described in item 15 above. 17. A method for producing therapeutic-grade human Müller cells, a) A step of culturing stem cells in a plate-based suspension in xeno-free and serum-free medium for at least 15 days in the presence of a ROCK signaling pathway inhibitor and a Wnt inhibitor; b) Adding a synthetic cell adhesion promoter to the xeno-free and serum-free media of step (a) and culturing the cells for at least 8 days; c) Add synthetic growth factor and a smoothed protein agonist of the Hedgehog signaling pathway to the xeno-free and serum-free media of step (b), and culture the cells for at least 3 days; d) Adding retinoic acid to the xeno-free and serum-free medium from step (c) and culturing the cells from step (c) for a further 2-300 days until retinal organoids are visible; and e) The step of dissociating the retinal organoids and isolating the Müller cells. A method that includes this. 18. The method according to 17, wherein the synthetic cell adhesion promoter in step (b) is a synthetic vitronectin-based glycoprotein. 19. The method according to 17 or 18 above, wherein the synthetic enrichment growth factor in step (c) is human platelet lysate. 20. The method according to any one of 17 to 19 above, wherein the cell medium of stage (d) further comprises human transferrin. 21. A method for producing Müller cells, a) The step of culturing retinal organoids in human platelet lysates; and b) Steps to dissociate the retinal organoids without incision and isolate the Müller cells. A method that includes this. 22. A method for producing a pure population of Müller cells: a) A step of culturing retinal organoids; b) A step of dissociating the retinal organoids and isolating the Müller cell suspension enriched with them; c) the step of culturing the Müller cells on a surface coated with fibronectin; and d) The step of isolating the Müller cells on fibronectin to form a pure population thereof. A method that includes this. 23. The method according to 22, wherein a further step (e) comprises growing the pure population of cells from step (d) in a medium supplemented with fibroblast growth factor (FGF) or epidermal growth factor (EGF) or FGF and EGF. 24. A method for producing therapeutic-grade pure human Müller cells, a) A step of culturing stem cells in a plate-based suspension in xeno-free and serum-free medium for at least 15 days in the presence of a ROCK signaling pathway inhibitor and a Wnt inhibitor; b) Adding a synthetic cell adhesion promoter to the xeno-free and serum-free media of step (a) and culturing the cells for at least 8 days; c) Add synthetic growth factor and a smoothed protein agonist of the Hedgehog signaling pathway to the xeno-free and serum-free media of step (b), and culture the cells for at least 3 days; d) Adding retinoic acid to the xeno-free and serum-free medium from step (c) and culturing the cells from step (c) for a further 2-300 days until retinal organoids are visible; e) The step of dissociating the retinal organoids and isolating the Müller cell suspension enriched with them; f) the step of culturing the Müller cells on a surface coated with fibronectin; and g) The step of isolating the Müller cells on fibronectin to form a pure population thereof. A method that includes this. 25. The method according to 24, wherein a further step is to grow the pure population of cells from step (g) in a medium supplemented with fibroblast growth factor (FGF) or epidermal growth factor (EGF) or FGF and EGF. 26. The method according to any one of 1 to 25 above, wherein the plate base system is a V-bottom well plate. 27. Müller cells obtained by any of the methods described in 17-26 above. 28. Use of Müller cells as described in 27 above in the manufacture of a pharmaceutical product for the treatment of a condition associated with cell loss or cell injury. 29. Use of Müller cells as described in 27 above in the manufacture of a pharmaceutical product for the treatment of any one of the following: age-related macular degeneration, proliferative diabetic retinopathy, proliferative vitreoretinopathy, retinal detachment, retinitis pigmentosa, glaucoma, or damage and degeneration of the optic nerve. 30. A cell medium for Müller cell differentiation, essentially consisting of minimal essential synthetic basal media, a carbon source, non-essential amino acids, a ROCK inhibitor, human platelet lysate, a Wnt inhibitor, and GMP-compliant synthetic vitronectin, a synthetic vitronectin-based substrate, or a hydrogel scaffold. 31. The cell medium according to 30 above, comprising an agonist of a smoothed protein of the Hedgehog signaling pathway. 32. A cell medium for Müller cell differentiation, essentially consisting of minimal essential synthetic basal medium, N2 auxiliary, retinoic acid, and human platelet lysate. [Examples]

