Methods and Compositions for Treating Vision Loss

Abstract

Described herein are methods for differentiating human pluripotent stem cells into populations of retinal cells, including photoreceptor neuronal cells (PNCs), in dynamic culture. Also provided are cells and cell compositions obtained by such methods, and uses of the cells in suspension or embedded within a scaffold, which may be administered to a subject suffering from a retinal disease or disorder.

Description

[Technical field]

[0001] Related Applications This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 334,593, filed April 25, 2022. The contents of the aforementioned patent application are incorporated herein by reference in their entirety. [Background technology]

[0002] At least 2.2 billion people worldwide have vision impairment. In the United States alone, cases of early age-related macular degeneration in people over 50 years of age are expected to double by 2050, from 9.1 million to 17.8 million, and cases of diabetic retinopathy in people over 65 years of age are expected to quadruple by 2050, from 2.5 million to 9.9 million (www.cdc.gov / visionhealth / pdf / vision_brief.pdf). The leading causes of blindness and vision loss in the United States are eye diseases, primarily age-related, such as age-related macular degeneration and diabetic retinopathy, although genetic disorders also play a role. In many cases, the cause of vision loss is related to the degeneration and death of cells in the retina and associated structures.

[0003] Retinal cells, such as photoreceptor neuronal cells (PNC), are intended as cell therapy for neurodegenerative patients who suffer from significant loss of vision. Transplanted retinal cells must exhibit high viability and connectivity with the surrounding functional layers, including functional neural connections, for vision restoration. Cell therapy using retinal cells may be used for patients with partial or complete blindness with widespread damage, which may include irreversible damage to photoreceptor cells, as well as for patients with neurodegenerative diseases with direct retinal damage to photoreceptor cells, such as retinitis pigmentosa or Stargardt's disease. Both of these diseases are genetic disorders that cause significant damage at a relatively young age. Thus, improved methods and compositions comprising populations of retinal cells are needed. Summary of the Invention

[0004] Provided herein is a method for differentiating human pluripotent stem cells into a population of cells, including retinal cells, such as photoreceptor neuronal cells (PNC), in dynamic culture. The method can be adapted for commercial scale processing. Also provided herein is a composition comprising cells and cell populations obtained by the methods described herein. Also provided herein is a method for treating retinal diseases, including, for example, vision loss, using cells and cell populations obtained by the methods described herein.

[0005] The disclosure provides a pharmaceutical composition comprising a population of retinal cells, where (a) 10% or more of the cells in the population express cone-rod homeobox (Crx), (b) 3% or more of the cells in the population express recoverin, (c) 3% or more of the cells in the population express cone arrestin (CAR), and (d) 1% or less of the cells in the population express TRA-1-60 and / or SSEA5, and the pharmaceutical composition comprises a pharma- ceutically acceptable carrier.

[0006] In some embodiments of the pharmaceutical compositions of the present disclosure, 15% or more of the cells in the population express Crx. In some embodiments, 5% or more of the cells in the population express recoverin. In some embodiments, 6% or more of the cells in the population express CAR.

[0007] In some embodiments, the population of retinal cells is a population of retinal cells expressing SIX homeobox 3 (six3), SIX homeobox 6 (six6), phosphodiesterase 6H (PDE 6H), visual system homeobox 2 (CHX10 or VSX2), pre-melanosome protein (PMEL), protein kinase C alpha (PKCa), ELAV-like RNA binding protein 3 / 4 (HuC / D), orthodenticle homeobox 2 (Otx2), neuronal differentiation 1 (NeuroD), B-lymphocyte-induced maturation protein-1 (Blimp1), transducin, phosducin (PdC), retinoid X receptor gamma (RXRy), thyroid hormone receptor isoform (Tr-32), atonal bHLH transcription factor 7 (Atoh7), insulin gene enhancer protein (Isl-1), retinal homeobox gene 1 (Rx1), paired box 6 (Pax6), LIM homeobox 2 (LHX2), and retina and anterior neural fold homeobox (RAX).

[0008] In some embodiments of the pharmaceutical compositions of the disclosure, (e) 30% or more of the cells in the population express Pax6, (f) 40% or less of the cells in the population express beta-tubulin 3, (g) 30% or less of the cells in the population express PMEL, (h) 30% or less of the cells in the population express PKCa, or (i) 10% or less of the cells in the population express HuCD. In some embodiments, 0.1% or less of the cells in the population express TRA-1-60 and / or SSEA5.

[0009] In some embodiments of the pharmaceutical compositions of the present disclosure, (a) between about 10% and 70% of the cells in the population express Crx, (b) between about 3% and 90% of the cells in the population express recoverin, (c) between about 3% and 90% of the cells in the population express CAR, and (d) between 0 and about 0.1% of the cells in the population express TRA-1-60 and / or SSEA5.

[0010] In some embodiments of the pharmaceutical composition of the present disclosure, the population of retinal cells comprises early eye field cells, embryonic retinal cells, progenitor cells of photoreceptor neuronal cells (PNC), mature photoreceptor neuronal cells, or any combination thereof. In some embodiments, (i) the early eye field cells comprise cells expressing Six3, Six6, Rx1, Pax6, RXRy, LHX2, and / or RAX, (ii) the embryonic retinal cells comprise cells expressing Otx2, NeuroD, Blimp1, transducin, phosducin (PdC), RXRy, and Tr-32, Atoh7, and / or Isl-1, or (iii) the progenitor cells of PNC and mature PNC comprise cells expressing Crx, recoverin, and / or cone arrestin (CAR). In some embodiments, the population of retinal cells comprises neuronal retinal cells (NRC), Müller glial cells, retinal pigment epithelium (RPE) cells, or any combination thereof. In some embodiments, the NRCs comprise PNCs, retinal ganglion cells, horizontal neurons, amacrine neurons, rod bipolar cells, cone bipolar cells, rod photoreceptor cells, cone photoreceptor cells, or any combination thereof.

[0011] In some embodiments of the pharmaceutical composition of the present disclosure, the pharmaceutical composition comprises a cryopreservation medium. In some embodiments, the cryopreservation medium comprises a cryoprotectant selected from the group consisting of glycerol, sucrose, dextran, and dimethyl sulfoxide (DMSO). In some embodiments, the pharmaceutical composition comprises about 0.1% to about 40% cryoprotectant. In some embodiments, the pharmaceutical composition comprises about 1% to about 10% cryoprotectant.

[0012] In some embodiments of the pharmaceutical compositions of the present disclosure, the population of retinal cells comprises between 5,000 cells and 25 million cells. In some embodiments, the population of retinal cells comprises between 1×10 5 cells / mL ~ approx. 100×10 6 The concentration is in cells / mL. In some embodiments, the population of cells is in suspension.

[0013] In some embodiments of the pharmaceutical compositions of the present disclosure, the population of cells is present in a scaffold. In some embodiments, the scaffold is biocompatible and / or biodegradable.

[0014] Provided herein is a method for obtaining a population of neural retinal cells (NRCs) from undifferentiated pluripotent stem cells. The method includes (a) growing undifferentiated pluripotent stem cells in static adherent culture or, optionally, dynamic culture; (b) seeding the undifferentiated pluripotent stem cells as single cells in a culture vessel at a cell concentration of about 100,000 cells / mL to about 2,000,000 cells / mL; and (c) differentiating the undifferentiated pluripotent stem cells in the culture vessel under conditions to obtain a population of NRCs. In some embodiments, the retinal cells are neural retinal cells, such as PNCs, retinal ganglion cells, horizontal neurons, amacrine neurons, rod bipolar cells, cone bipolar cells, rod and / or cone photoreceptors, as well as Müller glial cells, retinal pigment epithelial (RPE) cells. In embodiments, the NRCs include PNCs. In embodiments, the retinal cells include retinal pigment epithelial cells (RPE). In embodiments, the retinal cells include a mixture of PNCs and RPEs. In embodiments, the retinal cells include one or more of PNCs, RPE, bipolar cells, retinal ganglion cells, horizontal cells, and amacrine cells.

[0015] In embodiments, the undifferentiated pluripotent stem cells are human embryonic stem cells (hESCs). In other embodiments, the undifferentiated pluripotent stem cells are human induced pluripotent stem cells (hiPSCs).

[0016] In some embodiments, undifferentiated pluripotent stem cells are seeded for differentiation as single cells and grown in suspension as cell aggregates for at least 14-18 weeks.

[0017] In some embodiments, the aggregates have a size that is controlled by the rotation speed of the culture vessel. In embodiments, the size of the aggregates is about 100 μm to about 800 μm. For example, the rotation speed may be increased during the differentiation process.

[0018] In some embodiments, differentiating the undifferentiated pluripotent stem cells comprises: The method includes the steps of: (i) culturing the undifferentiated pluripotent stem cells for a first period of time under culture conditions sufficient to induce the stem cells to form cell aggregates (e.g., organoids), the cell aggregates comprising cells expressing one or more early eye field markers selected from Rxl, Pax6, RXRy, LHX2, Six3, Six6, Otx2, and RAX; (ii) culturing the cell aggregates from step i) for a second period of time under culture conditions sufficient to differentiate the cell aggregates into cells expressing one or more embryonic retinal markers selected from Otx2, NeuroD, Blimp 1, transducin, phosducin (PdC), RXRy, and Tr-32, Atoh7, or Isl-1; and (iii) culturing the cells expressing one or more embryonic retinal markers from step (ii) for a third period of time under culture conditions sufficient to differentiate the cells into precursor cells of PNC and mature photoreceptor neurons, the PNC expressing Crx, recoverin, PDE. and expressing one or more proteins selected from 6H (phosphodiesterase 6H) and cone arrestin (CAR). In some embodiments, the first period of time is about 3 days to about 120 days, or about 5 days to about 15 days, or about 3 days. In embodiments, the second period of time is about 1 day to about 120 days, or about 5 days to about 15 days, or about 11 days.

[0019] In an embodiment, differentiating the undifferentiated pluripotent stem cells includes: (a) differentiating with a Rock inhibitor (RI), nicotinamide (NIC) and a Wnt inhibitor (e.g., IWRel) in a first period; (b) differentiating with a NIC and a Wnt inhibitor (e.g., IWRel) in a second period; (c) differentiating with a NIC and insulin growth factor-1 (IGF-1) in a third period; (d) differentiating with a NIC, IGF-1 and a Notch inhibitor (e.g., DAPT) in a fourth period; and (e) differentiating with IGF-1, retinoic acid (RA), taurine and NT4 in a fifth period. In an embodiment, the first period is 0-7 days, and / or the second period is 2-20 days, and / or the third period is 10 days-8 weeks, and / or the fourth period is 4-10 weeks, and / or the fifth period is 12-18 weeks.

[0020] The disclosure provides a method for producing a pharmaceutical composition of the disclosure, the method comprising: (i) differentiating cells within a population of undifferentiated pluripotent stem cells into retinal cells; and (ii) culturing the population of undifferentiated cells under culture conditions sufficient to produce cell aggregates, the cell aggregates having a diameter of between 100 μm and 800 μm.

[0021] The disclosure provides a method for producing a composition comprising a population of retinal cells, the method comprising: (a) culturing a population of undifferentiated pluripotent stem cells for a first period of time in a first cell culture medium comprising nicotinamide (NIC), rel-4-[(3aR,4S,7R,7aS)-1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl]-N-8-quinolinylbenzamide (IWRe) and a Rock inhibitor (RI); and (b) culturing the cells produced in step (a) for a first period of time. (c) culturing the population of cells generated in step (b) in a third cell culture medium comprising NIC and insulin-like growth factor 1 (IGF-1) for a third period of time; and (d) collecting the population of cells, thereby producing a composition comprising a population of retinal cells, wherein the population of cells is cultured in a culture vessel for at least 14 weeks under conditions sufficient to produce cell aggregates having a diameter of 100 μm to 800 μm.

[0022] In some embodiments of the disclosed methods, the cells are cultured under dynamic culture conditions. In some embodiments, the culture vessel is a bioreactor. In some embodiments, the culture vessel comprises a vertical wheel bioreactor, a wave bioreactor, or a gas permeable rapid expansion bioreactor. In some embodiments, the culture vessel comprises a wheel bioreactor, and the rotational speed of the wheel bioreactor is increased over time, thereby generating cell aggregates of diameters between 100 μm and 800 μm suspended within the wheel bioreactor. In some embodiments, the rotational speed of the wheel bioreactor applies a controlled shear stress to the cell aggregates. In some embodiments, the rotational speed is between 35 and 80 revolutions per minute (rpm). In some embodiments, the method comprises an initial rotational speed of about 30 to 50 rpm, which is increased to about 60 to 80 rpm by the end of the differentiation process.

[0023] In some embodiments of the methods of the present disclosure, the first period of time is 1-15 days, the second period of time is 1-30 days, and / or the third period of time is 5 days to 14 weeks.

[0024] In some embodiments of the methods of the present disclosure, the method includes a step prior to step (d) that includes (i) culturing the population of cells generated in step (c) in a fourth cell culture medium comprising IGF-1, NIC, and tert-butyl (S)-{(2S)-2-[2-(3,5-difluorophenyl)acetamido]propanamide}phenylacetate (DAPT) for a fourth period of time. In some embodiments, the fourth period of time is between 1 week and 18 weeks.

[0025] In some embodiments of the methods of the present disclosure, the method comprises a step (ii) following step (i), where step (ii) comprises culturing the population of cells of step (i) in a fifth cell culture medium comprising IGF-1, retinoic acid (RA), taurine (TA), brain-derived neurotrophic factor (BDNF), and neurotrophin-4 (NT4) for a fifth period of time. In some embodiments, the fifth period of time is between 4 weeks and 24 weeks.

[0026] In some embodiments, RI is at a concentration of about 1-20 μM in the first cell culture medium. In some embodiments, NIC is at a concentration of about 1-50 mM in the first, second, third and / or fourth cell culture medium. In some embodiments, IWRe is at a concentration of about 0.01-20 μM in the first cell culture medium and / or second cell culture medium. In some embodiments, IGF-1 is at a concentration of about 0.5-20 ng / mL in the third, fourth and / or fifth cell culture medium. In some embodiments, DAPT is at a concentration of about 1-50 μM in the fourth cell culture medium. In some embodiments, BDNF is at a concentration of about 5-50 ng / mL in the fifth cell culture medium. In some embodiments, NT4 is at a concentration of about 2-200 ng / mL in the fifth cell culture medium. In some embodiments, TA is at a concentration of 10-400 μM in the fifth cell culture medium.

[0027] The present disclosure provides a method of generating a composition comprising a population of retinal cells, the method comprising: (a) culturing a population of undifferentiated pluripotent stem cells in a first cell culture medium comprising nicotinamide (NIC) at a concentration of 10 mM, IWRe at a concentration of 3 μm, and Rock inhibitor (RI) at a concentration of 10 μm for at least 1 day; (b) culturing the population of cells generated in step (a) in a second cell culture medium comprising NIC at a concentration of 10 mM, and IWRe at a concentration of 3 μm for at least 2 days; (c) culturing the population of cells generated in step (b) in a third cell culture medium comprising NIC at a concentration of 10 mM, and IGF-1 at a concentration of 5 ng / mL for at least 10 days; and (d) collecting the population of cells, thereby generating a composition comprising a population of retinal cells, the population of cells being cultured in a culture vessel for at least 14 weeks under conditions sufficient to generate aggregates having a diameter of 100 μm to 800 μm.

[0028] The present disclosure provides a method for producing a composition comprising a population of retinal cells, the method comprising the steps of: (a) culturing a population of undifferentiated pluripotent stem cells in a first cell culture medium comprising nicotinamide (NIC) at a concentration of 10 mM, IWRe at a concentration of 3 μm, and Rock inhibitor (RI) at a concentration of 10 μm for at least 1 day; (b) culturing the population of cells produced in step (a) in a second cell culture medium comprising NIC at a concentration of 10 mM, and IWRe at a concentration of 3 μm for at least 2 days; and (c) culturing the population of cells produced in step (b) in a second cell culture medium comprising NIC at a concentration of 10 mM, IWRe at a concentration of 3 μm, and Rock inhibitor (RI) at a concentration of 10 μm for at least 2 days. and 5 ng / mL IGF-1 for at least 10 days; (d) culturing the population of cells generated in step (c) in a fourth cell culture medium containing IGF-1 at a concentration of 5 ng / mL, NIC at a concentration of 10 mM, and DAPT at a concentration of 10 μM for at least 2 weeks; and (e) collecting the population of cells, thereby generating a composition comprising a population of retinal cells, wherein the population of cells is cultured in a culture vessel for at least 14 weeks under conditions sufficient to generate aggregates having a diameter of 100 μm to 800 μm.

[0029] The present disclosure provides a method for producing a composition comprising a population of retinal cells, the method comprising the steps of: (a) culturing a population of undifferentiated pluripotent stem cells in a first cell culture medium comprising nicotinamide (NIC) at a concentration of 10 mM, IWRe at a concentration of 3 μm, and Rock inhibitor (RI) at a concentration of 10 μm for at least 1 day; (b) culturing the population of cells produced in step (a) in a second cell culture medium comprising NIC at a concentration of 10 mM, and IWRe at a concentration of 3 μm for at least 2 days; (c) culturing the population of cells produced in step (b) in a third cell culture medium comprising NIC at a concentration of 10 mM, and IGF-1 at a concentration of 5 ng / mL for at least 10 days; and (d) culturing the population of cells produced in step (c) in a third cell culture medium comprising IGF-1 at a concentration of 5 ng / mL, IGF-1 at a concentration of 1 ng / mL, and IGF-1 at a concentration of 1 ng / mL for at least 10 days. (e) culturing the population of cells generated in step (d) for at least 2 weeks in a fourth cell culture medium containing NIC at a concentration of 0 mM and DAPT at a concentration of 10 μM; (e) culturing the population of cells generated in step (d) for at least 5 weeks in a fifth cell culture medium containing IGF-1 at a concentration of 5 ng / mL, retinoic acid (RA) at a concentration of 0.5 μM, taurine (TA) at a concentration of 100 μM, brain-derived neurotrophic factor (BDNF) at a concentration of 20 ng / mL, and neurotrophin-4 (NT4) at a concentration of 20 ng / mL; and (f) collecting the population of cells, thereby generating a composition comprising a population of retinal cells, wherein the population of cells is cultured in a culture vessel for at least 14 weeks under conditions described in claim 52 sufficient to generate aggregates having a diameter of 100 μm to 800 μm.

[0030] In some embodiments of the method of the present disclosure, the undifferentiated pluripotent stem cells comprise human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs). In some embodiments, the hESCs comprise HADC102 cells.

[0031] In some embodiments of the methods of the present disclosure, the methods include, prior to step (a), seeding the undifferentiated pluripotent stem cells as single cells into a culture vessel at a density of between 50,000 cells / mL and about 2,000,000 cells / mL.

[0032] In some embodiments of the method of the present disclosure, the population of retinal cells comprises early eye field cells, embryonic retinal cells, progenitor cells of photoreceptor neuron cells (PNC), mature photoreceptor neuron cells, or any combination thereof. In some embodiments, the population of retinal cells comprises neuronal retinal cells (NRC), Muller glial cells, retinal pigment epithelium (RPE) cells, or any combination thereof. In some embodiments, NRC comprises PNC, retinal ganglion cells, horizontal neurons, amacrine neurons, rod bipolar cells, cone bipolar cells, rod photoreceptor cells, cone photoreceptor cells, or any combination thereof.

[0033] In some embodiments of the methods of the present disclosure, prior to step (d), the method includes the steps of: (i) collecting the cell aggregates, (ii) dispersing the cell aggregates to generate dispersed cells, (iii) seeding the dispersed cells into tissue culture flasks, and (iv) culturing the dispersed cells under adherent static conditions for at least one week. In some embodiments, the dispersed cells are cultured for one to three weeks.

[0034] In embodiments, cells generated at any step may be cryopreserved. In embodiments, cells are cryopreserved in a freezing solution. For example, the freezing solution may include CRYOSTEM™. In other embodiments, CRYOSTEM™ may be used as a freezing solution for freezing cell aggregates, and in other embodiments, for freezing hESC subcolonies.

[0035] In embodiments, the cells may be cryopreserved from days 14 to 120 of the differentiation process. In embodiments, the step of cryopreserving the cells may occur at or near the completion of the first period.

[0036] In some embodiments of the methods of the present disclosure, the method includes cryopreserving a population of retinal cells. In some embodiments, cryopreserving includes suspending the population of cells in a cryopreservation medium to form a cell suspension and storing the cell suspension at -80°C or below, or at -140°C or below. In some embodiments, cryopreserving includes a cryopreservation medium suitable for administration to the subject's eye. In some embodiments, the cryopreservation medium includes a cryoprotectant selected from the group consisting of glycerol, sucrose, dextran, and dimethyl sulfoxide (DMSO). In some embodiments, the cryopreservation medium includes about 0.1% to about 40% cryoprotectant. In some embodiments, the pharmaceutical composition includes about 1% to about 10% cryoprotectant.

[0037] In some embodiments, the cryopreservation medium comprises CRYOSTOR® CS2, CRYOSTOR® CS5, CRYOSTOR® CS10, or CRYOSTEM™. In some embodiments, the cryopreservation medium comprises 2% DMSO, 5% DMSO, or 10% DMSO, or any range therebetween.

[0038] In some embodiments, the cryopreserved cells are thawed and the thawed cells are cultured, e.g., the thawed cells are cultured according to step (iii) above, or in other embodiments, the cells from step (iii) are cryopreserved after a third period of time.

[0039] In some embodiments, a method for preparing a photoreceptor neuronal cell (PNC) composition for administration to a subject immediately after thawing is provided herein. The method includes (a) suspending cells (e.g., retinal cells, including PNC) prepared according to the method herein in a cryopreservation medium to form a cell suspension, (b) storing the cell suspension at a cryopreservation temperature, and (c) thawing the cryopreserved suspension. In an embodiment, the cryopreservation medium includes one or more of adenosine, dextran-40, lactobionic acid, HEPES (N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid)), sodium hydroxide, L-glutathione, potassium chloride, potassium bicarbonate, potassium phosphate, dextrose, sucrose, mannitol, calcium chloride, magnesium chloride, potassium hydroxide, sodium hydroxide, dimethyl sulfoxide (DMSO), or water. In an embodiment, the freezing solution includes methylcellulose. In an embodiment, the freezing solution includes dimethyl sulfoxide (DMSO). In embodiments, the freezing solution comprises about 0.1% to about 20% DMSO, in other examples, the freezing solution comprises about 0.1 to 100%, or about 1 to about 100%, or about 10 to 100%, or about 20 to 100%, or about 50 to 100%, or about 0.5 to 1%, or about 0.1 to 1%, or about 0.1 to about 10% DMSO.

[0040] The present disclosure provides pharmaceutical compositions produced by the methods of the present disclosure.

[0041] In embodiments, provided herein is a pharmaceutical composition for administration to a subject, the composition comprising mature PNC(s) prepared according to any one of the methods described herein, and in embodiments, the pharmaceutical composition further comprises a cryopreservation medium. For example, in embodiments, the cryopreservation medium comprises one or more of adenosine, dextran-40, lactobionic acid, HEPES (N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid)), sodium hydroxide, L-glutathione, potassium chloride, potassium bicarbonate, potassium phosphate, dextrose, sucrose, mannitol, calcium chloride, magnesium chloride, potassium hydroxide, sodium hydroxide, dimethylsulfoxide (DMSO), and water.

[0042] In embodiments, the composition comprises about 1×10 5 cells / mL ~ approx. 100×10 6 Include cells (e.g., retinal cells including PNCs) at a concentration of 100 cells / mL.

[0043] In embodiments, the pharmaceutical composition is stored in a volume of about 100 μI, to about mL, or about 250 μL, or about 600 μL. In embodiments, the cryopreservation medium in the pharmaceutical composition comprises a freezing solution. In embodiments, the freezing solution comprises DMSO. In embodiments, the freezing solution comprises CRYOSTOR® CS2, CRYOSTOR® CS5, CRYOSTOR® CS10, or CRYOSTEM™.

[0044] In some embodiments, the pharmaceutical composition comprising mature PNC(s) prepared according to any one of the methods described herein comprises cells expressing one or more markers selected from Crx, recoverin, cone arrestin (CAR), or combinations thereof, hi other embodiments, the pharmaceutical composition comprises about 1%-30% RPE cells.

[0045] In other embodiments, the composition includes seeding PNCs onto a scaffold (optionally biodegradable), preparing an implant from the scaffold containing the PNCs, and cryopreserving the implant to treat an eye-related disease.

[0046] In other embodiments, a method for treating an eye-related disease, for example, a method for treating vision loss in a subject is provided herein. The method comprises administering to the subject a therapeutically effective amount of a composition comprising mature PNC(s) prepared according to any one of the methods described herein. In embodiments, administering comprises administering the composition to the retina of the subject or in close proximity to the retina. In embodiments, administering is by injection, implantation, or implantation. The present disclosure provides a method for treating a subject with a visual condition, comprising administering to the eye of the subject a therapeutically effective amount of a pharmaceutical composition of the present disclosure.

[0047] The present disclosure provides a method of treating a subject having a visual condition, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising a population of retinal cells, where (a) between about 10% and 70% of the cells in the population express Crx, (b) between about 3% and 90% of the cells in the population express recoverin, (c) between about 3% and 90% of the cells in the population express CAR, and (d) between 0 and about 0.1% of the cells in the population express TRA-1-60 and / or SSEA5, and the composition is administered to an eye of the subject.

[0048] In some embodiments of the methods of the present disclosure, the composition is administered by injection into the retina. In some embodiments, the injection comprises an intravitreal, subretinal or suprachoroidal injection of a suspension comprising the population of cells. In some embodiments, the method comprises implantation of a scaffold comprising the population of cells. In some embodiments, the scaffold is implanted in the subretinal space.

[0049] In some embodiments of the method of the present disclosure, the visual pathology comprises retinal neurodegenerative disease or retinal damage.In some embodiments, the retinal neurodegenerative disease comprises Stargardt's disease, diabetic retinopathy, macular degeneration, retinitis pigmentosa, Leber's congenital amaurosis, cone-rod dystrophy, choroideremia or X-linked retinoschisis.In some embodiments, the macular degeneration comprises age-related macular degeneration.

[0050] Other aspects of the invention are disclosed below.

[0051] Some embodiments of the present invention are described herein, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the details shown are exemplary and are intended to illustratively discuss embodiments of the invention. In this regard, when described in conjunction with the drawings, it will become apparent to those skilled in the art how embodiments of the present invention may be practiced. [Brief description of the drawings]

[0052] [Figure 1] FIG. 1 is a schematic diagram showing a bioretina differentiation flow chart, tracing major cell populations, inducers, and exemplary identity markers at each stage. [Figure 2A] 2A is an image showing a study design (Bioret-Pro-01 study design, also called PR-44) designed to induce differentiation of retinal cells from undifferentiated pluripotent cells, showing the seeding density and rotation speed, incubation period, and IGF-1 concentration tested in the study. FIG. 2B is an image showing a study with an initial hESC (human embryonic stem cell) density of 250,000 cells / mL and rotation speeds of 35, 50, and 70 rpm revolutions per minute (rpm). [Figure 2B]2A is an image showing a study design (Bioret-Pro-01 study design, also called PR-44) designed to induce differentiation of retinal cells from undifferentiated pluripotent cells, showing the seeding density and rotation speed, incubation period, and IGF-1 concentration tested in the study. FIG. 2B is an image showing the study with an initial hESC density of 150,000 cells / mL and rotation speeds of 35, 50, and 70 rpm. [Figure 2C] 2A-C are images showing a study design designed to induce differentiation of retinal cells from undifferentiated pluripotent cells (Bioret-Pro-01 study design, also called PR-44), showing the seeding density and rotation speed, incubation period, and IGF-1 concentration tested in the study. FIG. 2C is an image showing a study with an initial hESC density of 250,000 cells / mL and rotation speeds of 40, 60, and 80 rpm. [Figure 2D] FIG. 2C is an image showing a study design (also called Bioret-Pro-01 study design, PR-44) designed to induce differentiation of retinal cells from undifferentiated pluripotent cells, showing the seeding density and rotation speed, incubation period, and IGF-1 concentration tested in the study. FIG. 2D is an image showing an initial hESC cell density of 150,000 cells / mL, and rotation speeds of 40, 60, and 80 rpm. [Figure 3A] 2A-2D are phase contrast images showing aggregate morphology in various culture conditions from the study of FIG. 2A-D. All images are displayed to the same scale. [Figure 3B] 2A-2D are phase contrast images showing aggregate morphology in various culture conditions from the study of FIG. 2A-D. All images are displayed to the same scale. [Figure 3C] 2A-2D are phase contrast images showing aggregate morphology in various culture conditions from the study of FIG. 2A-D. All images are displayed to the same scale. [Figure 3D] 2A-2D are phase contrast images showing aggregate morphology in various culture conditions from the study of FIG. 2A-D. All images are displayed to the same scale. [Diagram 2]Images showing an estimate of cell mass at week 7. Numbers below the matrix (bottom panel) indicate the rectangles containing aggregates, and letters (A-D) labeling the bottom images correspond to the conditions shown in panels A-D of Figures 2A-2D, respectively. [Diagram 3] 13 is an image showing an estimate of cell mass at week 16. Numbers below the matrix indicate squares containing aggregates. [Figure 4] Paired graphs showing glucose (bottom) and lactate (top) levels throughout the differentiation process. [Diagram 5] A series of depictions from confocal imaging of cells. Retinal cells grown in PBS Wheel at 16 weeks were harvested and plated on PDL laminin-coated coverslips, and immunostained for CRX (red) and recoverin (green) 14 days after plating. [Figure 6] A series of depictions from confocal imaging of cells. Retinal cells grown in PBS Wheel at 16 weeks were harvested and plated on PDL laminin-coated coverslips and immunostained for rhodopsin (red) and cone arrestin (green) 14 days after plating. [Figure 7] A series of depictions from confocal imaging of cells. Eight and 16 week old retinal cells grown in PBS Wheel or static control were harvested and plated on PDL laminin coated coverslips and immunostained for rhodopsin, HuCD, CRX, βTub3, CHX10, OTX2 (red) and cone arrestin, OTX2, Six6, rhodopsin, and recoverin, which are the colors indicated in each image. [Figure 8] 1 is a table showing quantification of retinal marker expression throughout the differentiation process. [Figure 9] FIG. 13 is a table showing quantification of non-target retinal marker expression throughout the differentiation process. [Figure 10] 1 is a table showing quantification of marker expression of non-target cell populations throughout the differentiation process. [Figure 11] FIG. 13 is a table showing quantification of non-target retinal marker expression throughout the differentiation process. [Figure 12] 1 is a table showing quantification of marker expression of non-target cell populations throughout the differentiation process. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0053] Provided herein is a method for differentiating human pluripotent stem cells into populations of retinal cells, such as photoreceptor neuronal cells (PNC), among others, in dynamic culture. The method may be adapted for commercial scale processing. Also provided herein are compositions comprising cells and cell populations obtained by the methods described herein. Also provided herein are methods for treating retinal diseases, including, for example, vision loss, using cells and cell populations obtained by the methods described herein. Embodiments herein generally relate to methods, compositions, and methods for treating ocular diseases using neural retinal cells (NRC) or photoreceptor neuronal cells (PNC) obtained by the methods described herein.

[0054] The methods described herein offer several advantages over other methods, including, for example, 1) an extended period or timing when undifferentiated pluripotent stem cells are seeded for differentiation as single cells and grown in suspension as cell aggregates for at least 14-18 weeks (e.g., at least 4 months), 2) no isolation step is required, for example, when the target cells are routinely isolated using known techniques, 3) cells are grown in dynamic culture rather than non-adherent or adherent static culture, and 4) the aggregate size of the differentiated cell aggregates can be controlled by rotation speed (which can be important for commercial viability and even homogeneity of the resulting cell population). Other advantages include increased efficiency (e.g., increased efficiency of the culture process), improved cell quality, and increased percentage of viable cells.

[0055] As described herein, a method for generating retinal cells, such as photoreceptor neurons, is provided. This method may be capable of forming a reconstituted retina with high viability and neural connectivity to the surrounding functional layer. Moreover, the manufacturing process of the cells and compositions described herein may be adapted for large-scale production (e.g., closed systems) with improvements to enable industrial manufacturing. Clinically compatible characteristics of photoreceptor neurons have been established using the protocols herein, generating positive identity assays for key markers of both rod and cone photoreceptor populations. Furthermore, retinal cell populations generated using the methods described herein may contain markers indicative of the presence of additional retinal cell types, examples of which include, but are not limited to, retinal pigment epithelium (RPE) cells, glia, and precursor cells of photoreceptor neurons.

[0056] Furthermore, the data herein demonstrate that single cell suspensions of photoreceptor progenitor cells have the potential to survive and mature following transplantation (eg, in rodent models of retinal degeneration).

[0057] The methods described herein offer new opportunities for clinical (and commercial) production of photoreceptor cells in areas of great unmet need, including, but not limited to, retinitis pigmentosa, Stargardt's disease, retinal detachment and retinal tears.

[0058] After reading this description, it will be clear to a person skilled in the art how to implement the present disclosure in various alternative embodiments and applications. However, not all of the various embodiments of the present invention are described herein. It will be understood that the embodiments presented herein are presented merely as examples and are not limiting. Therefore, the detailed description of various alternative embodiments should not be construed as limiting the scope or breadth of the present disclosure described herein.

[0059] Before the present technology is disclosed and described, it is to be understood that the embodiments described below are not limited to particular compositions, methods of preparing such compositions, or uses thereof, which may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0060] The detailed description is divided into various sections solely for the convenience of the reader, and disclosures set forth in any section may be combined with those set forth in another section. Headings or subheadings may be used herein for the convenience of the reader, but are not intended to affect the scope of the disclosure.

[0061] definition As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise.

[0062] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes cases where the event or circumstance occurs and cases where it does not occur.

[0063] The term "about" when used prior to numerical designations (including ranges) of temperature, time, amounts, concentrations, and the like, indicates approximations that may vary by (+) or (-) 10%, 5%, 1%, or any subrange or subvalue therebetween. Preferably, the term "about" when used in reference to an amount means that the amount may vary by ±10%.

[0064] "Comprising" or "comprise" is intended to mean that the compositions and methods include the recited elements but do not exclude other elements. "Consisting essentially of" when used to define compositions and methods is intended to mean excluding other elements of any essential importance to the combination for the purposes defined. Thus, a composition consisting essentially of the elements defined herein does not exclude other materials or steps that do not materially affect the basic and novel property(ies) of the claimed invention. "Consisting of" means excluding more than trace elements and substantial method steps of other components. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0065] When a parameter range is provided, it is understood that all integers and tenths thereof within that range are also provided by the present invention. For example, "0.2 to 5 mg" is a disclosure of 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, etc., up to and including 5.0 mg.

[0066] In the description and claims herein, a conjunctive list of elements or features may appear following a phrase such as "at least one" or "one or more." Also, the term "and / or" may appear following a list of two or more elements or features. Unless otherwise implicitly or explicitly contradicted by the context in which it is used, such phrases are intended to mean any of the listed elements or features individually, or any of the listed elements or features in combination with any of the other listed elements or features. For example, the phrases "at least one of A and B," "one or more of A and B," and "A and / or B" are intended to mean "A alone, B alone, or A and B together," respectively. A similar interpretation is intended for lists containing three or more items. For example, the phrases "at least one of A, B, C," "one or more of A, B, C," and "A, B, and / or C" are intended to mean "A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B and C together." Additionally, use of the term "based on" above and in the claims is intended to mean "based at least in part on," thereby allowing for unrecited features or elements.

[0067] "Control" or "control experiment" is used according to its plain and ordinary meaning to refer to an experiment in which an experimental subject or agent is treated as in a parallel experiment, except for the omission of an experimental procedure, agent, or variable. In some cases, a control is used as a standard of comparison in the evaluation of an experimental effect. In some embodiments, a control is a measurement of a protein's activity in the absence of a composition described herein (including the embodiments and examples).

[0068] As used herein, "treating" or "treatment" of a condition, disease or disorder, or a symptom associated with a condition, disease or disorder, refers to an approach to obtain a beneficial or desired result, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, reduction in the severity of a condition, disorder or disease, stabilization of the condition, disorder or disease state, prevention of the onset of a condition, disorder or disease, prevention of the spread of a condition, disorder or disease, delaying or slowing the progression of a condition, disorder or disease, delaying or slowing the onset of a condition, disorder or disease, improvement or palliative of the condition, disorder or disease state, and partial or total remission. "Treatment" can mean inhibiting the progression of a condition, disorder or disease, slowing the progression of a condition, disorder or disease temporarily, but in some cases also includes permanently halting the progression of a condition, disorder or disease.

[0069] As used herein, the terms "treatment" and "prevention" are not intended as absolute terms. In various embodiments, treatment can refer to reducing the severity of an established disease, condition, or symptom of a disease or condition by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. In embodiments, a method of treating a disease is considered to be a treatment if one or more symptoms of the disease are reduced by 10% in a subject compared to a control. Thus, this reduction can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percentage reduction between 10% and 100% compared to the native or control level. It is understood that treatment does not necessarily refer to a cure or complete elimination of a disease, condition, or symptom of a disease or condition. In embodiments, reference to decrease, reduction, or inhibition includes a change of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to control levels, and such terms may, but do not necessarily, include complete elimination. In embodiments, the severity of the disease is reduced by at least 10%, for example, compared to the individual prior to administration or to a control individual not receiving treatment. In some aspects, the severity of the disease is reduced by at least 25%, 50%, 75%, 80%, or 90%, and in some cases, is no longer detectable using standard diagnostic techniques.

[0070] An "effective amount" is an amount sufficient for the composition to achieve a stated purpose (e.g., achieve the effect of administration, treat a disease, reduce enzyme activity, increase enzyme activity, reduce signal transduction pathways, or reduce one or more symptoms of a disease or condition) compared to the absence of the composition. An example of an "effective amount" is an amount sufficient to contribute to the treatment, prevention, or reduction of a symptom or symptoms of a disease, which may also be referred to as a "therapeutically effective amount." A "reduction" of a symptom or symptoms (and grammatical equivalents of this phrase) refers to a reduction in the severity or frequency of the symptom(s), or the elimination of the symptom(s). A "prophylactically effective amount" of a drug (e.g., a cell described herein) is an amount of a drug that, when administered to a subject, has an intended prophylactic effect, e.g., prevents or delays the onset (or recurrence) of an injury, disease, condition, or condition, or reduces the likelihood of the onset (or recurrence) of an injury, disease, condition, or condition, or a symptom thereof. A complete preventive effect may not necessarily occur by administering one dose, but may occur only after administering a series of doses. Thus, a prophylactically effective amount may be administered in one or more doses. For any composition described herein, a therapeutically effective amount or a prophylactically effective amount can be determined first from cell culture assays and / or in vivo experiments in animal models. The target concentration is the concentration (e.g., cell concentration or cell number) of the active composition(s) that can achieve the method described herein, as measured using the method described herein or known in the art.

[0071] As used herein, an "activity-reducing amount" refers to the amount of antagonist required to reduce the activity of an enzyme compared to the absence of the antagonist. As used herein, a "function-disrupting amount" refers to the amount of antagonist required to destroy the function of an enzyme or protein compared to the absence of the antagonist. The actual amount will depend on the purpose of the treatment and will be ascertainable by those skilled in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0072] For any of the compositions described herein, the therapeutically effective amount can be initially determined from cell culture assays. The target concentration is the concentration (e.g., cell concentration or number) of the active composition(s) that can achieve the methods described herein, measured using the methods described herein or known in the art.