[0120] [Example 1] Stage 1: Maintenance of RC-9 hES (human embryonic stem) cells Maintenance: PSC (pluripotent stem cell) cultures were maintained as feeder-free colonies on 6-well plates coated with human ESC-qualified Matrigel® in 2 mL of TeSR-E8® medium supplemented with 50 μM gentamicin or penicillin / streptomycin. Matrigel® is the trade name for a gel-like protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, produced and marketed by Corning Life Sciences and BD Biosciences. The main components of Matrigel® are type IV collagen, laminin, heparan sulfate proteoglycan, and entactin.

[0121] Cell lines were observed daily to examine PSC-like morphology, differentiated cells, and the presence of dense clusters. Any differentiated cells were isolated under an EVOS XLCore microscope in a tissue culture hood using a 200 μL pipette tip and sterile conditions. Other microscopes can be used if they have a good ×40 objective lens and can be used in a culture hood.

[0122] Cells were nourished daily by carefully aspirating the culture medium from the wells and gently adding 2 mL of fresh TeSR-E8® medium containing gentamicin or penicillin / streptomycin. Weekend nourishment consisted of a medium change to essential 8Flex medium on Friday, which was then replaced with TeSR-E8® medium on Monday. An alternative form of this method is to use iPS Brew with TGF-β and perform triple nourishment on Friday.

[0123] Growth: After daily observation, when the colonies were approximately 70% dense, colonies with distinct edges were separated in a 1:6 ratio. To achieve this, the culture medium was aspirated from each well, and the cells were first washed with 1 mL of PBS (phosphate-buffered saline). 1 mL of 0.5 mM EDTA (ethylenediaminetetraacetic acid) in PBS was then added, and the cells were incubated at room temperature for 4–6 minutes until small holes appeared throughout the colonies. The EDTA solution was then aspirated, 2 mL of TeSR-E8® medium was added, and the cells were gently washed from the plate by gently repeating pipetting operations to remove the colonies. The cell suspension was then diluted to the desired separation ratio and seeded into a 6-well plate coated with Matrigel®.

[0124] A variation of this method is to use a 6-well plate coated with vitronectin instead of a plate coated with Matrigel®. Another variation of this method is to use iPS-Brew and TGF beta instead of TeSR-E8® and Essential 8Flex medium. A further variation is to use TrypLE instead of EDTA to remove cells.

[0125] Freezing of PSCs. After the cells reached 70% compaction, the medium was aspirated and the cells were washed with 1 mL of PBS. The cells were then dissociated by incubation (as described above) at room temperature for 4–6 minutes using 1 mL of 0.5 mM EDTA in PBS. After aspirating the EDTA, the cells were gently dissociated using mFreSR® cryopreservation medium. mFreSR® is a standard serum-free cryopreservation medium designed for the cryopreservation of human embryonic and induced pluripotent stem cells (iPSCs). mFreSR® contains dimethyl sulfoxide (DMSO). Each 6-well plate generates enough cells for 6 vials of 1 mL cell suspension in mFreSR® cryopreservation medium. The vials containing 1 mL of cell suspension were then frozen at -80°C using a cell freezing container filled with isopropanol, and then transferred to liquid nitrogen for long-term storage.

[0126] A variation of this method is to replace the mFreSR® cryopreservation medium with Stemcell Technologies' CS10 or CS2. CS10 and CS2 are serum-free, animal-component-free standard cryopreservation media containing 10% or 2% dimethyl sulfoxide (DMSO), respectively.