[0073] As used herein, "implantation" or "transplantation" refers to administering a cell population to a target tissue using a suitable delivery technique (e.g., using an injection device). Transplantation or implantation may refer to administering a cell population as a liquid, e.g., administering a liquid containing a population of cells in suspension. Alternatively, implantation of a transplant may refer to administering a cell population to a target tissue when the cells are part of (contained within or attached to) a device such as a scaffold.

[0074] "Patient" or "subject" refers to a living member of the animal kingdom who suffers from or may suffer from the indicated disorder. In embodiments, the subject is a member of a species that includes individuals who may naturally suffer from the disease. In embodiments, the subject is a mammal. Non-limiting examples of mammals include rodents (e.g., mice, rats), primates (e.g., lemurs, galagos, monkeys, apes, humans), rabbits, dogs (e.g., companion dogs, service dogs, or working dogs such as police dogs, military dogs, racing dogs, or show dogs), horses (race horses, working horses, etc.), cats (e.g., house cats), livestock (pigs, cows, donkeys, mules, bison, goats, camels, sheep, etc.), and deer. In embodiments, the subject is a human.

[0075] As used herein, a "patient in need thereof" or a "subject in need thereof" refers to an animal or human with retinal damage or degeneration. Retinal degeneration can be caused by genetic disorders (such as Stargardt's disease, Leber's congenital amaurosis, retinitis pigmentosa, etc.), or by environmental factors (age, diet, smoking, trauma), or even underlying conditions such as diabetes (diabetic retinopathy).

[0076] "Pharmaceutically acceptable excipient" and "pharmaceutically acceptable carrier" refer to substances that aid in the administration and absorption of an active agent by a subject and can be included in the compositions of the present disclosure without causing significant adverse toxic effects to the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, saline, lactated Ringer's solution, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coating agents, sweeteners, flavorings, salt solutions (such as Ringer's solution), alcohols, oils, gelatin, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidine, and coloring agents. Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts that affect osmotic pressure, buffers, coloring agents, and / or aromatic substances that do not adversely react with the compositions of the present disclosure. Those skilled in the art will recognize that other pharmaceutical excipients are also useful in the present disclosure.

[0077] As used herein, "cell" refers to a cell that performs metabolic or other functions sufficient to preserve or replicate genomic DNA. Cells can be identified by methods well known in the art, including, for example, the presence of an intact membrane, staining with a particular dye, the ability to produce progeny, or, in the case of gametes, the ability to combine with a second gamete to produce viable progeny. Cells may include prokaryotic and eukaryotic cells. Prokaryotic cells include, but are not limited to, bacteria. Eukaryotic cells include, but are not limited to, yeast cells and cells derived from plants and animals, such as mammalian cells, insect (e.g., Spodoptera) cells, and human cells. Cells may be useful if they are naturally non-adherent or have been treated to prevent them from adhering to surfaces, such as by trypsinization.

[0078] "Biocompatibility" refers to the ability of the materials of a device, such as the scaffolds described herein, to elicit an appropriate host response in a particular situation (e.g., when implanted in a subject's retina). For example, a scaffold that is biocompatible with the retina may induce a reduced or no immune response upon implantation.

[0079] "Biodegradable" refers to a material that degrades when implanted in a subject, such as the materials of the scaffolds described herein. Biodegradable materials may degrade over a period of hours, days, weeks, months, or even years, depending on the material.

[0080] As used herein, "dynamic culture" includes any cell culture method that is not static (e.g., in a Petri dish). For example, dynamic culture includes, but is not limited to, culture on a rotor or shaker, wave bioreactor, stirred tank bioreactor, rocker, airlift bioreactor, vertical wheel bioreactor, and fixed bed bioreactor. Dynamic culture is performed by deliberate active motion to enhance mass transfer and mechanical transduction effects (e.g., using a bioreactor). This often results in more functional cells. For example, in a dynamic differentiation process, a bioreactor applies mechanical forces directly to generate physiological conditions and enhance differentiation into specific cell lineages. In dynamic culture, cells may also have a more homogenous environment that cannot be provided by diffusion alone in static culture. For example, cells growing around blood vessels versus cells growing in the inner regions of blood vessels. Additionally, static culture may generate different biologically isolated niches because it sustains microenvironments with different cell densities that cannot be sustained in dynamic culture.

[0081] As used herein, "pluripotent stem cells", "pluripotent cells" or "stem cells" refer to cells that have the ability to differentiate into all types of cells in an organism. Pluripotent cells can form teratomas and contribute to ectodermal, mesodermal, or endodermal tissues in vivo. Examples of pluripotent stem cells include embryonic stem (ES) cells, embryonic germ stem (EG) cells, induced pluripotent stem (iPS) cells, adult stem cells, mesenchymal stem cells, and hematopoietic stem cells. Stem cells can also remain in an undifferentiated state (e.g., pluripotent or multipotent stem cells) for long periods of time in culture until induced to differentiate into other cell types (e.g., fully differentiated cells) with specific specialized functions. Embryonic germ stem cells are cells that (a) are capable of self-renewal, (b) can differentiate to generate all types of cells in an organism, and (c) are derived from germ cells and germ cell progenitors, e.g., primordial germ cells, i.e., cells that become sperm and eggs. Embryonic germ cells (EG cells) are believed to have the same characteristics as the above-mentioned embryonic stem cells.Examples of methods for generating and characterizing EG cells are described in, for example, U.S. Patent No. 7,153,684; Matsui, Y., et al., (1992) Cell 70:841; Shamblott, M., et al. (2001) Proc. Natl. Acad. Sci. USA 98: 113; Shamblott, M., et al. (1998) Proc. Natl. Acad. Sci. USA, 95:13726; and Koshimizu, U., et al. (1996) Development, 122:1235, the disclosures of which are incorporated herein by reference.

[0082] NIC, also known as "niacinamide," "nicotinamide," or "NA," is an amide derivative of vitamin B3 (niacin) that is believed to maintain and improve beta cell function. The chemical formula for NIC is C6H6N20. NIC is essential for growth and the conversion of food to energy and has been used to treat arthritis, as well as to treat and prevent diabetes. [ka]

[0083] According to certain embodiments, nicotinamide is a nicotinamide derivative or a nicotinamide mimic.The term "nicotinamide derivative" as used herein refers to a compound that is a chemically modified derivative of natural NA.In one embodiment, chemical modification may be the replacement of the pyridine ring of the basic NA structure with the nitrogen atom or oxygen atom of the amide moiety (by a carbon ring member or a nitrogen ring member).

[0084] When substituted, one or more hydrogen atoms may be replaced by a substituent and / or the substituent may be bonded to the N atom to form a tetravalent positively charged nitrogen. Thus, the nicotinamide of the present invention includes substituted or unsubstituted nicotinamide. In another embodiment, the chemical modification may be, for example, the deletion or substitution of a single group to form a thiobenzamide analog of NA. All of these are as understood by those familiar with organic chemistry. Derivatives in the context of the present invention also include nucleoside derivatives of NA (e.g., nicotinamide adenine). Various derivatives of NA have been described, some of which are related to the inhibitory activity of PDE4 enzyme (WO03 / 068233, WO02 / 060875, GB2327675A) or as VEGF receptor tyrosine kinase inhibitors (WOO1 / 55114). For example, the process for preparing 4-aryl nicotinamide derivatives (WO05 / 014549). Other exemplary nicotinamide derivatives are disclosed in WOO1 / 55114 and EP2128244.

[0085] Nicotinamide mimetics include modified forms of nicotinamide and chemical analogs of nicotinamide that reproduce the effect of nicotinamide in differentiation and maturation of RPE cells from pluripotent cells.Exemplary nicotinamide mimetics include benzoic acid, 3-aminobenzoic acid, and 6-aminonicotinamide.Another class of compounds that can act as nicotinamide mimetics are inhibitors of poly(ADP-ribose) polymerase (PARP).Exemplary PARP inhibitors include 3-aminobenzamide, iniparib (BSI 201), olaparib (AZD-2281), rucaparib (AG014699, PF-01367338), veliparib (ABT-888), CEP9722, MK4827, BMN-673.

[0086] As used herein, "induced pluripotent stem cells" or "iPSCs" are cells that can be generated from somatic cells by genetic manipulation of the somatic cells, examples of which include retroviral transduction of transcription factors such as Oct-3 / 4, Sox2, c-Myc, and KLF4 into somatic cells such as fibroblasts, hepatocytes, and gastric epithelial cells (Yamanaka S, Cell Stem Cell. 2007, 1 (1):39-49; Aoi T, et al., Generation of Pluripotent Stem Cells from Adult Mouse Liver and Stomach Cells. Science, 2008 Feb 14; IH Park, Zhao R, West JA, et al. Reprogramming of human somatic cells to pluripotency with defined factors, Nature 2008;451:141-146; K Takahashi, Tanabe K, Ohnuki M, et al. Induction of pluripotent stem cells from adult human fibroblasts by (see defined factors, Cell 2007;131:861-872). Other embryonic-like stem cells can be generated by nuclear transfer into oocytes, fusion with embryonic stem cells, or nuclear transfer into zygotes when the recipient cell is arrested in mitosis. Additionally, non-integrative methods may be used to generate iPSCs, examples of which include the use of small molecules or RNA. Thus, iPSCs are cells that (a) are capable of self-renewal, (b) can differentiate to generate all cell types within an organism, and (c) are derived from somatic cells. iPS cells can have ES cell-like morphology, growing as flat colonies with a large nucleus-to-cytoplasm ratio, well-defined borders, and prominent nuclei.Additionally, iPS cells express one or more key pluripotency markers known to those skilled in the art, including, but not limited to, alkaline phosphatase, SSEA3, SSEA4, SRY-box transcription factor 2 (Sox2), Oct-4, Nanog, TRA-1-60, TRA-1-81, teratocarcinoma-derived growth factor 1 (TDGF1), DNA methyltransferase 3 beta (Dnmt3b), forkhead box D3 (FoxD3), growth differentiation factor 3 (GDF3), cytochrome P450 family 26 subfamily A member 1 (Cyp26a1), telomerase reverse transcriptase (TERT), and ZFP42 zinc finger protein (zfp42). iPS cells may be generated by providing the cells with one or more, i.e., a mixture of "reprogramming factors," i.e., biologically active factors that act on the cells to alter transcription, thereby reprogramming the cells to pluripotency. These reprogramming factors may be provided to the cells individually or as a single composition of reprogramming factors, i.e., a premixed composition. The factors may be provided at the same molar ratio or at different molar ratios. The factors may be provided one or more times during the course of culturing the cells of the subject invention. Examples of methods for generating and characterizing iPS cells are described, for example, in application numbers US20090047263, US20090068742, US20090191159, US20090227032, US20090246875, and US20090304646, the disclosures of which are incorporated herein by reference. Pluripotent stem cells express markers known to those of skill in the art, including, but not limited to, the TRA-1-60 antigen (TRA-1-60) and the SSEA-5 glycan (SSEA5 or SSEA-5).

[0087] The term "embryonic stem cells" refers to embryonic cells that can differentiate into cells of all three embryonic germ layers (i.e., endoderm, ectoderm, mesoderm) or remain undifferentiated. The term "embryonic stem cells" includes cells obtained from embryonic tissues (e.g., blastocysts) formed after conception before implantation of the embryo (i.e., pre-implantation blastocysts), expanded blastocyst cells (EBCs) obtained from blastocysts at the post-implantation / pre-gastrulation stage (see WO2006 / 040763), and embryonic germ (EG) cells obtained from fetal reproductive tissues at any time during conception, preferably before 10 weeks of conception. In embodiments, embryonic stem cells are obtained using well-known cell culture methods. For example, human embryonic stem cells can be isolated from human blastocysts.

[0088] It is also understood that commercially available stem cells can be used in aspects and embodiments of the present disclosure. Human ES cells may be purchased from the NIH human embryonic stem cell registry www.grunts.nih.govstem_cells / or other hESC registries. Suitable human ES cell lines will be apparent to those skilled in the art and are described in more detail below.

[0089] As used herein, "somatic cell" refers to any cell in an organism that does not normally give rise to all types of cells in the organism in the absence of experimental manipulation. In other words, a somatic cell is a cell that is sufficiently differentiated that it does not naturally give rise to cells of all three germ layers of the body, namely ectoderm, mesoderm, and endoderm. For example, somatic cells include both neurons and neural progenitor cells. The latter may be able to self-renew and naturally give rise to all or some cell types of the central nervous system, but cannot give rise to cells of mesodermal or endodermal lineages.

[0090] As used herein, the term "retina" refers to a layer of light-sensitive cells that covers the inner posterior wall of the eye. The cells of the retina receive images through light stimuli and transmit this information as electrical signals through the optic nerve to the brain. The retina and optic nerve are considered to be extensions of the brain and therefore part of the central nervous system (CNS). The retina is composed of the retinal pigment epithelium (RPE) and neural retina (NR), which are described as interconnected layers of specialized cells. From closest to furthest from the vitreous, that is, from closest to the front outer side of the head toward the inside and back of the head, the retinal layers include: The retina is made up of three layers: (i) the inner limiting membrane, which is made up of the stapes bases of Müller cells; (ii) the nerve fiber layer, which contains the axons of the ganglion cell nuclei; (iii) the ganglion cell layer, which contains the nuclei of the ganglion cells, whose axons become the optic nerve fibers; (iv) the inner plexiform layer, which contains the synapses between the bipolar cell axons and the dendrites of the ganglion cells and amacrine cells; (v) the inner nuclear layer, which contains the nuclei and surrounding cell bodies (perikarya) of the bipolar cells; (vi) the outer plexiform layer, which contains the processes of the rods and cones that terminate in the rod spherules and cone stalks, respectively; (vii) the outer nuclear layer, which contains the cell bodies of the rods and cones; (viii) the outer limiting membrane, which separates the inner segmental portion of the photoreceptors from their nuclei; (xi) the photoreceptor layer; and (x) the retinal pigment epithelium (RPE), which is a single layer of cuboidal cells. Some cells in the retina are photoreceptive, among them the rods, cones, and ganglion cells, which are directly sensitive to light. Rods function primarily in providing black and white vision in the dark. Cones function primarily in daytime vision and color perception. Photoreceptors are light-sensitive ganglion cells that are important for reflex responses to bright sunlight. Retinal structure and retinal cell types are described in US20190085287, the contents of which are incorporated herein by reference in their entirety.

[0091] As used herein, "retinal cells" refers to all cell types found in the retina, in any combination. Retinal cells include, but are not limited to, neural retinal cells (NRCs), Müller glial cells, retinal pigment epithelial cells (RPE), or any combination thereof. As used herein, the term "retinal cells" also encompasses "retinal progenitor cells" or "RPCs." The vertebrate retina develops from a domain of the anterior neural plate (eye field). In this region, bilateral optic vesicles form and develop by invagination into the optic cup. The retina forms from the posterior wall of the optic cup. Retinal progenitor cells (RPCs) within the optic cup actively proliferate and generate the future RPE and NR layers of the retina. Retinal progenitor cells in the outer layer of the optic cup become RPE progenitor cells, while retinal progenitor cells in the inner layer become NR progenitor cells. RPCs are multipotent and can generate any retinal cell type when under the appropriate conditions that induce differentiation.

[0092] As used herein, "neural retinal cells," "neuronal retinal cells," and similar abbreviations, NRCs, refer to neuronal cells that arise from NR precursor cells. There are six main types or neural retinal (NR) cells, all of which are neurons, including rod and cone photoreceptor cells, bipolar cells, horizontal cells, amacrine cells, and ganglion cells. Muller glial cells (astroglial cells), microglial cells (resident tissue-specific macrophages), astrocytes, and oligodendrocytes are non-neuronal cells that are incorporated into the NR layer of the retina.

[0093] As used herein, "photoreceptor cell", "photoreceptor neuron" or "PNC" and the like refer to a specialized type of neuroepithelial cell found in the retina capable of visual light signal transduction. Currently, three types of photoreceptor cells are known in the mammalian eye: rod cells, cone cells, and intrinsically light-sensitive retinal ganglion cells. Rod and cone photoreceptor cells are the primary photoreceptor cells, while light-sensitive retinal ganglion cells are thought to play a role in non-visual responses to light (such as circadian rhythms). Rod photoreceptor cells primarily mediate scotopic vision (i.e., under dim light conditions), whereas cone photoreceptor cells primarily mediate photopic vision (i.e., under bright conditions). Rod and cone photoreceptor cells have the same basic structure. The outer segment is closest to the visual field and contains stacks of membranous discs that contain photopigments. This is followed by the inner segment, which contains mitochondria, nuclei, and finally synaptic bodies.

[0094] "Photoreceptor cell progenitor cells," "progenitor cells of photoreceptor neuronal cells" refer to postmitotic progenitor cells that can differentiate into cone or rod photoreceptor cells and ultimately express various photopigments. Photoreceptor cells and photoreceptor cell progenitor cells can express markers, including, but not limited to, cone-rod homeobox (Crx), recoverin, and cone arrestin (CAR).

[0095] Bipolar cells are retinal interneurons that provide one of the major pathways connecting photoreceptor cells to ganglion cells. Based on the type of photoreceptor cell they synapse with, bipolar cells can be divided into rod bipolar cells and cone bipolar cells.

[0096] Horizontal and amacrine cells are interneurons primarily responsible for lateral interactions within the retina. Amacrine cells receive input from bipolar cells, while horizontal cells receive input from photoreceptor cells. Amacrine cells operate in the inner plexiform layer, while horizontal cells operate in the outer plexiform layer. Both cell types are found in the inner nuclear layer of the retina.

[0097] Retinal ganglion cells transmit visual signals from photoreceptor cells to the visual center of the brain. The axons of retinal ganglion cells project from the retina to the brain. There are various subtypes of retinal ganglion cells, which can be classified by those skilled in the art based on the size and morphology of the cell body and dendrites, as well as the extent of the dendritic field, for example, the layer of the inner plexiform layer into which the dendrites extend.

[0098] Glial cells, also called neuroglia, are non-neuronal cells located within the central and peripheral nervous systems that provide physical and metabolic support to neurons. Muller glial cells are the main type of retinal glial cells. Without wishing to be bound by theory, it is believed that Muller glia are responsible for maintaining the structural and functional stability of retinal neurons. This includes, for example, regulating the extracellular environment by uptake of neurotransmitters, removal of cellular debris, regulating ion levels, and storing glycogen, as well as providing electrical insulation and mechanical support to neural retinal cells.

[0099] As used herein, "retinal pigment epithelium," "retinal pigment epithelial cells," or "RPE" refers to a single layer of cuboidal cells that is the layer furthest from the vitreous of the eye. In other words, the RPE is between the NR layer and the choroid. RPE cells play an important role in maintaining visual function. RPE cells are phagocytic and can engulf and eliminate photoreceptor outer segments. RPE cells help maintain normal regeneration of visual cells. RPE cells express various markers during development, which can be categorized into early, middle, and late RPE markers. Early RPE markers include, but are not limited to, melanocyte-inducing transcription factor (MITF), tyrosinase-related protein 1 (TYRP1), pre-melanosome protein (PMEL), and transmembrane protein with EGF-like and two follistatin-like domains (TMEFF2). Middle RPE markers include, but are not limited to, tyrosinase (TYR) and retinaldehyde-binding protein (1RLBP1). Late RPE markers include, but are not limited to, retinoid isomerohydrolase RPE65 (RPE65), bestrophin 1 (BEST1), and retinal G protein-coupled receptor (RGR).