[0127] Thawing of PSCs. Before thawing the cells, a 6-well plate coated with Matrigel® was prepared at room temperature for 1 hour. The cell vials were thawed for 3 minutes using a 37°C water bath. The cell suspension was then collected in 2 mL of TeSR-E8® medium containing 10 μM ROCKi (Rock inhibitor) and centrifuged at 300 g to obtain a cell pellet. The supernatant was removed, and the cell pellet was resuspended in 2 mL of TeSR-E8® containing 10 μM ROCKi and 50 μM gentamicin for plate culture in pre-coated Matrigel® wells.

[0128] A variation of this protocol is to use iPS-Brew without ROCKi instead of TeSR-E8. Another variation is to use vitronectin instead of Matrigel®. Further variations include using penicillin / streptomycin instead of gentamicin, or not using any type of antibiotic at all.

[0129] Stage 2: Differentiation To create retinal organoids, a method based on that of Tokushige Nakano et al. (Cell Stem Cell 10, 771-785, June 14, 2012) is used. Nakano does not describe Müller glial identification, but it has been reported in other contexts; Xiufeng Zhong, et al. Nature Communications. Volume 5, Article number: 4047 (2014) and Chen HY et al. Molecular Vision. 09 Sep 2016, 22:1077-1094.

[0130] Three different culture media are used in Stage 2 as follows: Culture medium 1 384.5 mL of GMEM (Glasgow Minimum Essential Medium) containing L-glutamine 100 mL of KOSR (Knockout Serum Replacement) 5 mL of 100x sodium pyruvate 5 mL of 100x non-essential amino acids 5 mL of 100x penicillin / streptomycin Culture medium 2 334.5 mL of GMEM containing L-glutamine 100mL KOSR 50 mL HPL 5 mL of 100x sodium pyruvate 5 mL of 100x non-essential amino acids 5 mL of 100x penicillin / streptomycin Culture medium 3 439.5 mL of DMEM (Dulbecc's Modified Eagle Medium) / F12-Glutamax 50 mL HPL 5 mL of 100 × N2 auxiliary agent 5 mL of 100x penicillin / streptomycin / amphotericin 0.5 μM retinoic acid

[0131] Other factors added to the culture medium at various concentrations as indicated in the text below include: ROCK inhibitor in medium 1, 20 μM Wnt antagonist IWR-1-endo in medium 1, 3 μM Medium 2: SAG (Smoothed Agonist), 100 nM Human-grade Matrigel® in medium 1 on day 2, 2% (w / v) Matrigel® (final concentration of 1% Matrigel®), and 1% (w / v) Matrigel® on days 5-18.

[0132] In the modified form of the culture medium shown above, antibiotics can be omitted.

[0133] In the modified form of the culture medium shown above, Synthemax is a human-compatible Matrigel® substitute.

[0134] Nutritional support on day 0 As shown above, PSCs grown in a compacted monolayer in a 6-well plate were used for differentiation into retinal organoids. After compaction, the cells were washed with 1×PBS and dissociated at 37°C by 1 mL of TrypLE(×1) trypsin substitution containing 10 μM ROCKi and 0.5 mg / mL DNase. TrypLE inactivation was performed by adding 5 mL of "Medium 1". The cells were then pelleted by centrifugation at 300 g for 5 minutes.

[0135] The supernatant was discarded, and the cells were suspended in 2 mL of "Medium 1" containing 10 μM ROCKi for cell counting. The cells were then refrozen in "Medium 1" containing 20 μM ROCKi and 3 μM Wnt antagonist, resulting in a count of 9 × 10⁶ cells. 4 The concentration was then adjusted to / mL. Aliquots of 100 μL (9,000 cells) were placed in each well of a 96-well V-bottom plate, and the cells were cultured at 37°C in 5% CO2 and atmospheric O2.

[0136] Nutritional supplementation on days 2, 5, and 9 Two days after incubation, 100 μL of "Culture Medium 1," containing 20 μM ROCKi, 3 μM Wnt antagonist, and 2% (w / v) Matrigel® (final Matrigel® concentration 1%), was added to each well of a 96-well V-bottom plate.

[0137] After 5 and 9 days, partial replacement of the culture medium was performed by removing 100 μL of the medium and replacing it with 100 μL of "Medium 1" containing 20 μM ROCKi, 3 μM Wnt antagonist, and 1% (w / v) Matrigel™.