[0100] As used herein, "early eye field" refers to the domain of the anterior neural plate that forms during gastrulation and gives rise to the optic vesicle. The early eye field is a single cohesive cell population whose identity can be determined through the expression of early eye field markers, including, but not limited to, SIX homeobox 3 (Six3), SIX homeobox 6 (Six6), retina homeobox gene 1 (Rx1), paired box 6 (Pax6), retinoid X receptor gamma (RXRγ), LIM homeobox 2 (LHX2), retina and anterior neural fold homeobox (RAX). Early eye field cells differentiate into embryonic retinal cells, which in turn give rise to retinal progenitor cells, which differentiate into the various cell types found in the retina. Embryonic retinal cells express markers, including but not limited to orthodenticle homeobox 2 (Otx2), neuronal differentiation 1 (NeuroD), B-lymphocyte-induced maturation protein 1 (Blimp1), transducin, phosducin (PdC), RXRy and thyroid hormone receptor isoform (Tr-32), atonal bHLH transcription factor 7 (Atoh7), and insulin gene enhancer protein (ISl-1). Without wishing to be bound by theory, it is believed that retinal differentiation occurs when retinal progenitor cells undergo a series of changes in competence in response to developmental signals, with RPCs at early developmental stages giving rise to early retinal cell types (e.g., retinal ganglion cells), whereas later retinal progenitor cells give rise to later cell types (e.g., Müller cells). However, because there is a temporal overlap in the development of various retinal cell types, multiple retinal cell types may form simultaneously, and retinal progenitor cells may be heterogeneous.

[0101] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used in the description of the disclosure herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure.

[0102] All publications, patent applications, patents, and other references cited herein are incorporated by reference in their entirety.

[0103] Unless the context indicates otherwise, it is expressly intended that the various features of the present disclosure described herein can be used in any combination. Moreover, the present disclosure also contemplates that in some embodiments of the present disclosure, any feature or combination of features described herein can be excluded or omitted.

[0104] The methods disclosed herein may include one or more steps or acts for achieving the described method. Method steps and / or acts may be interchanged without departing from the scope of the invention. In other words, unless a particular order of steps or acts is required for proper operation of an embodiment, the order and / or use of specific steps and / or acts may be modified without departing from the scope of the invention.

[0105] Methods for generating retinal cell populations The present disclosure provides a method for generating a population of retinal cells from undifferentiated pluripotent stem cells. The method includes culturing a population of undifferentiated pluripotent stem cells with various combinations of growth factors and growth factor inhibitors in a series of steps that induce the differentiation of the undifferentiated pluripotent stem cells into a retinal cell fate. In an exemplary differentiation pathway, human embryonic stem cells (hESCs) are induced to differentiate into early eye field cells, which are induced to differentiate into embryonic retinal cells, which are then induced to differentiate into progenitor cells and mature photoreceptor neuronal cells (PNCs). The resulting population of cells may contain a mixture of cell types. However, cells from later stages of the pathway may predominate, and residual hESCs may be minimal or absent.

[0106] Without wishing to be bound by theory, it is believed that compositions containing mixed cell populations, especially when delivered in combination with a scaffold, may be suitable treatments for vision disorders and diseases because the range of cell types increases the number of niches into which the cells can engraft when administered to a subject, and increases the number of fates the cells can adopt upon administration.

[0107] In some aspects, a method is provided herein for obtaining a population of neural retinal cells, e.g., neural retinal cells, from undifferentiated pluripotent stem cells. In an aspect, the method includes growing undifferentiated pluripotent stem cells in dynamic culture, seeding the undifferentiated pluripotent stem cells as single cells in a culture vessel, and culturing the undifferentiated pluripotent stem cells in the culture vessel under differentiation conditions to obtain a population of retinal cells, e.g., a population comprising NRCs. In an embodiment of the present invention, the culture vessel is a bioreactor, e.g., a PBS vertical wheel bioreactor.

[0108] Method for inducing differentiation of retinal cells In accordance with the present disclosure, human pluripotent stem cells (hPSCs) can be differentiated into various populations of cells by culture in a variety of different media that include growth factors and growth factor inhibitors. In some embodiments, the undifferentiated pluripotent stem cells are under conditions sufficient for induced differentiation to generate a composition comprising a population of retinal cells. The population of retinal cells can be, for example, a population of cells comprising eye field cells, embryonic retinal cells, precursor cells of photoreceptor neuronal cells, mature photoreceptor neuronal cells, neuronal retinal cells (NRCs) (e.g., retinal ganglion cells, horizontal neurons, amacrine neurons, rod bipolar cells, cone bipolar cells, rod photoreceptor cells, cone photoreceptor cells), Müller glial cells, retinal pigment epithelial (RPE) cells, or any combination thereof. In some embodiments, the method includes culturing the population of hPSCs in three, four, or five different culture media (each comprising a combination of growth factors and / or growth factor inhibitors) for three, four, or five periods under conditions sufficient to drive the population cells into a target cell type, thereby generating a population of cells comprising the target cell type.

[0109] In some embodiments, undifferentiated pluripotent stem cells are seeded into a culture vessel for differentiation as single cells and grown in suspension as cell aggregates for at least 14 weeks. In some embodiments, undifferentiated pluripotent stem cells are seeded into a culture vessel for differentiation as single cells and grown in suspension as cell aggregates for 14-18 weeks. In examples, the cell concentration at seeding and the agitation speed of the vessel along the differentiation process may be adjusted to achieve optimal yield and quality. In further examples, the feeding frequency may be determined according to the lactate and glucose levels along the differentiation process to achieve optimal yield and quality.

[0110] In some embodiments, the undifferentiated pluripotent stem cells are seeded at a density of 50,000 cells / mL to about 2,000,000 cells / mL, about 100,000 cells / mL to about 2,000,000 cells / mL, or about 200,000 cells / mL to about 500,000 cells / mL. In some embodiments, the cells are seeded at a density of about 200,000 cells / mL to about 300,000 cells / mL. In some embodiments, the cells are seeded at a density of about 150,000 cells / mL to about 250,000 cells / mL. In some embodiments, the cells are seeded at a density of 250,000 cells / mL.

[0111] In some embodiments, the culture vessel is a bioreactor. In embodiments, the culture vessel is a vertical wheel bioreactor. In other embodiments, the bioreactor is a vertically oriented bioreactor, e.g., a VERTICAL-WHEEL® bioreactor (PBS Biotech, Camarillo, CA). In some embodiments, the bioreactor is a wave bioreactor. In some embodiments, the bioreactor is a gas permeable rapid expansion bioreactor (e.g., a G-REX®, Wilson Wolf Corp., St. Paul, MN) bioreactor. PBS Wheel dynamic culture offers the unique advantage of being able to control the size of the aggregates while avoiding numerous manual steps (seeding microwells for aggregate formation, magnetic separation of undifferentiated cells, aggregate size filtration, etc.) that would otherwise impede the transition of the technology to industrial grade. Although cell growth in bioreactors has been performed for many years with transformed mammalian cell lines, it is not a trivial practice to grow differentiated cells in bioreactors and achieve a controlled differentiation process as well. Because such differentiation requires a specific microenvironment to occur within the 3D structure niche of the cell aggregates. Tight control is required to keep the aggregates at the correct size. The aggregates need to be small enough to allow differentiation factors access to the required cell layers, but the rate at which the aggregate size is controlled needs to maintain minimal shear forces to avoid cell damage. Moreover, the biological state of origin of the undifferentiated cells can greatly affect the differentiation potential of the cells, affecting the yield, quality and cell viability of the final product.

[0112] In some embodiments, culture conditions (e.g., optimal culture conditions) may be utilized in larger vessels for differentiation process and scale-up (e.g., to be commercially viable). In yet other examples, the cell aggregates (e.g., organoids) described herein are grown in dynamic suspension and differentiated directly into PNCs without excluding pigment cell aggregates (e.g., organoids) containing retinal epithelial cells (RPE). In examples, the cells after the differentiation process (e.g., mature PNCs or PNCs) contain about 1% to 30% RPE. In examples, the cells after the differentiation process contain less than about 50% RPE. In examples, the cells after the differentiation process contain less than about 40% RPE. In examples, the cells after the differentiation process contain less than about 30% RPE. In examples, the cells after the differentiation process contain about 5% to about 30%, about 10% to about 30%, or about 20% to about 30% RPE. In embodiments, the cells after the differentiation process contain about 1% to about 25%, about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, or about 1% to about 5% RPE. The percentage of RPE can be any value or subrange within the stated ranges, including the endpoints.

[0113] In some embodiments, the aggregates have a size controlled by the rotation speed of the culture vessel. In some embodiments, the size of the aggregates is about 50 μm to about 900 μm in diameter. In some embodiments, the size of the aggregates is about 100 μm to about 800 μm in diameter. In some embodiments, the size of the aggregates is about 50 μm to about 500 μm in diameter, about 50 μm to about 300 μm in diameter, about 100 μm to about 250 μm in diameter, about 150 μm to about 350 μm in diameter, about 300 μm to about 800 μm in diameter, about 200 μm to about 600 μm in diameter, or about 250 μm to about 450 μm in diameter. For example, as the size of the aggregates increases, the speed is increased to keep the aggregates in suspension and generate a controlled shear stress to control the size of the aggregates. For example, the speed can start at about 30 rpm (also called revolutions per minute, rpm) and can be increased (e.g., scaled) to about 50 rpm, about 60 rpm, about 70 rpm, about 80 rpm, about 90 rpm, or about 100 rpm. For example, the rotation speed is increased during the differentiation process. The speed can be increased by 1 rpm, 5 rpm, 10 rpm, 15 rpm, 20 rpm, or more at various stages throughout the differentiation process.

[0114] In embodiments, the size of the aggregates is from about 100 μm to about 800 μm in diameter, examples of which include 50-100 microns, 50-150 microns, 100-250 microns, or 150-350 microns or 250-450 microns. The size may be any value or subrange within the recited ranges, including the endpoints.

[0115] In some embodiments, the culture conditions sufficient to generate aggregates of a desired size (e.g., 100 μm to 800 μm in diameter) include culturing cells in a culture vessel. In some embodiments, the culture vessel is a vertical wheel bioreactor, a wave bioreactor, or a gas permeable rapid expansion bioreactor. In some aspects, the rotational speed in the wheel bioreactor is increased over time, thereby generating cell aggregates of 100 μm to 800 μm in diameter suspended in the bioreactor. In some aspects, the rotational speed of the wheel bioreactor applies a controlled shear stress to the cell aggregates to maintain optimal aggregate size. Without wishing to be bound by theory, it is believed that the culture conditions described herein allow for uniform differentiation of all cells in the cell aggregates by allowing the cells in the aggregates to be uniformly exposed to differentiation factors in the cell culture medium.

[0116] The size of the aggregates can be controlled by the rotation speed of the culture vessel (see Borys et al. Stem Cell Research & Therapy, 2021, 12:55, Overcoming bioprocess bottlenecks in the large-scale expansion of high-quality hiPSC aggregates in vertical-wheel stirred suspension bioreactors, doi.org / 10.1186 / s13287-020-02109-4). In an embodiment, the size of the aggregates is about 100 μm to about 800 μm. For example, the rotation speed may be increased during the differentiation process. In an exemplary embodiment, 300 micron sized aggregates of hESC-derived embryoid bodies were found to be optimal for the generation of CRX (cone-rod homeobox) positive cells (see Yanai et al. 2013, Differentiation of Human Embryonic Stem Cells Using Size-Controlled Embryoid Bodies and Negative Cell Selection in the Production of Photoreceptor Precursor Cells, Tissue Engineering: Part C Volume 19, Number 10).

[0117] In some embodiments, the rotation speed of the bioreactor is between 25 and 90 revolutions per minute (RPM, or rpm). In some embodiments, the rotation speed of the bioreactor is between 35 and 80 rpm. In some embodiments, the initial rotation speed, i.e., the rotation speed at the beginning of the differentiation process, is about 30 to 50 rpm, and is increased to about 60 to 80 rpm by the end of the differentiation process. In some embodiments, the initial rotation speed is about 30 to about 50 rpm, about 30 to about 45 rpm, or about 35 to about 40 rpm. In some embodiments, the initial rotation speed is about 30 rpm. In some embodiments, the initial rotation speed is about 35 rpm. In some embodiments, the initial rotation speed is about 40 rpm. In some embodiments, the initial rotation speed is about 45 rpm. In some embodiments, the final rotation speed, i.e., the rotation speed at the end of the differentiation process and before the cell aggregates are optionally disaggregated and seeded into static culture, is about 60 to about 90 rpm. In some embodiments, the final rotation speed is about 60 rpm. In some embodiments, the final rotation speed is about 70 rpm. In some embodiments, the final rotation speed is about 80 rpm. In some embodiments, the final rotation speed is about 90 rpm.

[0118] In some embodiments, the method includes an initial rotation speed of about 35-40 rpm during the first 4 weeks of the differentiation process, a second rotation speed of about 50-60 rpm during the 5th-7th week of the differentiation process, and a third rotation speed of about 70-80 rpm during the 8th week of the differentiation process and thereafter. One of skill in the art will appreciate that the rotation speeds provided herein are exemplary and are suitable for use with, for example, PBS Wheel and similar wheel bioreactors to generate aggregates of 100-800 microns in diameter. Alternative wheel bioreactors may have different rotation speeds to achieve the desired aggregate size, which can be readily determined by one of skill in the art.

[0119] In some embodiments, culturing the cells comprises the step of i) culturing the undifferentiated pluripotent stem cells for a first period under culture conditions sufficient to induce the stem cells to form cell aggregates (e.g., organoids), the cell aggregates comprising cells expressing one or more early eye field markers selected from retinal homeobox gene 1 (Rx1), paired box 6 (Pax6), retinoid X receptor gamma (RXRy), LIM homeobox 2 (LHX2), and retina and anterior neural fold homeobox (RAX).

[0120] In other embodiments, culturing the cells comprises culturing the cell aggregates from step i) above for a second period of time under culture conditions sufficient to differentiate the cell aggregates into cells expressing one or more embryonic retinal markers selected from orthodenticle homeobox 2 (Otx2), neuronal differentiation 1 (NeuroD), B-lymphocyte-induced maturation protein 1 (Blimp1), transducin, phosducin (PdC), RXRy and thyroid hormone receptor isoform (Tr-32), atonal bHLH transcription factor 7 (Atoh7) or insulin gene enhancer protein (Isl-1). In other embodiments, the differentiation process further comprises culturing the cells expressing one or more embryonic retinal markers from step ii) above for a third period of time under culture conditions sufficient to differentiate the cells into precursor cells of photoreceptor neurons (PNCs) and mature P, wherein the PNCs express one or more proteins selected from cone-rod homeobox (Crx), recoverin or cone arrestin (CAR).

[0121] In other embodiments, different differentiation factors may be further introduced at different stages of the differentiation process.

[0122] In some embodiments, the disclosure relates to a method of generating a composition comprising a population of retinal cells. In some embodiments, the method comprises: (a) culturing a population of undifferentiated pluripotent stem cells for a first period of time in a first cell culture medium comprising nicotinamide (NIC), a Wnt inhibitor, such as rel-4-[(3aR,4S,7R,7aS)-1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl]-N-8-quinolinylbenzamide (IWRe), and a Rock inhibitor (RI). In some embodiments, the RI is Y-27632 (trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide dihydrochloride). In some aspects, the undifferentiated pluripotent stem cells comprise human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs). In some embodiments, the hESCs comprise HADC102 cells. In some embodiments, the method comprises culturing the population of cells generated in step (a) for a second period of time in a second cell culture medium comprising NIC and a Wnt inhibitor (e.g., IWRe). In some embodiments, the method comprises culturing the population of cells generated in step (b) for a third period of time in a third cell culture medium comprising NIC and insulin-like growth factor 1 (IGF-1). In some embodiments, the method comprises (d) collecting the population of cells, thereby producing a composition comprising a population of retinal cells. In some embodiments, the population of cells is cultured in a culture vessel for at least 14 weeks under conditions sufficient to produce cell aggregates about 100 μm to 800 μm in diameter.

[0123] In some embodiments, the method of producing a composition comprising a population of retinal cells comprises step (i) prior to step (d), where step (i) comprises culturing the population of cells produced in step (c) in a fourth cell culture medium comprising IGF-1, NIC, and tert-butyl (S)-{(2S)-2-[2-(3,5-difluorophenyl)acetamido]propanamide}phenylacetate (DAPT) for a fourth period of time.

[0124] In some embodiments, the method of producing a composition comprising a population of retinal cells comprises step (ii) following step (i), where step (ii) comprises culturing the population of cells of step (i) in a fifth cell culture medium comprising IGF-1, retinoic acid (RA), taurine (TA), brain-derived neurotrophic factor (BDNF), and neurotrophin-4 (NT4) for a fifth period of time.

[0125] In some embodiments, the first period is 1 to 25 days, 1 to 15 days, 1 to 7 days, or 1 to 3 days. In some embodiments, the second period is 1 to 30 days, 2 to 20 days, 11 to 20 days, or 3 to 14 days. In some embodiments, the third period is 5 days to 14 weeks, 10 days to 3 weeks, 10 days to 8 weeks, or 2 weeks to 5 weeks. In some embodiments, the fourth period is 2 weeks to 20 weeks, 2 weeks to 18 weeks, 2 weeks to 12 weeks, 4 weeks to 10 weeks, or 5 weeks to 6 weeks. In some embodiments, the fifth period is 4 weeks to 18 weeks, 4 weeks to 12 weeks, 5 weeks to 9 weeks, 10 weeks to 25 weeks, 6 weeks to 24 weeks, 12 weeks to 18 weeks, 7 weeks to 16 weeks, or 7 weeks to 12 weeks. The times may be any value or subrange within the recited ranges, including the endpoints.

[0126] In some embodiments, the first period is 1-7 days. In some embodiments, the second period is 2-20 days. In some embodiments, the third period is 10 days-8 weeks. In some embodiments, the fourth period is 2-10 weeks. In some embodiments, the fifth period is 5-18 weeks. In some embodiments, the fifth period is 9-18 weeks.

[0127] In some embodiments, the first period is 3-4 days, the second period is 10-11 days, the third period is 20-21 days, the fourth period is 7-8 days, and the fifth period is 35-36 days, in other embodiments, the fifth period is 63-64 days.

[0128] In some embodiments, the first period is about 1-7 days, the second period is about 2-20 days, the third period is about 10 days-8 weeks, the fourth period is about 2-10 weeks, and the fifth period is about 5-18 weeks.

[0129] In some embodiments, the methods include (i) collecting the cell aggregates, (ii) dispersing the cell aggregates to generate dispersed cells, and (iii) cryopreserving the dispersed cells using the methods described herein.

[0130] In alternative embodiments, the method includes, prior to step (d), (i) collecting the cell aggregates, (ii) dispersing the cell aggregates to generate dispersed cells, (iii) seeding the dispersed cells into tissue culture flasks, and (iv) culturing the dispersed cells under adherent static conditions for at least 1 week. In some embodiments, the dispersed cells are cultured for 1-5 weeks, 1-4 weeks, 1-3 weeks, or 1-2 weeks. In some embodiments, the cells are collected from the tissue culture flasks and cryopreserved using the methods described herein.

[0131] The cell aggregates can be collected by any method known in the art, including, but not limited to, centrifugation and filtration, or a combination thereof.

[0132] The cell aggregates can be dispersed by any method known in the art, including mechanical dispersion, chemical dispersion, and enzymatic digestion (eg, with trypsin, such as TrpLE).

[0133] In some embodiments, for example, when the dispersed cells are cultured under adherent static conditions, the dispersed cells are seeded at a density of 50,000 cells / mL to about 2,000,000 cells / mL, about 100,000 cells / mL to about 2,000,000 cells / mL, or about 200,000 cells / mL to about 500,000 cells / mL. In some embodiments, the cells are seeded at a density of about 200,000 cells / mL to about 300,000 cells / mL. In some embodiments, the cells are replated at a density of about 150,000 cells / mL to about 250,000 cells / mL. In some embodiments, the cells are seeded at a density of 250,000 cells / mL.

[0134] In some embodiments, the population of retinal cells comprises early eye field cells, embryonic retinal cells, photoreceptor cells, which may include precursor cells of photoreceptor neuron cells (PNC) or mature photoreceptor neuron cells, or any combination thereof. In some embodiments, the population of retinal cells comprises neuronal retinal cells (NRC), Muller glial cells, retinal pigment epithelial (RPE) cells, or any combination thereof. In some embodiments, the NRC comprises PNC, retinal ganglion cells, horizontal neurons, amacrine neurons, rod bipolar cells, cone bipolar cells, rod photoreceptor cells, cone photoreceptor cells, or any combination thereof.