[0138] Nutritional supplementation on day 12 Using a 1,000 μL pipette tip, the formed embryoid bodies (EBs) were gently transferred to each well of a 25-well square low-adhesion plate, with the tip cut to avoid any damage to the EBs. Individual EBs were moved in approximately 100–200 μL of culture medium. Each well was then filled with 1 mL of "Medium 2" containing 1% (w / v) Matrigel® and 100 nM SAG. The plates were then incubated at 37°C as described above.

[0139] Nutritional supplementation on day 15 The culture medium was replaced with fresh "Medium 2" containing 1% (w / v) Matrigel™ and 100 nM SAG. EB is an indicator of retinal organoids and was examined by inverted microscopy for the clear appearance of the "mantle" visible from day 15.

[0140] Nutritional support from day 18 onwards The culture medium was removed from each well and replaced with "Culture Medium 3". EB was given nutritional supplements twice a week.

[0141] From days 15 to 90, retinal organoids were dissected under microscopic conditions using a microblade. This procedure was performed to purify optic cup structures from EB. The organoids were then transferred to new 25-well square low-adhesion plates and kept in "Medium 3" for long-term culture with two medium changes per week. Müller cells were isolated from the retinal organoids 30 to 300 days after the start of the differentiation protocol.

[0142] A variation of this method involves dissecting all organoids on a single day between day 50 and day 57.

[0143] Stage 3: Differentiation and proliferation Retinal organoids containing human Müller cells were collected 30 to 300 days after the onset of retinal differentiation. A variation of this method involves dissociating all organoids on day 70.

[0144] Organoids were dissociated using a papain dissociation kit protocol supplied by the manufacturer (Worthington Biochemical) and used according to the manufacturer's instructions for use. Single or multiple pooled organoids were isolated using this protocol.

[0145] First, 32 mL of Earle's equilibrium salt solution (EBSS) (Vial 1) was added to the albumin ovomucoid inhibitor mixture (Vial 4), and the contents were dissolved while the other components were prepared. Next, 5 mL of EBSS (Vial 1) was added to the papain vial (Vial 2), and the mixture was placed in a 37°C water bath for 10 minutes, or until the papain was completely dissolved. During dissociation, this solution was used immediately at room temperature.

[0146] 500 μL of EBSS was added to the DNase vial (Vial 3) and gently mixed. 250 μL of this solution was added to the vial containing papain. This preparation contained approximately 20 units / mL of papain and a final concentration of 0.005% DNase. The tissue was placed in the papain solution. The vial containing the tissue was left at 37°C for 30 minutes to 1.5 hours, while being agitated by pipetting and dispensing every 10 minutes.

[0147] After incubation with papain, the mixture was then crushed using a 1 mL pipette. Any remaining undissociated tissue after crushing was allowed to settle at the bottom of the tube. The turbid cell suspension was carefully removed and placed in a sterile screw-cap tube, and centrifuged at 300 g for 5 minutes at room temperature. The supernatant was discarded, and the cell pellet was immediately resuspended in a DNase-diluted albumin-inhibitor solution [2.7 mL of EBBS (Earle's equilibrium salt solution) (Vial 1); 300 μL of recomposed albumin-ovomucoid inhibitor solution (Vial 4); 150 μL of DNase solution (Vial 3)].

[0148] A discontinuous density gradient was prepared as follows: 5 mL of albumin-inhibitor solution (vial 4) was added to a centrifuge tube, the cell suspension was carefully placed on top, and the tube was centrifuged at 70 g for 6 minutes at room temperature. The interface between the two layers of the gradient was clearly visible. The supernatant was discarded, leaving the cell pellet.

[0149] A variation of this method involves using a Gentle Cell Dissociation reagent, such as one supplied by Worthington Biochemical, instead of a papain dissociation kit. In this method using the Gentle Cell Dissociation reagent, 200 μL is added per organoid, the organoid is ground, and the organoid is placed in an incubator. It is typically ground for only 10 minutes at 5-minute intervals, but this can be up to 2 hours for complete dissociation.

[0150] After dissociation from retinal organoids, the cells were centrifuged to obtain a pellet and immediately resuspended in a cell medium consisting of DMEM (Dulbecco's Modified Eagle Medium) containing 10% (v / v) FBS, 20 ng / mL FGF, and 20 ng / mL EGF. The plates and flasks used to culture the Müller cells were pre-coated with fibronectin (50 μg / mL, 37°C for 2 hours).

[0151] Cells isolated from a single retinal organoid were first cultured in the wells of a 24-well plate, grown to density, and then subcultured in a single well of a 6-well plate. After reaching density in the 6-well plate, the cells were transferred to a T25 tissue culture flask and then to a T75 culture flask. Subsequent subculturing was consistently performed in the T75 flask at a 1:3 dilution until a sufficient number of cells for gene and protein expression analysis were obtained.

[0152] Approximately 1 x 10 6 It takes approximately 1-2 weeks from the day of isolation to obtain cells. The inventors also have evidence that the yield of Müller cells cultured and expanded using the GMP-compliant method of the present invention is significantly higher and faster than the non-GMP / research-grade method (yield of approximately 1e6 cells after about 28-30 days) (yield of approximately 1e6 cells per colony in 7 days, as estimated from pooling, and approximately 60e6 cells within 14 days). Cell counts may be increased in 10-degree increments as needed.

[0153] 1 x 10 per vial 6 This was further increased over an additional four weeks to create a bank of frozen cell vials.

[0154] [Example 2] Cell characterization The inventors of the present invention induce allogeneic Müller cells from hESCs (RC-9 cell line) using an optimized GMP-compliant method of the present invention, and these cells • To express important Müller markers, • Does not express stem cell markers. • Secreting important neurotrophins BDNF and PEDF, • To increase RGC survival in vitro. • To increase the function of RGCs in vivo This was shown.

[0155] Müller cells differentiated from RC-9 cells do not express the stem marker Tra-1-60. The expression of the stem cell marker Tra-1-60 was measured on the surface of undifferentiated RC-9 cells using flow cytometry and compared to its expression on the surface of Müller cells differentiated from RC-9 cells (generated using a GMP-compliant protocol). Undifferentiated RC-9 cells were highly positive for Tra-1-60 (99.44% of the population were positive), indicating the stem cell state of the cells. As shown in Figure 4, Tra-1-60 expression is lost after differentiation of RC-9 cells into Müller cells.

[0156] Müller cells differentiated from RC-9 cells express markers associated with Müller cells. Müller cells differentiated from RC-9 cells using a GMP-compliant protocol were characterized for the expression of Müller markers using flow cytometry. Compared to negative isotype controls, vimentin, CD29, CD44, and nestin were highly expressed by the induced Müller cells. Figure 5 shows that Müller cells differentiated from RC-9 cells express Müller cell-associated markers.

[0157] Müller cells differentiated from RC-9 cells secrete neuroprotective factors and antioxidants known to support RGC function to a higher degree than differentiated hESCs. Müller cells differentiated from RC-9 cells using a GMP-compliant protocol were characterized for neuroprotective factor and antioxidant secretion using ELISA. The concentrations of BDNF, PEDF, and PRDX6 measured by ELISA in the supernatant from Müller cells were significantly higher than those observed in hESCs of undifferentiated RC-9 cells.

[0158] Müller cells differentiated from RC-9 cells express and secrete Müller cell-related markers to a higher degree than Müller cells differentiated from iPS cells. Mueller cells differentiated from RC-9 cells using a GMP-compliant protocol were characterized for Mueller marker expression using transcriptomics and quantification of secretion levels in the cell supernatant using ELISA. Mueller cells generated using the GMP-compliant protocol of the present invention expressed and secreted more BDNF (Figure 7) and more PEDF (Figure 8) than the published cells differentiated from iPS cells by Eastlake et al. 2019.