[0135] As described herein, in an embodiment, the pluripotent stem cell may be a human embryonic stem cell (hESC) or a human induced pluripotent stem cell (hiPSC). In an aspect, culturing the cell comprises the step of i) i.e., i) culturing the undifferentiated pluripotent stem cell for a first period under culture conditions sufficient to induce the stem cell to form a cell aggregate (e.g., an organoid), the cell aggregate comprises cells expressing one or more early eye field markers selected from Rx1, Pax6, RXRy, LHX2 and RAX. In other aspects, various differentiation factors may be further introduced at various stages of the differentiation process.

[0136] In other aspects, culturing the cells further comprises culturing the cell aggregates from step i) above for a second period of time under culture conditions sufficient to differentiate the cell aggregates into cells expressing one or more embryonic retinal markers selected from Otx2, NeuroD, Blimp1, transducin, phosducin (PdC), RXRy, and Tr-32, Atoh7, or Isl-1. In other embodiments, the differentiation process further comprises culturing the cells expressing one or more embryonic retinal markers from step ii) above for a third period of time under culture conditions sufficient to differentiate the cells into photoreceptor neuronal (PNC) precursor cells and mature photoreceptor neurons, where the PNC express one or more proteins selected from Crx, recoverin, or cone arrestin (CAR).

[0137] In an exemplary differentiation protocol, embryonic stem cells are differentiated using a first differentiation lineage and then further differentiated into photoreceptor cells using IGF-1, retinoic acid (RA), BDNF, and NT4, thereby generating progenitor cells and mature photoreceptor neuronal cells.

[0138] Additional contemplated differentiation agents include, for example, Noggin, FGF antagonists (Dkkl or IWR1e), Nodal antagonists (Lefty-A), retinoic acid, taurine, GSK3b inhibitors (CHIR99021), Notch inhibitors (DAPT), retinoic acid receptor (RAR) agonists or antagonists, FGF signaling pathway agonists (aFGF, bFGF), Hedgehog pathway agonists (Shh), Insulin growth factor pathway agonists (IGF), PI3 kinase pathway, EGF pathway, BMP pathway, and Hippo pathway agonists, and Rho kinase (Rock) inhibitors.

[0139] Rho kinase inhibitors (Rho-associated protein kinase inhibitors or ROCK inhibitors) are a group of compounds that target Rho kinase (ROCK) and inhibit the ROCK pathway.The ROCK inhibitors used in the methods described herein can be any ROCK inhibitor, including but not limited to Y-27632 (trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide dihydrochloride), HA-1077 (fasudil), HA-1100 (hydroxyfasudil), H-1152, 3-(4-pyridyl)-1H-indole, N-(4-pyridyl)-N'-(2,4,6-trichlorophenyl)urea, aurothioglucose, LY294002 or its salt, base, ester or prodrug.

[0140] Wnt inhibitors can be small molecules or proteins that act to antagonize Wnt signaling, generally by preventing ligand-receptor interaction or Wnt receptor maturation, including sFRP, Dkk, WIF, Wise / SOST, Cerberus, IGFBP, Shisa, Waif1, APCDD1, and Tiki1. Exemplary Wnt inhibitors include rel-4-[(3aR,4S,7R,7aS)-1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl]-N-8-quinolinylbenzamide (IWRe) and IWP-4.

[0141] Such differentiation agents may be added at any stage of the differentiation procedure, examples include before the first period, during or after the first period, during or after the second period, during or after the third period, during or after the fourth period, or during or after the fifth period.

[0142] In some embodiments of the method, the cells are cultured with a Rock inhibitor (RI, e.g., Y-27632), nicotinamide (NIC), and a Wnt inhibitor (e.g., IWRe1) for a first period of time. In some embodiments, the Rock inhibitor can be at a concentration of about 0.01-100 μM, or about 0.1-100 μM, or about 1-100 μM, or about 1-50 μM, or about 1-40 μM, or about 1-30 μM, or about 1-20 μM, or about 1-10 μM. In some embodiments, the Rock inhibitor can be at a concentration of about 1-10 μM. In some embodiments, the Rock inhibitor can be at a concentration of about 10 μM. Nicotinamide can be at a concentration of about 0.1-100 mM, or about 1-100 mM, or about 1-90 mM, or about 1-80 mM, or about 1-70 mM, or about 1-60 mM, or about 1-50 mM, or about 1-40 mM, or about 1-30 mM, or about 1-20 mM. In some embodiments, nicotinamide can be at a concentration of about 1-50 mM. In some embodiments, Rock inhibitor can be at a concentration of about 10 mM. In other embodiments, Wnt inhibitor can include any Wnt inhibitor, such as IWRe1. In some embodiments, the Wnt inhibitor can be at a concentration of about 0.01 to about 100 μM, or about 0.5 to about 100 μM, or about 0.5 to about 50 μM, or about 0.5 to about 40 μM, or about 0.5 μM to about 30 μM, or about 0.5 to about 20 μM. In some embodiments, the Wnt inhibitor can be at a concentration of about 0.5 to about 20 μM. In some embodiments, the Wnt inhibitor can be at a concentration of about 3 μM. The concentration may be any value or subrange within the recited ranges, including the endpoints.

[0143] In some embodiments of the methods described herein, the cells are cultured with NIC and a Wnt inhibitor for a second period of time. In some embodiments, the NIC can be at a concentration of about 0.1-100 mM, or about 0.1-50 mM, or about 1-100 mM, or about 1-50 mM. In some embodiments, the nicotinamide can be at a concentration of about 1-50 mM. In some embodiments, the NIC can be at a concentration of about 10 mM. The Wnt inhibitor can include IWRe1 or Dkkl, sFRP, Dkk, WIF, Wise / SOST, Cerberus, IGFBP, Shisa, Waifl, APCDD1, and Tiki1. In some embodiments, the Wnt inhibitor includes IWRe1. The Wnt inhibitor can be at a concentration of about 0.01 to about 100 μM, or about 0.5 to about 100 μM, or about 0.5 to about 50 μM, or about 0.5 to about 40 μM, or about 0.5 μM to about 30 μM, or about 0.5 to about 20 μM. In some embodiments, the Wnt inhibitor can be at a concentration of about 0.5 to about 20 μM. In some embodiments, the Wnt inhibitor can be at a concentration of about 3 μM. The concentration may be any value or subrange within the recited ranges, including the endpoints.

[0144] In some embodiments of the methods described herein, the cells are cultured with NIC and insulin growth factor 1 (IGF-1) for a third period. In some embodiments, nicotinamide can be at a concentration of about 0.1-100 mM, or about 1-100 mM, or about 1-90 mM, or about 1-80 mM, or about 1-70 mM, or about 1-60 mM, or about 1-50 mM, or about 1-40 mM, or about 1-30 mM, or about 1-20 mM. In some embodiments, nicotinamide can be at a concentration of about 1-50 mM. In some embodiments, nicotinamide can be at a concentration of 10 mM. In some embodiments, IGF-1 can be at a concentration of about 0.01 to about 100 ng / mL, or about 0.5 to about 100 ng / mL, or about 0.5 to about 50 ng / mL, or about 0.5 to about 40 ng / mL, or about 0.5 ng / mL to about 30 ng / mL, or about 0.5 to about 20 ng / mL. In embodiments, IGF-1 can be at a concentration of about 0.5 to about 20 ng / mL. In some embodiments, IGF-1 can be at a concentration of about 5 ng / mL. The concentration can be any value or subrange within the recited ranges, including the endpoints.

[0145] In further embodiments of the methods described herein, culturing the cells further comprises differentiating for a fourth period with NIC, IGF-1 and a Notch inhibitor. Nicotinamide can be at a concentration of about 0.1-100 mM, or about 1-100 mM, or about 1-90 mM, or about 1-80 mM, or about 1-70 mM, or about 1-60 mM, or about 1-50 mM, or about 1-40 mM, or about 1-30 mM, or about 1-20 mM. In some embodiments, nicotinamide can be at a concentration of about 1-50 mM. In some embodiments, NIC can be at a concentration of about 10 mM. In some embodiments, IGF-1 can be at a concentration of about 0.01 to about 100 ng / mL, or about 0.5 to about 100 ng / mL, or about 0.5 to about 50 ng / mL, or about 0.5 to about 40 ng / mL, or about 0.5 ng / mL to about 30 ng / mL, or about 0.5 to about 20 ng / mL. In some embodiments, IGF-1 can be at a concentration of about 0.5 to about 20 ng / mL. In some embodiments, IGF-1 can be at a concentration of about 5 ng / mL. In embodiments, the Notch inhibitor is DAPT, valproic acid, RO4929097, YO-1027, BT-GSI, CB-10, IMR-1, psoralidin, avagacestat, J1051, or the like. In embodiments, the Notch inhibitor may be at a concentration of about 0.1-100 μM, or about 1-100 μM, or about 1-90 μM, or about 1-80 μM, or about 1-70 μM, or about 1-60 μM, or about 1-50 μM, or about 1-40 μM, or about 1-30 μM, or about 1-20 μM. In some embodiments, the Notch inhibitor may be at a concentration of about 1-50 μM. In some embodiments, the Notch inhibitor may be at a concentration of about 10 μM. The concentration may be any value or subrange within the recited ranges, including the endpoints.

[0146] In further embodiments of the methods described herein, culturing the cells further comprises differentiating for a fifth period with IGF-1, retinoic acid (RA), taurine, BDNF, and NT4. For example, the IGF can be at a concentration of about 1-100 ng / mL, or about 1-50 ng / mL, or about 1-20 ng / mL, or about 5-100 ng / mL, or about 5-50 ng / mL, or about 10-50 ng / mL, or about 10.5-20 ng / mL. In some embodiments, the IGF can be at a concentration of about 0.5-20 ng / mL. In some embodiments, the IGF can be at a concentration of about 5 ng / mL. In other embodiments, retinoic acid can be at a concentration of about 0.01-10 μM, or about 0.01-5 μM, or about 0.01-1 μM, or about 0.1-10 μM, or about 0.1-5 μM, or about 0.1-20 μM. In other embodiments, retinoic acid can be at a concentration of about 0.1-2 μM. In some embodiments, RA can be at a concentration of about 0.5 μM. In other embodiments, taurine (TA) may be at a concentration of about 0.1 to 1000 μM, or about 1 μM to 1000 μM, or about 10 to about 1000 μM, or about 10 to 900 μM, or about 10 to 800 μM, or about 10 to 700 μM, or about 10 to 600 μM, or about 10 to 500 μM, or about 10 to 400 μM, or about 10 to 300 μM, or about 10 to 200 μM, or about 10 to 100 μM, or about 10 to 400 μM. In other examples, taurine (TA) may be at a concentration of about 10 to 400 μM. In some embodiments, the concentration of TA may be at a concentration of about 100 μM. In other embodiments, the BDNF can be at a concentration of about 1-500 ng / mL, or about 1-400 ng / mL, or about 1-300 ng / mL, or about 1-200 ng / mL, or about 1-100 ng / mL, or about 2-500 mg / mL, or about 2-400 ng / mL, or 2-300 ng / mL, or about 2-200 ng / mL. In other embodiments, the BDNF can be at a concentration of about 2-200 ng / mL. In some embodiments, the concentration of BDNF can be at a concentration of 20 ng / mL.In some embodiments, NT4 can be at a concentration of about 1-500 ng / mL, or about 1-400 ng / mL, or about 1-300 ng / mL, or about 1-200 ng / mL, or about 1-100 ng / mL, or about 2-500 mg / mL, or about 2-400 ng / mL, or about 2-300 ng / mL, or about 2-200 ng / mL. In some embodiments, NT4 can be at a concentration of about 2-200 ng / mL. In some embodiments, the concentration of NT4 can be at a concentration of 20 ng / mL. The concentration can be any value or subrange within the recited ranges, including the endpoints.

[0147] In some embodiments, the disclosure relates to a method of generating a composition of retinal cells, the method comprising: (a) culturing a population of undifferentiated pluripotent stem cells in a first cell culture medium comprising nicotinamide (NIC) at a concentration of 10 mM, rel-4-[(3aR,4S,7R,7aS)-1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl]-N-8-quinolinylbenzamide (IWRe) at a concentration of 3 μM, and a Rock inhibitor (RI) at a concentration of 10 μM for at least one day. In some embodiments, the method comprises: (b) culturing the population of cells generated in step (a) in a second cell culture medium comprising NIC at a concentration of 10 mM, and IWRe at a concentration of 3 μM for at least two days. In some embodiments, the method includes (c) culturing the population of cells generated in step (b) in a third cell culture medium containing NIC at a concentration of 10 mM and insulin-like growth factor 1 (IGF-1, or IGF1) at a concentration of 5 ng / mL for at least 10 days. In some embodiments, the method includes (d) harvesting the population of cells, thereby producing a composition comprising a population of retinal cells. In some embodiments, the population of cells is cultured in a culture vessel for at least 14 weeks under conditions sufficient to produce aggregates having a diameter of 100 μm to 800 μm.

[0148] In other embodiments, the disclosure relates to a method of generating a composition of retinal cells, the method comprising: (a) culturing a population of undifferentiated pluripotent stem cells in a first cell culture medium comprising nicotinamide (NIC) at a concentration of 10 mM, IWRe at a concentration of 3 μm, and Rock inhibitor (RI) at a concentration of 10 μm for at least 1 day. In some embodiments, the method comprises: (b) culturing the population of cells generated in step (a) in a second cell culture medium comprising NIC at a concentration of 10 mM, and IWRe at a concentration of 3 μm for at least 2 days. In some embodiments, the method comprises: (c) culturing the population of cells generated in step (b) in a third cell culture medium comprising NIC at a concentration of 10 mM, and insulin-like growth factor 1 (IGF-1) at a concentration of 5 ng / mL for at least 10 days. In some embodiments, the method includes (d) culturing the population of cells generated in step (c) in a fourth cell culture medium comprising IGF-1 at a concentration of 5 ng / mL, NIC at a concentration of 10 mM, and DAPT at a concentration of 10 μM for at least 2 weeks. In some embodiments, the method includes (e) harvesting the population of cells, thereby producing a composition comprising a population of retinal cells. In some embodiments, the population of cells is cultured in a culture vessel for at least 14 weeks under conditions sufficient to produce aggregates having a diameter of 100 μm to 800 μm.

[0149] In other embodiments, the disclosure relates to a method of generating a composition of retinal cells, the method comprising: (a) culturing a population of undifferentiated pluripotent stem cells in a first cell culture medium comprising nicotinamide (NIC) at a concentration of 10 mM, IWRe at a concentration of 3 μm, and Rock inhibitor (RI) at a concentration of 10 μm for at least 1 day. In some embodiments, the method comprises: (b) culturing the population of cells generated in step (a) in a second cell culture medium comprising NIC at a concentration of 10 mM, and IWRe at a concentration of 3 μm for at least 2 days. In some embodiments, the method comprises: (c) culturing the population of cells generated in step (b) in a third cell culture medium comprising NIC at a concentration of 10 mM, and insulin-like growth factor 1 (IGF-1) at a concentration of 5 ng / mL for at least 10 days. In some embodiments, the method includes (d) culturing the population of cells generated in step (c) in a fourth cell culture medium comprising IGF-1 at a concentration of 5 ng / mL, NIC at a concentration of 10 mM, and DAPT at a concentration of 10 μM for at least 2 weeks. In some embodiments, the method includes (e) culturing the population of cells generated in step (d) in a fifth cell culture medium comprising IGF-1 at a concentration of 5 ng / mL, retinoic acid (RA) at a concentration of 0.5 μM, taurine (TA) at a concentration of 100 μM, brain-derived neurotrophic factor (BDNF) at a concentration of 20 ng / mL, and neurotrophin-4 (NT4) at a concentration of 20 ng / mL for at least 5 weeks. In some embodiments, the method includes (f) harvesting the population of cells, thereby producing a composition comprising a population of retinal cells. In some embodiments, the population of cells is cultured in a culture vessel for at least 14 weeks under conditions sufficient to produce aggregates having a diameter of 300 μm to 800 μm.

[0150] In some embodiments, the methods described herein result in a population of cells in which at least 50% of the cells are viable, at least 60% of the cells are viable, at least 70% of the cells are viable, at least 80% of the cells in the population are viable, at least 90% of the cells in the population are viable, or at least 99% of the cells in the population are viable. In some embodiments, at least 60% of the cells in the population are viable. In some embodiments, at least 70% of the cells in the population are viable. In some embodiments, at least 80% of the cells in the population are viable. In some embodiments, at least 90% of the cells in the population are viable. In some embodiments, at least 95% of the cells in the population are viable. In some embodiments, about 50% to about 99% of the cells in the population are viable, about 60% to about 90% of the cells in the population are viable, about 70% to about 90% of the cells in the population are viable, about 70% to about 80% of the cells in the population of cells are viable, or about 80% to about 95% of the cells in the population are viable. In some embodiments, about 85% to about 95% of the cells in the population are viable. In some embodiments, about 50% to about 70% of the cells in the population are viable. In some embodiments, about 60% to about 80% of the cells in the population are viable. Cell viability can be determined by any suitable assay known in the art, including vital dye-based assays (e.g., MitoTracker Red, TMRE, calcein and esterase-based assays, etc.).

[0151] Suitable basal cell culture media for culturing cell populations are known to those skilled in the art and include commercially available basic media (i.e., chemically defined media or CDM), such as NUTRISTEM® (without TGFbeta and FGF2 for ESC differentiation, with TGFbeta and FGF2 for ESC proliferation), NEUROBASAL™, KO-DMEM, DMEM, DMEM / F12, CELLGRO™ Stem Cell Growth Medium, or X-VIVO™. Basic media may be supplemented with various agents known in the art dealing with cell culture. The following is a non-limiting reference to various supplements that may be included in the cultures used in accordance with the present disclosure: serum or serum replacement containing media (examples of which include, but are not limited to, knockout serum replacement (KOSR), NUTRIDOMA-CS, TCH™, N2, N2 derivatives, or B27, or combinations), extracellular matrix (ECM) components (examples of which include, but are not limited to, fibronectin, laminin, collagen, and gelatin). In some embodiments, the cell culture medium comprises a chemically defined medium (CDM) supplemented with N2, B27, or a combination thereof, and optionally supplemented with BrainPhys™. As a result, the ECM may be used to retain one or more members of the TGFI3 superfamily of growth factors, antimicrobial agents (examples include, but are not limited to, L-glutamine, beta-mercaptoethanol, penicillin, and streptomycin), and non-essential amino acids (NEAAs), i.e., neurotrophic factors known to play a role in promoting survival of SCs in culture (examples include, but are not limited to, BDNF, NT3, NT4).

[0152] In some embodiments, the present disclosure relates to a pharmaceutical composition produced by any of the above methods.

[0153] In some embodiments, the present disclosure relates to a method of treating a subject having a visual condition comprising a therapeutically effective amount of a pharmaceutical composition produced by any of the methods described above.

[0154] Cryopreservation of retinal cells In some embodiments of the methods described herein, the population of retinal cells is cryopreserved and ready for administration to a subject upon thawing. In embodiments, the population of retinal cells is cryopreserved in a cryopreservation medium. In embodiments, the cryopreservation medium comprises a cryoprotectant, e.g., glycerol, sucrose, dimethylsulfoxide (DMSO), or other suitable cryoprotectant. In embodiments, the cryoprotectant comprises glycerol. In embodiments, the cryoprotectant comprises sucrose. In embodiments, the cryoprotectant comprises DMSO. In embodiments, the cryoprotectant comprises dextran.

[0155] In some embodiments, the cells are in suspension when cryopreserved, for example by dispersing cell aggregates and optionally culturing the dispersed cells under static culture conditions, followed by filtration and / or centrifugation and resuspension in cryopreservation medium.

[0156] In an alternative embodiment, the cells are placed on or contained within a scaffold and both the cells and the scaffold are cryopreserved.

[0157] In some embodiments, the cryopreservation medium comprises about 0.1% to about 40% cryoprotectant. In embodiments, the cryopreservation medium comprises about 0.1% to about 30% cryoprotectant. In embodiments, the cryopreservation medium comprises about 0.1% to about 20% cryoprotectant. In embodiments, the cryopreservation medium comprises about 0.1% to about 10% cryoprotectant. In embodiments, the cryopreservation medium comprises about 0.1% to about 5% cryoprotectant. In embodiments, the cryopreservation medium comprises about 1% to about 40% cryoprotectant. In embodiments, the cryopreservation medium comprises about 1% to about 30% cryoprotectant. In embodiments, the cryopreservation medium comprises about 1% to about 20% cryoprotectant. In embodiments, the cryopreservation medium comprises about 1% to about 10% cryoprotectant. In embodiments, the cryopreservation medium comprises about 1% to about 5% cryoprotectant. The percentages may be measured as weight of cryoprotectant per volume of medium. The ratio may be measured as volume of cryoprotectant per volume of medium. The ratio may be any value or subrange within the stated range, including the endpoints.

[0158] In some embodiments, cryopreservation includes CRYOSTEM™, CRYOSTOR® CS2, CRYOSTOR® CS5, CRYOSTOR® CS10.

[0159] In other embodiments, cells can be cryopreserved during the differentiation process. For example, cells from step ii), e.g., culturing for a second period to differentiate into cells expressing embryonic retinal markers, can be cryopreserved, e.g., on days 14-120. Cells can be cryopreserved in a freezing solution, e.g., CRYOSTEM™, CRYOSTOR® CS2, CRYOSTOR® CS5, CRYOSTOR® CS10. In embodiments, the freezing solution can be specialized for freezing cell aggregates, and the freezing solution comprises CRYOSTEM™.

[0160] In some embodiments, the cryopreservation comprises a cryopreservation medium suitable for administration to the eye of a subject.

[0161] In some embodiments, the cells can be cryopreserved after the first, second, third, fourth or fifth period. In some embodiments, a therapeutically effective amount of the cryopreserved cells can be thawed and the thawed cells can be immediately administered to the eye of a subject having a visual condition. In some embodiments, a therapeutically effective amount of the cryopreserved cells can be thawed and the thawed cells are cultured before being administered to the eye of a subject having a visual condition. In further embodiments, the cryopreserved cells are thawed and then the thawed cells are further cultured according to any of the methods described herein.

[0162] In other embodiments, the cells can be cryopreserved after the second period and differentiated into cells expressing embryonic retinal markers at or near the completion of the first period. In other embodiments, the cells can be cryopreserved after a third period and differentiated into cells expressing photoreceptor neuronal (PNC) precursors and mature photoreceptor neurons. For example, the cryopreserved cells can be thawed and cultured at any of the remaining steps of the method.

[0163] In other embodiments, methods are described herein for preparing a retinal cell composition (e.g., a composition comprising PNC) for administration to a subject immediately after thawing. For example, the method includes (a) suspending the cells prepared according to the methods herein in a cryopreservation medium to form a cell suspension; (b) storing the cell suspension at a cryopreservation temperature; and (c) thawing the cryopreserved suspension. For example, the cryopreservation medium can include one or more of adenosine, dextran-40, lactobionic acid, HEPES (N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid)), sodium hydroxide, L-glutathione, potassium chloride, potassium bicarbonate, potassium phosphate, dextrose, sucrose, mannitol, calcium chloride, magnesium chloride, potassium hydroxide, sodium hydroxide, dimethyl sulfoxide (DMSO), or water.

[0164] In some embodiments, the cryopreservation temperature is below −80° C., or below −140° C. In some embodiments of the methods described herein, the population of cryopreserved retinal cells is greater than or equal to 1×10 5 cells / mL ~ approx. 100×10 6 Concentration is in cells / mL.

[0165] In some embodiments of the methods described herein, the population of cryopreserved retinal cells is greater than or equal to 2×10 6 cells / mL ~ approx. 5×10 6 is the concentration of cells / mL cells. In some embodiments of the methods described herein, the population of cryopreserved retinal cells is stored in a volume of about 100 μL to about 1 mL. In some embodiments, the cryopreserved retinal cells are stored in a volume of about 250 μL. In some embodiments, the cryopreserved retinal cells are stored in a volume of about 600 μL.

[0166] Methods for Proliferating and Maintaining Pluripotent Cells Provided herein are methods for propagating and maintaining human embryonic stem cells (hESCs), such as HADC102 cells, in an undifferentiated pluripotent state. In some embodiments, the methods include (a) coating tissue culture flasks with LN521 for static propagation of human embryonic stem cells in NutriStem® Growth Medium supplemented with TGFbeta and FGF2, and (b) culturing the adherent hESCs for a period of time. In some embodiments, the hESCs are further propagated by repeating steps (a) and (b). In some embodiments, the methods include (c) harvesting the cultured human embryonic stem cells generated by static propagation using TrypLE Select. The harvested hESCs are then differentiated using the methods described herein.

[0167] Provided herein is a method for growing and maintaining human embryonic stem cells (hESCs), such as HADC102 cells, in an undifferentiated pluripotent state, comprising (a) simultaneously combining human embryonic stem cells and extracellular matrix components (ECM) in growth medium in tissue culture flasks for static growth, and (b) culturing the adherent hESCs for a period of time.

[0168] In one aspect, provided herein is a method for growing and maintaining human embryonic stem cells (hESCs) in an undifferentiated pluripotent state. The method comprises (a) simultaneously combining human embryonic stem cells and extracellular matrix components (ECM) in a growth medium in a tissue culture flask for static growth, and (b) culturing the adherent hESCs for a period of time. In some embodiments, the growth medium comprises NutriStem® supplemented with TGFbeta and FGF2.

[0169] In some embodiments, statically grown cultured human embryonic stem cells are non-enzymatically harvested using ReLeSR™ and cultured in mTeSR™ plus medium on iMatrix-511 coated vessels. In some embodiments, the hESCs are further expanded by repeating steps (a) and (b).

[0170] In some embodiments, statically grown cultured human embryonic stem cells are harvested and further differentiated.

[0171] In one aspect, provided herein is a method for growing and maintaining human embryonic stem cells (hESCs) in an undifferentiated pluripotent state, the method comprising the steps of (a) simultaneously combining human embryonic stem cells, extracellular matrix components (ECM), and microcarriers in a growth medium to form a suspendable growth complex, and (b) culturing the suspendable growth complex for a period of time.

[0172] In some embodiments, the cultured human embryonic stem cells in the suspendible growth complexes are harvested and further expanded by repeating steps (a) and (b).

[0173] In some embodiments, the cultured human embryonic stem cells of the suspendible growth complexes are harvested and further differentiated.

[0174] Human embryonic stem cells can be isolated from human blastocysts. Human blastocysts are typically obtained from human in vivo preimplantation embryos or in vitro fertilized (IVF) embryos. Alternatively, single-cell human embryos can be expanded to the blastocyst stage. To isolate human ES cells, the zona pellucida is removed from the blastocyst and the intact inner cell mass (ICM) is isolated by a procedure in which the trophectoderm cells are lysed and removed from the intact ICM by gentle pipetting. The ICM is then seeded into tissue culture flasks containing an appropriate medium that allows its growth. After 9-15 days, the ICM-derived outgrowths are dispersed into clumps, either by mechanical dispersion or enzymatic dissociation, and the cells are then reseeded in fresh tissue culture medium. Colonies that show undifferentiated morphology are individually selected with a micropipette, mechanically dispersed into clumps, and seeded again. The resulting ES cells are then split, typically every 4-7 days. For further details regarding the details of methods for preparing human ES cells, see Reubinoff et al. Nat Biotechnol 2000, May: 18(5): 559; Thomson et al., [US Patent No. 5,843,780; Science 282: 1145, 1998; Curr. Top. Dev. Biol. 38: 133, 1998; Proc. Natl. Acad. Sci. USA 92: 7844, 1995]; Bongso et al., [Hum Reprod 4: 706, 1989]; and Gardner et al., [Fertil. Steril. 69: 84, 1998].

[0175] In addition, ES cells can be obtained from other species, including mice (Mills and Bradley, 2001), golden hamsters [Doetschman et al., 1988, Dev Biol. 127: 224-7], rats [Iannaccone et al., 1994, Dev Biol. 163: 288-92], rabbits [Giles et al. 1993, Mol Reprod Dev. 36: 130-8;Graves & Moreadith, 1993, Mol Reprod Dev. 1993, 30 36: 424-33], and several domestic animal species [Notarianni et al., 1991, J Reprod Fertil Suppl. 43: 255-60;Wheeler 1994, Reprod Fertil Dev. 6: 563-8;Mitalipova et al. al., 2001, Cloning. 3: 59-67], and non-human primate species (rhesus monkeys and marmosets) [Thomson et al., 1995, Proc Natl Acad Sci US A. 92: 7844-8; Thomson et al., 1996, Biol Reprod. 55: 254-9].

[0176] Expanded blastocyst cells (EBCs) can be obtained from blastocysts at least 9 days after fertilization, at the pre-gastrulation stage. Prior to culturing the blastocysts, the zona pellucida is digested (e.g., with Tyrode's acid solution (Sigma Aldrich, St. Louis, MO, USA)) to expose the inner cell mass. The blastocysts are then cultured as whole embryos in vitro for at least 9 days and up to 14 days after fertilization (i.e., before the gastrulation event) using standard embryonic stem cell culture methods.

[0177] Another method for preparing ES cells is described in Chung et al., Cell Stem Cell, Volume 2, Issue 2, 113-117, 7 February 2008. This method involves removing a single cell from an embryo during the process of in vitro fertilization, without destroying the embryo in the process.

[0178] EG (embryonic germ) cells are prepared from primordial germ cells obtained from fetuses at approximately 8-11 weeks of gestation (for human fetuses) using laboratory techniques known to those skilled in the art. The genital ridges are dispersed, cut into small pieces, and then disaggregated into cells by mechanical dispersion. The EG cells are then grown in tissue culture flasks with appropriate medium. The cells are cultured with daily changes of medium until cell morphology consistent with EG cells is observed, usually after 7-30 days or 1-4 passages. For additional details regarding methods for the preparation of human EG cells, see Shamblott et al., [Proc. Natl. Acad. Sci. USA 95: 13726, 1998] and U.S. Patent No. 6,090,622.

[0179] Yet another method for preparing ES cells is parthenogenesis, a process that also does not destroy the embryo.

[0180] Cells may be grown in suspension or in monolayers, with or without microcarriers. Growth of mixed cell populations in monolayer or suspension cultures may be adapted to large-scale growth in bioreactors or multistacks / hyperstacks by methods well known to those skilled in the art.

[0181] According to some embodiments, the growth phase lasts for at least 1 week to 20 weeks, such as at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, or even 10 weeks. In embodiments, the growth phase lasts for 1 week to 10 weeks, examples of which include 2 weeks to 10 weeks, 3 weeks to 10 weeks, 4 weeks to 10 weeks, or 4 weeks to 8 weeks. The duration may be any value or subrange within the recited ranges, including the endpoints.

[0182] According to yet other embodiments, the proliferation phase continues until a suitable lactate concentration in the cell culture medium and / or percent confluence is achieved. Percent confluence is the percentage of the surface area of ​​the culture vessel that appears covered with a layer of cells when observed under a microscope. In some embodiments, the undifferentiated pluripotent stem cells are cultured until the lactate concentration in the cell culture medium is about 1.0-13.0 mM, or about 1.5-12.5 mM. In some embodiments, the cells are cultured until the lactate concentration in the cell culture medium is about 1.68-12.29 mM. In some embodiments, the percent confluence is 5%-85%.

[0183] According to yet other embodiments, the mixed population of cells is passaged at least once during the growth phase, at least twice during the growth phase, at least three times during the growth phase, at least four times during the growth phase, at least five times during the growth phase, at least six times during the growth phase, or at least seven times during the growth phase.

[0184] Once the cells are enzymatically harvested, they can continue to grow for 8 or more passages, 9 or more passages, or even 10 or more passages (e.g., 11-15 passages). The number of total cell doublings can be increased to more than 30 (e.g., 31, 32, 33, 34 or more). (See International Patent Application Publication No. WO2017 / 021973, which is incorporated herein by reference in its entirety).

[0185] The extracellular matrix (ECM) is a three-dimensional network composed of extracellular polymers and minerals, such as collagen, enzymes, glycoproteins, and hydroxylapatite, that provide structural and biochemical support to surrounding cells. Because multicellularity evolved independently in various multicellular lineages, the composition of the ECM varies among multicellular structures, but cell adhesion, cell-cell communication, and differentiation are common functions of the ECM.

[0186] The extracellular matrix of animals includes the interstitial matrix and basement membranes. The interstitial matrix is ​​present between the various animal cells (i.e., in the intercellular spaces). A gel of polysaccharides and fibrous proteins fills the interstitial spaces and acts as a compressive buffer against stresses on the ECM. The basement membrane is a sheet-like deposit of ECM on which the various epithelial cells rest. Each type of connective tissue in animals has one type of ECM. Collagen fibers and bone minerals comprise the ECM of bone tissue, reticular fibers and ground substance comprise the ECM of loose connective tissue, and plasma is the ECM of blood.

[0187] Suitable extracellular matrix components for use within the scope of the present disclosure may include, but are not necessarily limited to, Matrigel®, vitronectin, gelatin, collagen I, collagen IV, laminin (e.g., laminin 521), fibronectin poly-D-lysine, derivatives thereof, or combinations thereof. In a particular embodiment, the human laminin is human laminin 511 E8 fragment.

[0188] In some embodiments, the microcarrier may comprise one or more of polystyrene, cross-linked dextran, magnetic particles, microchips, cellulose, hydroxylated methacrylate, collagen, gelatin, polystyrene, plastic, glass, ceramic, or silicone. In some embodiments, the microcarrier is comprised of polystyrene, surface-modified polystyrene, chemically modified polystyrene, cross-linked dextran, cellulose, acrylamide, collagen, alginate, gelatin, glass, DEAE-dextran, or combinations thereof. In some embodiments, the microcarrier is comprised of polystyrene. In some embodiments, the microcarrier is comprised of surface-modified polystyrene. In some embodiments, the microcarrier is comprised of chemically modified polystyrene. In some embodiments, the microcarrier is comprised of cross-linked dextran. In some embodiments, the microcarrier is comprised of cellulose. In some embodiments, the microcarrier is comprised of acrylamide. In some embodiments, the microcarrier is comprised of collagen. In some embodiments, the microcarrier is comprised of alginate. In some embodiments, the microcarrier is comprised of gelatin. In some embodiments, the microcarrier is comprised of glass. In some embodiments, the microcarrier is comprised of DEAE-dextran. In some embodiments, the microcarriers are uncoated.

[0189] In some embodiments, the microcarriers are coated. In embodiments, the microcarriers may be coated with Matrigel®, laminin, vitronectin, collagen, derivatives thereof, or combinations thereof. In embodiments, the microcarriers may be coated with polylysine, poly-L-lysine, poly-D-lysine, fibronectin, tenascin, dextran, peptides, or combinations thereof. In some embodiments, the microcarriers are coated with laminin. In some embodiments, the microcarriers are coated with Matrigel®. In some embodiments, the microcarriers are coated with collagen. In some embodiments, the microcarriers are coated with polylysine. In some embodiments, the microcarriers are coated with poly-L-lysine. In some embodiments, the microcarriers are coated with poly-D-lysine. In some embodiments, the microcarriers are coated with vitronectin. In some embodiments, the microcarriers are coated with fibronectin. In some embodiments, the microcarriers are coated with tenascin. In some embodiments, the microcarriers are coated with dextran. In some embodiments, the microcarriers are coated with peptides.

[0190] In some embodiments, the microcarriers may be spherical, smooth, macroporous, rod-shaped, or a combination thereof. In some embodiments, the microcarriers may be conjugated with protamine or polylysine. In some embodiments, the microcarriers are spherical. In some embodiments, the microcarriers are ellipsoids. In some embodiments, the microcarriers are rod-shaped. In some embodiments, the microcarriers are disc-shaped. In some embodiments, the microcarriers are porous. In some embodiments, the microcarriers are non-porous. In some embodiments, the microcarriers are smooth. In some embodiments, the microcarriers are flat.

[0191] In some embodiments, the microcarriers are neutral. In some embodiments, the microcarriers are negatively charged. In some embodiments, the microcarriers are hydrophilic.

[0192] In some embodiments, the microcarriers are 25 cm 2 , 50cm 2 , 75cm 2 , 100cm 2 , 125cm 2 , 150cm 2 , 175cm 2 , 200cm 2 , 225cm 2 , 250cm 2 , 500cm 2 , 625cm 2 , 750cm 2 , 1,000cm 2 , 1,250cm 2 , 5,000cm 2 , or 7,500 cm 2 The surface area may be any value or subrange within the stated range, including the endpoints.

[0193] In certain embodiments, the microcarriers are surface treated to enhance cell attachment and maximize cell yield and viability. The microcarriers may be constructed of USP Class VI polystyrene material. This provides a consistent platform. In some embodiments, the microcarriers create a synthetic surface on the microcarrier for stem cell growth. The enhanced attachment surface treatment infuses oxygen to the surface of the microcarrier improving cell attachment. In some embodiments, the microcarriers are non-pyrogenic. In some embodiments, the microcarriers are optimized for mesenchymal stem cell applications. In certain embodiments, the bead size may vary from 125-212 μm. In certain embodiments, the density of the microcarriers may be 1.026±0.004. In certain embodiments, the microcarriers are 360 ​​cm 2 / gram may be used.

[0194] In some embodiments, the method includes combining hESCs with laminin or a derivative thereof to improve cell attachment to the support surface. In a particular embodiment, the laminin is human laminin 511. In alternative embodiments, a number of other extracellular matrices may be used for cell attachment, examples of which include, but are not necessarily limited to, vitronectin, fibronectin, collagen, Matrigel®, or derivatives thereof.

[0195] In some embodiments, the cells may be cultured for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days.

[0196] In some embodiments, the cells may be cultured in a working volume of 10 mL to 3,000 mL, e.g., about 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 100 mL, 250 mL, 500 mL, 750 mL, 1,000 mL, or 3,000 mL. The volume may be any value or subrange within the stated ranges, including the endpoints.

[0197] In some embodiments, the cultured cells may be further expanded.

[0198] In some embodiments, the cultured cells may remain undifferentiated. Undifferentiated cells may be identified by the expression of various markers, examples of which include, but are not limited to, SSEA-5, TRA-1-60, Oct-4, and Nanog. In some embodiments, undifferentiated cells express SSEA-5. In some embodiments, undifferentiated cells express TRA-1-60. In some embodiments, undifferentiated cells express Oct-4. In some embodiments, undifferentiated cells express Nanog. In some embodiments, undifferentiated cells express both SSEA-5 and TRA-1-60. In some embodiments, undifferentiated cells express both Oct-4 and Nanog. In some embodiments, undifferentiated cells express SSEA-5, TRA-1-60, Oct-4, and Nanog (IPC#0).

[0199] In some embodiments, the cells may be cultured in feeder cell conditioned medium. ES culture methods may include the use of a feeder cell layer that secretes factors necessary for the proliferation of stem cells and at the same time inhibits their differentiation. Culturing is usually performed on a solid surface, for example, a surface coated with gelatin or vimentin. Exemplary feeder layers include human embryonic fibroblasts, adult fallopian tube epithelial cells, primary mouse embryonic fibroblasts (PMEF), mouse embryonic fibroblasts (MEF), mouse fetal fibroblasts (MFF), human embryonic fibroblasts (HEF), human fibroblasts obtained from the differentiation of human embryonic stem cells, human fetal muscle cells (HFM), human fetal skin cells (HFS), human adult skin cells, human foreskin fibroblasts (HFF), human umbilical cord fibroblasts, human cells obtained from the umbilical cord or placenta, and human bone marrow stromal cells (hMSC). Growth factors may be added to the medium to maintain the ESCs in an undifferentiated state. Such growth factors include bFGF and / or TGF. In another embodiment, agents may be added to the medium to maintain hESCs in a naive, undifferentiated state, see, e.g., Kalkan et al., 2014, Phil. Trans. R. Soc. B, 369: 20130540.