[0159] Therefore, the Müller cells of the present invention not only express the genes for both BDNF and PEDF-Müller cells, two important neurotrophins related to the mechanism of action of Müller cells, but also express these important neurotrophins to a higher degree than Müller cells known in the art that are produced by other means.

[0160] Müller cells differentiated from RC-9 cells do not express the pluripotency markers LIN28, SOX2, OCT3 / 4, NANOG, ESRG, and DPPA4. The expression of pluripotency markers LIN28, SOX2, OCT3 / 4, NANOG, ESRG, and DPPA4 was measured in undifferentiated RC-9 cells using transcriptomics and compared to their expression on the surface of Müller cells differentiated from RC-9 cells (generated using a GMP-compliant protocol). Undifferentiated RC-9 cells highly expressed all markers, indicating the stem cell state of the cells. As shown in Figure 9, the expression of all markers was lost after differentiation of RC-9 cells into Müller cells.

[0161] Müller cells differentiated from RC-9 express the marker POU5F1(OCT3) at a considerably lower level than Müller cells differentiated from iPS cells. Gene expression of the pluripotency marker OCT3 was measured using transcriptomics in Müller cells differentiated from iPS cells according to the Eastlake et al. 2019 protocol and compared with expression in Müller cells differentiated from RC-9 cells (generated using the GMP-compliant protocol of the present invention). Müller cells differentiated from iPS cells according to the Eastlake et al. 2019 protocol expressed significantly higher levels of OCT3 than Müller cells of the present invention (Figure 10). OCT3 is a pluripotency marker that is expected to decrease after differentiation. Therefore, products with lower levels of this gene / protein would be considered safer for administration to patients.

[0162] Mueller cells differentiated from RC-9 enhance RGC survival in vitro. Mueller cells differentiated from RC-9 cells and generated using the GMP-compliant protocol of the present invention enhance RGC survival after treatment with excess glutamate, as demonstrated by increased neurite length (Figure 11A and B).

[0163] Müller cells differentiated from RC-9 enhance RGC survival in vivo. The Müller cells of this invention are effective in an NMDA rodent model of RGC loss, consistent with previously published data, and improved RGC function (nSTR) as measured by ERG in the NMDA model. Treatment of rat eyes with NMDA mimics the RGC loss observed in glaucoma. NMDA erases the RGC layer and worsens vision, making it an ideal model for investigating the effects of transplanted human Müller cells in vivo. As measured by electroretinography, NMDA suppresses b-waves and nSTR (negative dark adaptation response). nSTR is directly related to RGC function. Figure 12 shows that Müller cells differentiated from RC-9 cells and generated using the GMP-compliant protocol of this invention partially restore nSTR and improve RGC function.

[0164] In conclusion, Müller cells can be derived from hESCs such as RC-9 cells and cultured in vitro and in vivo to provide biochemical and metabolic support. The Müller cells of the present invention are characterized by Müller cell markers and express appropriate neurotrophins and antioxidants at biologically significant levels. Furthermore, the Müller cells of the present invention exhibit improved properties compared to published Müller cells. In addition, the Müller cells of the present invention enhance RGC neurite proliferation in vitro and enhance RGC function as measured by ERG after in vivo transplantation into injured eyes.

Claims

1. Müller cells derived from RC-9 human embryonic stem cells, (a) expressing detectable levels of CD29, vimentin, CD44, and nestin, and not expressing detectable levels of Tra-1-60, (b) Müller cells capable of secreting neurotrophins BDNF and PEDF.

2. A purified, substantially homogeneous population of two or more Müller cells as described in claim 1.

3. A method for producing therapeutic-grade human Müller cells, a) A step of culturing RC-9 human embryonic stem cells in a plate-based suspension in xeno-free and serum-free medium for at least 15 days in the presence of a ROCK signaling pathway inhibitor and a Wnt inhibitor. b) Adding a synthetic cell adhesion promoter to the xeno-free and serum-free media of step (a) and culturing the cells for at least 8 days, c) Adding synthetic enrichment growth factor and a smoothed protein agonist of the Hedgehog signaling pathway to the xeno-free and serum-free media of step (b), and culturing the cells for at least three days. d) Adding retinoic acid to the xeno-free and serum-free medium from step (c) and culturing the cells from step (c) for a further 2 to 300 days until the retinal organoids are visible. e) The step of dissociating the retinal organoids and isolating the Müller cells. A method that includes this.

4. A pharmaceutical composition comprising Müller cells as described in claim 1, or a population of Müller cells as described in claim 2, or Müller cells that can be derived from claim 3, and a pharmaceutically acceptable carrier.

5. (a) A step of culturing RC-9 human embryonic stem cells in a plate-based suspension in xeno-free and serum-free medium for at least 15 days in the presence of a ROCK signaling pathway inhibitor and a Wnt inhibitor. (b) Adding a synthetic cell adhesion promoter to the xeno-free and serum-free media of step (a) and culturing the cells for at least 8 days, (c) Add synthetic enrichment growth factor and a smoothed protein agonist of the Hedgehog signaling pathway to the xeno-free and serum-free media of step (b), and culture the cells for at least three days. (d) Adding retinoic acid to the xeno-free and serum-free medium from step (c) and culturing the cells from step (c) for a further 2 to 300 days until the retinal organoids are visible. (e) The step of dissociating the retinal organoid and isolating the Müller cells. A pharmaceutical composition comprising a population of Müller cells obtainable by a method including and a pharmaceutically acceptable carrier.

6. A pharmaceutical composition according to claim 4 for treating a disease or condition of the retina, or a composition comprising Müller cells according to claim 1, a population of Müller cells according to claim 2, or Müller cells that can be derived from claim 3 for treating a disease or condition of the retina.

7. A pharmaceutical composition for treating a disease or condition of the retina, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier and a population of Müller cells, the Müller cells being: (a) A step of culturing RC-9 human embryonic stem cells in a plate-based suspension in xeno-free and serum-free medium for at least 15 days in the presence of a ROCK signaling pathway inhibitor and a Wnt inhibitor. (b) Adding a synthetic cell adhesion promoter to the xeno-free and serum-free media of step (a) and culturing the cells for at least 8 days, (c) Add synthetic enrichment growth factor and a smoothed protein agonist of the Hedgehog signaling pathway to the xeno-free and serum-free media of step (b), and culture the cells for at least three days. (d) Adding retinoic acid to the xeno-free and serum-free medium from step (c) and culturing the cells from step (c) for a further 2 to 300 days until the retinal organoids are visible. (e) The step of dissociating the retinal organoid and isolating the Müller cells. A pharmaceutical composition obtained by a method including [a certain element].

8. The pharmaceutical composition or composition according to claim 6, wherein the disease or condition of the retina is loss of vision, blindness, glaucoma, optic nerve injury, optic nerve degeneration, a disease causing damage or degeneration of the optic nerve, dominant ocular atrophy, Leber hereditary optic neuropathy, congenital amaurosis, optic neuritis, mitochondrial disorder causing optic nerve injury, ganglion cell disease, optic nerve cell disease, or ischemic optic neuropathy.

9. The aforementioned glaucoma: (a) Primary glaucoma, including primary open-angle glaucoma, acute primary closed-angle glaucoma, chronic primary angle glaucoma, normal-tension glaucoma, childhood glaucoma and juvenile glaucoma; or (b) Developmental glaucoma, e.g., Axenfeld anomaly, Rieger anomaly, Rieger syndrome, aniridia and Peters anomaly, traumatic glaucoma, steroid-induced glaucoma, pseudo-epidermal exfoliative glaucoma, pigmentary glaucoma, uveitis glaucoma, neovascular glaucoma, mixed-mechanism glaucoma, iris-corneal endothelial syndrome, diseases causing optic nerve damage, Posner-Schlossman syndrome, juvenile chronic rheumatoid arthritis and secondary glaucoma, including ankylosing spondylitis with secondary uveitis. The pharmaceutical composition or composition according to claim 8.

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