[0200] hESCs are usually seeded on top of feeder cells after 1-4 days in supportive medium (e.g., NUT(+) with human serum albumin, mTeSR™ plus, or mTeSR™1 StemFit™). Additional factors such as bFGF and TGFβ3 may be added to the medium to prevent ESC differentiation. Once a sufficient amount of hESCs is obtained, the cells may be mechanically disrupted (e.g., by using a sterile tip or a disposable sterile stem cell tool; 14602 Swemed). Alternatively, the cells may be removed by enzymatic treatment (e.g., collagenase A or TrypLE™ Select). This process may be repeated several times until the required amount of hESCs is reached. According to some embodiments, after the first round of expansion, hESCs are removed using TrypLE™ Select, and after the second round of expansion, hESCs are removed using collagenase A.

[0201] Feeder cell-free systems have also been used in ES cell culture, which utilize matrices supplemented with serum replacement, cytokines and growth factors (including IL6 and soluble IL6 receptor chimeras) as an alternative to feeder cell layers. Stem cells can be grown on solid surfaces such as extracellular matrices (e.g., MATRIGEL®, laminin or vitronectin) in the presence of culture medium (e.g., Lonza L7™ system, mTeSR™, StemPro™, XFKSR, E8, NUTRISTEM®). Unlike feeder-based cultures, which require simultaneous growth of feeder cells and stem cells and may result in mixed cell populations, stem cells grown in feeder-free systems easily detach from the surface. Culture media used to grow stem cells contain factors that effectively inhibit differentiation and promote their growth, examples of which include MEF-conditioned medium and bFGF.

[0202] Also within the scope of the present disclosure is a method of expanding and maintaining human embryonic stem cells (hESCs) in an undifferentiated state, the method comprising culturing human pluripotent stem cells on a non-adherent surface to obtain a population of undifferentiated hESCs, combining the population of undifferentiated hESCs with microcarriers in a growth medium, and expanding the population of cells.

[0203] Examples of non-adherent cell culture plates include those manufactured by Nunc (e.g., Hydrocell Cat No. 174912), etc. In other embodiments, non-adherent suspension culture dishes (e.g., Corning) may be used.

[0204] According to some embodiments, when cells are cultured on a non-adherent substrate, such as a cell culture plate, the atmospheric oxygen conditions are 20%. However, it is contemplated that the atmospheric oxygen conditions can be manipulated to have an atmospheric oxygen percentage of less than about 20%, 15%, 10%, 9%, 8%, 7%, 6%, or even less than about 5% (e.g., 1%-20%, 1%-10%, or 0-5%). According to other embodiments, cells are initially cultured on a non-adherent substrate under normal atmospheric oxygen conditions, and then reduced to less than normal atmospheric oxygen conditions.

[0205] Although the above methods are directed to methods of expanding and maintaining hESCs, similar methods directed to induced pluripotent stem cells (iPSCs) are also within the scope of this disclosure. iPSCs are a type of stem cell derived from somatic cells that have been reprogrammed to a pluripotent state by the introduction of pluripotency-associated genes, and are available from a variety of sources. One of skill in the art will understand the modifications necessary to adapt the above hESC methods for use with iPSCs, etc.

[0206] It is also understood that commercially available stem cells can be used in aspects and embodiments of the present disclosure. Human ES cells may be purchased from the NIH human embryonic stem cell registry www.grunts.nih.govstem_cells / or other hESC registries. Non-limiting examples of commercially available embryonic stem cell lines include HADC102, ESI, BGO 1, BG02, BG03, BG04, CY12, CY30, CY92, CY1O, TE03, TE32, CHB-4, CHB-5, CHB-6, CHB-8, CHB-9, CHB-10, CHB-11, CHB-12, HUES 1, HUES 2, HUES 3, HUES 4, HUES 5, HUES 6, HUES 7, HUES 8, HUES 9, HUES 10, HUES 11, HUES 12, HUES 13, HUES 14, HUES 15, HUES 16, HUES 17, HUES 18, HUES 19, HUES 20, HUES 21, HUES 22, HUES 23, HUES 24, HUES 25, HUES 26, HUES 27, HUES 28, CyT49, RUES3, WA01, UCSF4, NYUES 1, NYUES2, NYUES3, NYUES4, NYUESS, NYUES6, NYUES7, UCLA 1, UCLA 2, UCLA 3, WA077 (H7), WA09 (H9), WA13 (H13), WA14 (H14), HUES 62, HUES, 63, HUES 64, CT1, CT2, CT3, CT4, MA135, Eneavour-2, WIBR 1, WIBR2, WIBR3, WIBR4, WIBRS, WIBR6, HUES 45, Shef3, Shef6, BINhem19, BJNhem20, SAGO 1 and SA001.

[0207] composition The present disclosure provides a population of cells that expresses the markers described herein.A skilled artisan will understand that the population of cells described herein can include a population of mixed cell types, and the identity of the cell type is reflected by the percentage of cells in the population that express one or more markers described herein.Each cell in the population may express only a single marker described below, or each cell may express a combination of markers described below, depending on the differentiation state of the cell.

[0208] In some embodiments, the cells in the population are endogenous to SIX homeobox 3 (six3), SIX homeobox 6 (six6), phosphodiesterase 6H (PDE 6H), visual system homeobox 2 (CHX10 or VSX2), premelanosome protein (PMEL), protein kinase C alpha (PKCa), ELAV-like RNA binding protein 3 / 4 (HuC / D), orthodenticle homeobox 2 (Otx2), neuronal differentiation 1 (NeuroD), B lymphocyte-induced maturation protein-1 (Blimp1), transducin, phosducin (PdC), retinoid X receptor gamma (RXRy), thyroid hormone receptor isoform (Tr-32), atonal bHLH transcription factor 7 (Atoh7), insulin gene enhancer protein (Isl-1), retinal homeobox gene 1 (Rx1), paired box 6 (Pax6), LIM homeobox 2 (LHX2), or retinal and anterior neural fold homeobox (RAX).

[0209] In some embodiments, cells in the population express one or more early eye field markers selected from the group consisting of Six3, Six6, Rxl, Rax, Pax6, RXRy, and Lhx2.

[0210] In some embodiments, the cells in the population express one or more embryonic retinal markers selected from the group consisting of Otx2, NeuroD, Blimp1, transducin, phosducin (PdC), RXRy and Trf-32, Atoh7 and Isl-1.

[0211] In some embodiments, cells in the population express one or more photoreceptor neuron cell markers selected from the group consisting of cone-rod homeobox (Crx), recoverin, cone arrestin (CAR) and rhodopsin.

[0212] In some embodiments, cells in the population express one or more photoreceptor neuron cell markers selected from the group consisting of cone-rod homeobox (Crx), recoverin, and cone arrestin (CAR).

[0213] In some embodiments, 1% or more of the cells, 3% or more of the cells, 20% or more of the cells, 50% or more of the cells, 70% or more of the cells, 90% or more of the cells, or 99% or more of the cells in the population express one or more early eye field markers. In some embodiments, about 1% to about 99%, about 3% to about 90%, about 20% to about 70%, about 5% to about 50%, about 10% to about 50%, or about 10% to about 80% of the cells in the population express one or more early eye field markers.

[0214] In some embodiments, 1% or more of the cells, 3% or more of the cells, 20% or more of the cells, 50% or more of the cells, 70% or more of the cells, 90% or more of the cells, or 99% or more of the cells in the population express one or more embryonic retinal markers. In some embodiments, about 1% to about 99%, about 3% to about 90%, about 20% to about 70%, about 5% to about 50%, about 10% to about 50%, or about 10% to about 80% of the cells in the population express one or more embryonic retinal markers.

[0215] In some embodiments, 10% or more of the cells in the population express cone-rod homeobox (Crx), 3% or more of the cells in the population express recoverin, 3% or more of the cells in the population express cone arrestin (CAR), and 1% or less of the cells in the population express TRA-1-60 and / or SSEA5.

[0216] In some embodiments, more than 50% of the cells in the population express cone-rod homeobox (Crx), more than 12% of the cells in the population express recoverin, more than 60% of the cells in the population express cone arrestin (CAR), and less than 1% of the cells in the population express TRA-1-60 and / or SSEA5.

[0217] In some embodiments, 60% or more of the cells in the population express cone-rod homeobox (Crx), 70% or more of the cells in the population express recoverin, 75% or more of the cells in the population express cone arrestin (CAR), and 1% or less of the cells in the population express TRA-1-60 and / or SSEA5.

[0218] In some embodiments, about 45% to about 65% of the cells in the population express cone-rod homeobox (Crx), about 10% to about 15% of the cells in the population express recoverin, about 50% to about 70% of the cells in the population express cone arrestin (CAR), and about 0.001% to about 1.5% of the cells in the population express TRA-1-60 and / or SSEA5.

[0219] In some embodiments, about 50% to about 70% of the cells in the population express cone-rod homeobox (Crx), about 60% to about 80% of the cells in the population express recoverin, about 70% to about 80% of the cells in the population express cone arrestin (CAR), and about 0.001% to about 1.5% of the cells in the population express TRA-1-60 and / or SSEA5.

[0220] In some embodiments, 5% or more of the cells in the population, 10% or more of the cells, 50% or more of the cells, 70% or more of the cells, or greater than 90% of the cells express Crx. In some embodiments, 10% or more of the cells in the population express Crx. In some embodiments, 15% or more of the cells in the population express Crx. In some embodiments, about 5% to about 90% of the cells, about 10% to about 70% of the cells, or about 50% to about 60% of the cells in the population express Crx. In some embodiments, about 40% to about 60% of the cells express Crx. In some embodiments, about 60% to about 80% of the cells express Crx.

[0221] In some embodiments, 1% or more of the cells in the population, 3% or more of the cells, 10% or more of the cells, 20% or more of the cells, 50% or more of the cells, 70% or more of the cells, 90% or more of the cells, or 99% or more of the cells express recoverin. In some embodiments, 3% or more of the cells in the population express recoverin. In some embodiments, 5% or more of the cells in the population express recoverin. In some embodiments, about 1% to about 99%, about 3% to about 90%, about 20% to about 70%, or about 50% to about 60% of the cells in the population express recoverin. In some embodiments, about 10% to about 15% of the cells express recoverin. In some embodiments, about 65% to about 85% of the cells express recoverin.

[0222] In some embodiments, 3% or more of the cells in the population, 5% or more of the cells, 20% or more of the cells, 50% or more of the cells, 70% or more of the cells, 90% or more of the cells, or 99% or more of the cells express a CAR. In some embodiments, 3% or more of the cells in the population express a CAR. In some embodiments, 5% or more of the cells in the population express a CAR. In some embodiments, 6% or more of the cells in the population express a CAR. In some embodiments, 10% or more of the cells in the population express a CAR. In some embodiments, about 3% to about 99%, about 5% to about 90%, about 20% to about 70%, or about 50% to about 60% of the cells in the population express a CAR. In some embodiments, about 50% to about 70% of the cells in the population express a CAR. In some embodiments, about 65% to about 75% of the cells in the population express a CAR. In some embodiments, about 70% to about 80% of the cells in the population express a CAR.

[0223] In some embodiments, 3% or more of the cells in the population, 5% or more of the cells, 20% or more of the cells, 50% or more of the cells, 70% or more of the cells, 90% or more of the cells, or 99% or more of the cells express rhodopsin. In some embodiments, 3% or more of the cells in the population express rhodopsin. In some embodiments, 5% or more of the cells in the population express rhodopsin. In some embodiments, 6% or more of the cells in the population express rhodopsin. In some embodiments, 10% or more of the cells in the population express rhodopsin. In some embodiments, about 3% to about 99%, about 5% to about 90%, about 20% to about 70%, or about 50% to about 60% of the cells in the population express rhodopsin. In some embodiments, about 50% to about 70% of the cells in the population express rhodopsin. In some embodiments, about 65% to about 75% of the cells in the population express rhodopsin. In some embodiments, between about 70% and about 80% of the cells in the population express rhodopsin.

[0224] In some embodiments, 0.001% or less, 0.01% or less, 0.1% or less, 1% or less, or 5% or less of the cells in the population express TRA-1-60. In some embodiments, 0.1% or less of the cells in the population express TRA-1-60. In some embodiments, 0 to about 5%, about 0.001% to about 1%, or about 0.01% to about 0.1% of the cells in the population express TRA-1-60.

[0225] In some embodiments, 0.001% or less, 0.01% or less, 0.1% or less, 1% or less, or 5% or less of the cells in the population express SSEA5. In some embodiments, about 0.1% or less of the cells in the population express SSEA5. In some embodiments, 0 to about 5%, about 0.001 to about 1%, or about 0.01 to about 0.1% of the cells in the population express SSEA5.

[0226] In some embodiments, 10% or more, 20% or more, 30% or more, or 40% or more of the cells in the population express Pax 6. In some embodiments, about 3% to about 99%, about 5% to about 90%, about 20% to about 70%, or about 50% to about 60% of the cells in the population express Pax 6.

[0227] In some embodiments, no more than 50%, no more than 40%, no more than 30%, no more than 20%, or no more than 10% of the cells in the population express beta-tubulin 3. In some embodiments, no more than 40% of the cells in the population express beta-tubulin 3.

[0228] In some embodiments, no more than 50%, no more than 40%, no more than 30%, no more than 20%, or no more than 10% of the cells in the population express pre-melanosomal protein (PMEL), hi some embodiments, no more than 30% of the cells in the population express PMEL.

[0229] In some embodiments, no more than 50%, no more than 40%, no more than 30%, no more than 20%, or no more than 10% of the cells in the population express PKCa, hi some embodiments, no more than 30% of the cells in the population express PKCa.

[0230] In some embodiments, no more than 20%, no more than 15%, no more than 10%, no more than 5%, or no more than 3% of the cells in the population express HuCD, hi some embodiments, no more than 10% of the cells in the population express HuCD.

[0231] In some embodiments, between 30% and 90% of the cells in the population express Pax6, between 5% and 30% of the cells in the population express beta-tubulin 3, between 1% and 30% of the cells in the population express PMEL, between 3% and 30% of the cells in the population express PKCa, and / or between 0.5% and 10% of the cells in the population express HuCD.

[0232] Methods for determining marker expression are known to those of skill in the art and include methods based on flow cytometric analysis (FACS) and immunohistochemistry, among others.

[0233] In some embodiments, the population of cells comprises retinal cells. In some embodiments, the population of retinal cells comprises early eye field cells, embryonic retinal cells, photoreceptor cells, which may comprise precursor cells of photoreceptor neuron cells (PNC) or mature photoreceptor neuron cells, or any combination thereof. In some embodiments, the population of retinal cells comprises neuronal retinal cells (NRC), Müller glial cells, retinal pigment epithelium (RPE) cells, or any combination thereof. In some embodiments, NRC comprises PNC, retinal ganglion cells, horizontal neurons, amacrine neurons, rod bipolar cells, cone bipolar cells, rod photoreceptor cells, cone photoreceptor cells, or any combination thereof.

[0234] Pharmaceutical Compositions The present disclosure provides a pharmaceutical composition comprising a population of cells expressing a marker described herein and a pharma- ceutically acceptable carrier.

[0235] In some embodiments, compositions are provided herein that include cells and cell populations prepared by the methods described. For example, pharmaceutical compositions are provided herein for administration to a subject, the compositions include mature PNCs prepared according to the methods described herein. For example, the compositions can also include cryopreservation medium(s). In some embodiments, the cryopreservation medium includes a cryoprotectant that can include one or more of adenosine, dextran-40, lactobionic acid, HEPES (N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid)), sodium hydroxide, L-glutathione, potassium chloride, potassium bicarbonate, potassium phosphate, dextrose, sucrose, mannitol, calcium chloride, magnesium chloride, potassium hydroxide, sodium hydroxide, dimethylsulfoxide (DMSO), and water.

[0236] In an embodiment, the pharmaceutical composition comprises about 0.1% to about 40% cryoprotectant. In an embodiment, the pharmaceutical composition comprises about 0.1% to about 30% cryoprotectant. In an embodiment, the pharmaceutical composition comprises about 0.1% to about 20% cryoprotectant. In an embodiment, the pharmaceutical composition comprises about 0.1% to about 10% cryoprotectant. In an embodiment, the pharmaceutical composition comprises about 1% to about 10% cryoprotectant. In an embodiment, the pharmaceutical composition comprises about 0.1% to about 5% cryoprotectant. In an embodiment, the pharmaceutical composition comprises about 1% to about 40% cryoprotectant. In an embodiment, the cryopreservation medium comprises about 1% to about 30% cryoprotectant. In an embodiment, the pharmaceutical composition comprises about 1% to about 20% cryoprotectant. In an embodiment, the pharmaceutical composition comprises about 1% to about 10% cryoprotectant. In an embodiment, the pharmaceutical composition comprises about 1% to about 5% cryoprotectant. The percentage may be measured as weight of cryoprotectant per volume of medium. The percentage may be measured as volume of cryoprotectant per volume of medium. The percentage may be any value or subrange within the stated range, including the endpoints.

[0237] In some embodiments, the pharmaceutical composition comprises at least about 1,000 cells, at least about 5,000 cells, at least about 25,000 cells, 50,000 cells, at least about 250,0000 cells, at least about 500,000 cells, at least about 1 million cells, at least about 10 million cells, at least about 20 million cells, or at least about 25 million cells. In some embodiments, the pharmaceutical composition comprises about 5,000 cells to 25 million cells, about 50,000 cells to 10 million cells, about 100,000 cells to 1 million cells, about 500,000 cells to 10 million cells, about 1 million cells to 10 million cells, or about 1 million cells to 5 million cells. In some embodiments, the pharmaceutical composition comprises about 5,000 cells to 25 million cells. In some embodiments, the pharmaceutical composition comprises between about 50,000 cells and 10 million cells. In some embodiments, the pharmaceutical composition comprises between about 100,000 cells and 25 million cells.

[0238] In embodiments, the pharmaceutical composition comprises a population of cells described herein, or prepared according to the methods herein, the cells being at least about 1×10 5 cells / mL ~ approx. 100×10 6 cells / mL, or approximately 1 x 10 5 cells / mL ~ approx. 1×10 6 The pharmaceutical composition can be at a concentration of about 100,000 cells / mL to about 100 million cells / mL, about 250,000 cells to about 50 million cells / mL, about 500,000 cells to about 25 million cells / mL, or about 2 million to about 5 million cells / mL. In some embodiments, the pharmaceutical composition can be at a concentration of about 2×10 6 cells / mL ~ approx. 5×10 6 In some embodiments, the pharmaceutical composition comprises between about 100,000 cells and 1 million cells. In some embodiments, the pharmaceutical composition comprises between about 100,000 cells and 500,000 cells. In some embodiments, the pharmaceutical composition comprises between about 500,000 cells and 1 million cells.

[0239] In other embodiments, pharmaceutical compositions comprising cells prepared by the methods herein can be stored in volumes of about 100 μl to about 1 mL, or about 250 μl, or about 600 μl, or about 1000 mL. The pharmaceutical composition can further comprise a cryopreservation medium, which comprises a freezing solution, such as CryoStor® (CS2, CS5, or CS10) or CryoStem™.

[0240] In some embodiments, the present disclosure relates to a method of producing a pharmaceutical composition comprising a population of cells described herein. In embodiments, the pharmaceutical composition comprises cells prepared according to the methods herein, wherein the cells (e.g., PNC or mature PNC) express one or more markers selected from Crx, recoverin, cone arrestin (CAR), or a combination thereof. The pharmaceutical composition may also include other NRCs, such as RPE. In some embodiments, a method of producing a pharmaceutical composition comprising a population of retinal cells derived herein comprises culturing a population of undifferentiated pluripotent stem cells under culture conditions sufficient to differentiate the cells in the population of undifferentiated cells into retinal cells to generate cell aggregates, wherein the cell aggregates are between 100 μm and 800 μm in diameter.

[0241] In some embodiments, a pharmaceutical composition comprises a population of cells in suspension, for example suspended in a pharma- ceutically acceptable carrier, diluent or excipient.

[0242] In embodiments, the pharmaceutical composition can be formulated for administration to the eye by injection, for example, subretinal, intravitreal or suprachoroidal injection. The injectable preparation can be provided in unit dosage form (e.g., ampoules or multi-dose containers) with the addition of a preservative. The composition can take the form of a suspension, solution or emulsion in an oily or aqueous vehicle, and can contain formulating agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle (e.g., sterile pyrogen-free water) before use. In addition to the above formulations, the composition can also be formulated as a depot preparation. Such long-acting formulations can be administered by implantation (e.g., subcutaneously). Thus, for example, the composition can be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resin, or as a sparingly soluble derivative (e.g., a sparingly soluble salt).

[0243] The nature of the pharmaceutical compositions described herein depends on the mode of administration and can be easily determined by those skilled in the art. The pharmaceutical compositions described herein can contain carriers or excipients, many of which are known to the skilled artisan. Excipients that can be used include buffers (e.g., citrate buffer, phosphate buffer, acetate buffer, bicarbonate buffer), amino acids, urea, alcohol, ascorbic acid, phospholipids, polypeptides (e.g., serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol, glycerol. The modulating compounds can be formulated in various ways according to the corresponding route of administration. For example, liquid solutions can be prepared for administration by drops into the ear, injection or oral ingestion, and gels or powders can be prepared for oral ingestion or topical application. Methods for preparing such formulations are well known and are described, for example, in "Remington's Pharmaceutical Sciences".

[0244] Pharmaceutical compositions according to the present disclosure can further comprise a pharma- ceutically acceptable carrier. In embodiments, the compositions can be formulated to be thawed and administered directly to a subject (e.g., by injection) without further manipulation prior to administration.

[0245] Scaffold In some embodiments of the pharmaceutical compositions and methods of using the same of the present disclosure, the population of cells is contained within and / or disposed on a scaffold.

[0246] The scaffold can take any form suitable for implantation into the retina, such as a sheet, disk, capsule, mesh, or other two- and / or three-dimensional structure designed to guide the transplantation of a population of cells to a suitable location within the retina. Without wishing to be bound by theory, it is believed that the scaffold can be used to effectively target a population of cells to an area of ​​the retina that has been degenerated or damaged, facilitating the transplantation of the population of cells and the connection of the transplanted cells to host retinal cells. As an additional advantage, the scaffold can be used to deliver a population of cells to a large area of ​​the retina, such as an area that has been physically damaged (e.g., lacerations) or degenerated. For example, a two-dimensional scaffold can be used to create a planar sheet of retinal progenitor cells and / or retinal cells, which can then be implanted into the subretinal space (Singh, D. et al. Biomaterials 154: 158-168 (2018)). By fine-tuning the topographical properties of the scaffold and including micro- or nano-patterned structures on the scaffold with extracellular matrix (ECM) properties, the scaffold can be designed to effectively retain stem cell populations, retinal progenitor cell populations and / or retinal cell populations and deliver these cell populations as patches to the subretinal space (Nair et al., Appl Sci (Basel) 11:2154 (2021)). In general, a suitable two-dimensional scaffold should be thin enough to allow nutrient exchange between the choriocapillaris and the retina and should not physically distort the photoreceptor layer of the retina.

[0247] In some embodiments, the scaffold is biocompatible. In some embodiments, the scaffold is biodegradable. In some embodiments, the scaffold is both biocompatible and biodegradable. Suitable scaffold materials include, but are not limited to, materials designed to mimic natural components of the retinal extracellular matrix, examples of which include collagen, gelatin, chondroitin sulfate, hyaluronic acid (GCH), and combinations thereof. Such materials are generally non-immunogenic and promote cell attachment. Additional suitable scaffold materials include, but are not limited to, biocompatible materials, examples of which include polyphosphazenes, polyanhydrides, polyacetals, polyorthoesters, polyphosphates, polycaprolactones, polyurethanes, polypeptides, polycarbonates, polyamides, polysaccharides, polyamino acids, other polymers, proteins, metals, or ceramics. In some aspects, the scaffold can be formed in whole or in part from a biodegradable hydrogel, which can be hyaluronic acid based (e.g., Hystem®). Such hydrogels are described in WO2019 / 028088, the contents of which are incorporated herein by reference. Additionally, hyaluronic acid-based hydrogels (trade name) that mimic the natural extracellular matrix environment (ECM, Renevia®) may be suitable for use in the scaffolds described herein. As a further example, the scaffolds may include decellularized tissues, such as retinal tissue.

[0248] Methods for manufacturing scaffolds are known to those skilled in the art and include, among others, 3D printing using suitable materials. Alternatively or additionally, the scaffold material can be electrospun, deposited, coated, lyophilized, or crosslinked.

[0249] Treatment The present disclosure provides a method for treating a visual condition (e.g., an eye-related disease resulting in vision loss) in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein.

[0250] The present disclosure provides a method for preventing or treating an eye-related disease (e.g., a disease that results in vision loss) in a subject in need thereof. In a further embodiment, the method comprises administering to the subject an effective amount of a composition comprising cells prepared according to the methods described herein.

[0251] The present disclosure provides compositions for use in treating or preventing a visual condition in a subject, the use comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein.

[0252] The present disclosure provides compositions for use in the manufacture of a medicament for the treatment of a visual condition in a subject, comprising administering to a subject a therapeutically effective amount of a pharmaceutical composition described herein.

[0253] In some embodiments, a method for treating an eye-related disease (e.g., vision loss) comprises administering to a subject a composition comprising cells (e.g., PNCs, mature PNCs, or NRCs) prepared according to the methods described herein in combination with a method for controlling the onset of a condition. In particular, the combination treatment can include administering a pre-existing treatment known to those skilled in the art.

[0254] In some embodiments, the method comprises administering a therapeutically effective amount of a pharmaceutical composition comprising a population of retinal cells generated by the methods described herein. In some embodiments, the population of retinal cells comprises a population of cells, wherein (a) 10% or more of the cells in the population express cone-rod homeobox (Crx), (b) 3% or more of the cells in the population express recoverin, (c) 3% or more of the cells in the population express cone arrestin (CAR), and (d) 1% or less of the cells in the population express TRA-1-60 and / or SSEA5. In some embodiments, the population of retina comprises a population of cells, wherein (a) between about 10% and 70% of the cells in the population express Crx, (b) between about 3% and 90% of the cells in the population express recoverin, (c) between about 3% and 90% of the cells in the population express CAR, and (d) between 0% and about 0.1% of the cells in the population express TRA-1-60 and / or SSEA5. In some embodiments, the population of retinal cells comprises early eye field cells, embryonic retinal cells, progenitor cells of photoreceptor neuronal cells (PNC), mature photoreceptor neuronal cells, or any combination thereof. In some embodiments, the population of retinal cells comprises neuronal retinal cells (NRC), Müller glial cells, retinal pigment epithelial (RPE) cells, or any combination thereof. In some embodiments, NRC comprises progenitor cells of PNC, retinal ganglion cells, horizontal neurons, amacrine neurons, rod bipolar cells, cone bipolar cells, rod photoreceptor neuronal cells, cone photoreceptor neuronal cells, or any combination thereof. In some embodiments, the population of retinal cells comprises neuronal retinal cells (NRC). In some embodiments, the population of retinal cells comprises photoreceptor cells (e.g., rod and / or cone photoreceptor cells). In some embodiments, the population of retinal cells comprises progenitor cells of PNC and mature photoreceptor neuronal cells.

[0255] In some embodiments, the visual condition comprises (is the result of) a neurodegenerative disease of the retina or damage to the retina.

[0256] In some embodiments, retinal neurodegenerative diseases include genetic diseases such as Stargardt's disease, Leber's congenital amaurosis, retinitis pigmentosa, congenital color blindness, Best's disease, etc. Alternatively, or in addition, retinal neurodegenerative diseases may be caused by additional underlying conditions (e.g., diabetic retinopathy) and / or environmental factors, examples of which include diet, age (age-related macular degeneration), sun exposure, alcohol exposure, etc.

[0257] In some embodiments, the retinal neurodegenerative disease includes Leber's congenital amaurosis, congenital color blindness, Stargardt's disease, Best's disease, Doyne's disease, cone dystrophy, retinitis pigmentosa, X-linked retinoschisis, Usher's syndrome, age-related macular degeneration, atrophic age-related macular degeneration, neovascular AMD, diabetic maculopathy, proliferative diabetic retinopathy (PDR), cystoid macular edema, central serous retinopathy, retinal detachment, intraocular inflammation, glaucoma, or posterior uveitis. Retinal neurodegenerative diseases are described in US20210189430, the contents of which are incorporated by reference. As further examples, retinal neurodegenerative diseases that can be treated by the compositions and methods described herein include, but are not limited to, Stargardt's disease, diabetic retinopathy, macular degeneration, retinitis pigmentosa, Leber's congenital amaurosis, cone-rod dystrophy, choroideremia, and X-linked retinoschisis. In some embodiments, the macular degeneration comprises age-related macular degeneration.

[0258] In some embodiments, the visual condition is the result of retinal damage. Retinal damage can be the result of physical trauma. Alternatively or additionally, retinal damage can include, but is not limited to, retinal tears, retinal detachment, retinal wrinkles, epiretinal membranes, and macular holes.

[0259] The methods and compositions described herein can be used to treat macular pathologies. The macula is a small area in the center of the retina that provides higher visual acuity for straight-on vision. Exemplary macular pathologies include, but are not limited to, branch retinal vein occlusion, central retinal vein occlusion, central serous retinopathy, choroidal neovascular membrane, cytomegalovirus retinitis, diabetic retinopathy, histoplasmosis, macular degeneration (atrophic and exudative), macular edema, macular hole, premacular fibrosis, macular telangiectasia, retinal detachment, retinitis pigmentosa, retinoblastoma, retinopathy of prematurity, Stargardt's disease, and Usher syndrome. A skilled artisan will understand that the pathologies described herein may affect only the macula, or may affect the macula and areas of the retina other than the macula.

[0260] In some embodiments, the method described herein comprises administering the composition described herein to the eye of a subject.In some embodiments, the composition is administered to the retina of the subject or in close proximity to the retina.For example, the composition may be administered directly to the retinal layer.Alternatively or additionally, the composition may be administered in close proximity to the retina, for example, in the subretinal space.

[0261] In some embodiments, the compositions described herein are administered by injection, implantation, or implantation.

[0262] The described compositions can be administered as pharma- ceutically or physiologically acceptable preparations or compositions containing a physiologically acceptable carrier, excipient, or diluent, and can be administered to tissues of intended recipient organisms, including humans and non-human animals. In some embodiments, the compositions comprise a cell suspension, i.e., a cell population generated by the methods described herein, suspended in a pharma- ceutically acceptable carrier.

[0263] In alternative embodiments, the composition comprises a scaffold comprising the population of cells, and the composition is administered to the subject by implantation of the scaffold comprising the population of cells. In some embodiments, the scaffold comprises a sheet, disk, capsule, or mesh. In some embodiments, the scaffold is biocompatible, biodegradable, or both.

[0264] In some embodiments, such as those in which the composition comprises a cell suspension, the composition is administered to the retina by injection. In some embodiments, the injection comprises an intravitreal, subretinal or suprachoroidal injection of a suspension comprising a population of cells. Suprachoroidal injections are known to those skilled in the art and are described, for example, in U.S. Patent No. 9,956,114, as well as in U.S. Patent Application No. US2022-0280341, the contents of which are incorporated herein by reference. Subretinal and intravitreal injection methods are described, for example, in U.S. Patent Nos. 9,795,452, 9,567,376, 9,433,688, and 10,081,659, as well as in U.S. Patent Application No. 2022-0233768, the contents of which are incorporated herein by reference.

[0265] In some embodiments, the method includes administering a volume of the composition of about 0.1 μL to about 1 mL, including all values ​​therein, depending on the size of the area to be treated, the concentration of the cells, the route of administration, and the desired effect of the method. In some embodiments, the volume is about 50 μL. In some embodiments, the volume is about 70 μL. In some embodiments, the volume is about 100 μL. In some embodiments, the volume is about 125 μL. In some embodiments, the volume is about 150 μL. In some embodiments, the volume is about 175 μL. In some embodiments, the volume is about 200 μL. In some embodiments, the volume is about 250 μL. In some embodiments, the volume is about 300 μL. In some embodiments, the volume is about 450 μL. In some embodiments, the volume is about 500 μL. In some embodiments, the volume is about 600 μL. In some embodiments, the volume is about 750 μL. In some embodiments, the volume is about 850 μL. In some embodiments, the volume is about 1000 μL. In some embodiments, the volume is about 10 μL to about 800 μL, about 30 μL to 700 μL, about 50 μL to about 500 μL, about 50 μL to about 300 μL, about 50 μL to about 250 μL, about 100 μL to about 500 μL, about 100 to about 250 μL, about 100 μL to about 800 μL, about 200 μL to about 1 mL, about 300 μL to about 800 μL, about 400 μL to about 800 μL, or about 500 to about 1 mL.

[0266] In some embodiments, for example those in which the composition comprises a cell suspension, the population of cells is about 1×10 4 cells / mL ~ approx. 100×10 7 In some embodiments, the population of cells is administered at a concentration of about 1×10 5 cells / mL ~ approx. 100×10 6 cells / mL, or approximately 1 x 10 5 cells / mL ~ approx. 1×10 6 In some embodiments, the population of cells is about 1×10 5 cells / mL ~ approx. 100×10 6 Concentration is in cells / mL.

[0267] In some embodiments, the method comprises thawing a composition of a population of cells in a cryopreservation medium described herein and administering the thawed composition directly to a subject. In alternative embodiments, the method comprises thawing a population of cells in a cryopreservation medium, followed by culturing the cells and / or transferring the cells to a pharma- ceutically acceptable carrier, and administering the cells to a subject.

[0268] In embodiments, a therapeutically effective amount of a composition (e.g., cells (e.g., PNCs, mature PNCs, or NRCs)) in a human can be any therapeutically effective amount. In one embodiment, the composition is administered 3 times daily, twice daily, once daily, 14 days in a 3 week cycle (4 times daily, 3 times daily or 2 times daily, or once daily) with 7 days off, up to 5 days or 7 days in a 3 week cycle (4 times daily, 3 times daily or 2 times daily, or once daily) with 14-16 days off, or once every 2 days, or once a week, or once every 2 weeks, or once every 3 weeks. In examples, the composition is administered once a week, or once every two weeks, or once every three weeks, or once every four weeks for at least one week, and in some embodiments, the composition is administered for 1-4 weeks, 2-6 weeks, 2-8 weeks, 2-10 weeks, or 2-12 weeks, 2-16 weeks, or longer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 36, 48 weeks, or more). In some embodiments, a therapeutically effective amount of the composition is administered monthly, bimonthly, every three months, every four months, every six months, or yearly. In some embodiments, a single administration of the composition is sufficient to provide a therapeutic effect.

[0269] In some embodiments, administration of the compositions described herein reduces or eliminates the severity of one or more clinical signs of visual pathology in a subject. Clinical signs of visual pathology include, but are not limited to, reduced peripheral vision, reduced central vision (e.g., when reading), reduced night vision, loss of color vision, reduced visual acuity, reduced photoreceptor function, and pigmentary changes.

[0270] In some embodiments, administering the compositions described herein reduces or eliminates the severity of retinal degeneration or damage.For example, administering the compositions described herein can reduce the area of ​​retinal degeneration or damage, reduce the severity of degeneration or damage, or reduce the number of damaged, dead, or dying cells.Methods for measuring retinal damage are clear to the skilled artisan, and include, but are not limited to, optical coherence tomography (OCT), electroretinogram (ERG), fundus autofluorescence imaging, and the like.

[0271] Kits and Articles of Manufacture The present disclosure provides kits comprising a population of cells generated using the methods described herein, or a pharmaceutical composition comprising a population of cells. In some embodiments, the population of cells comprises NRCs, mature PNCs, and / or PNCs prepared according to the methods described herein. In some embodiments, the kits further comprise reagents (e.g., cryopreservation reagents and pharma- ceutically acceptable carriers or diluents).

[0272] The present invention also provides packages and kits comprising pharmaceutical compositions for use in the methods of the present invention. The kits can include one or more containers selected from the group consisting of bottles, vials, ampoules, blister packs, syringes, and cryovials. The kits can further include one or more instructions for use in the treatment and / or prevention of diseases, conditions, or disorders of the present invention (e.g., eye-related diseases, e.g., vision loss), one or more syringes, one or more applicators, or a suitable sterile solution for thawing and reconstituting the pharmaceutical compositions of the present invention.

[0273] Exemplary embodiments The present invention can be understood with reference to the embodiments listed below.

[0274] Embodiment 1. A method for obtaining a population of neural retinal cells (NRCs) from undifferentiated pluripotent stem cells, comprising: (a) growing the undifferentiated pluripotent stem cells in dynamic culture; (b) seeding the undifferentiated pluripotent stem cells as single cells into a culture vessel; (c) differentiating the undifferentiated pluripotent stem cells in the culture vessel under conditions to obtain a population of NRCs.

[0275] Embodiment 2. The method of embodiment 1, wherein the culture vessel is a bioreactor.

[0276] Embodiment 3. The method of embodiment 2, wherein the bioreactor is a vertical wheel bioreactor.

[0277] Embodiment 4. The method of embodiments 1-3, wherein the undifferentiated pluripotent stem cells are seeded for differentiation as single cells and grown in suspension as cell aggregates for at least 14-18 weeks.

[0278] Embodiment 5. The method according to embodiment 4, wherein the cell aggregates have a size controlled by the rotation speed of the culture vessel, and the size of the aggregates is about 100 μm to about 800 μm.

[0279] Embodiment 6. The method of embodiment 5, wherein the rotation speed is increased during the differentiation process.

[0280] Embodiment 7. Step (c) comprises: (i) culturing the undifferentiated pluripotent stem cells for a first period of time under culture conditions sufficient to induce the stem cells to form cell aggregates, the cell aggregates comprising cells expressing one or more early eye field markers selected from Rxl, Pax6, RXRy, LHX2 and RAX; (ii) culturing the cell aggregates from step i) for a second period of time under culture conditions sufficient to differentiate the cell aggregates into cells expressing one or more embryonic retinal markers selected from Otx2, NeuroD, Blimp1, transducin, phosducin (PdC), RXRy, and Tr-32, Atoh7, or IS1-1; (iii) culturing the cells expressing the one or more embryonic retinal markers from step (ii) for a third period under culture conditions sufficient to differentiate the cells into photoreceptor neuronal (PNC) progenitor cells and mature PNCs, wherein the PNCs express one or more proteins selected from Crx, recoverin, or cone arrestin (CAR).

[0281] Embodiment 8. The method of any one of embodiments 1 to 7, wherein the pluripotent stem cells are human embryonic stem cells (hESCs).

[0282] Embodiment 9. The method of any one of embodiments 1 to 7, wherein the pluripotent stem cells are human induced pluripotent stem cells (hiPSCs).

[0283] Embodiment 10. The method according to any one of embodiments 1 to 7, wherein step (c) comprises differentiating using a Rock inhibitor (RI), nicotinamide (NIC) and a Wnt inhibitor in a first period, differentiating using NIC and IWRel in a second period, differentiating using NIC and insulin growth factor-1 (IGF-1) in a third period, differentiating using NIC, IGF-1 and a Notch inhibitor in a fourth period, and differentiating using IGF-1, retinoic acid (RA), taurine and NT4 in a fifth period.

[0284] Embodiment 11. The method of embodiment 10, wherein the first period of time is 0-7 days, and / or the second period of time is 2-20 days, and / or the third period of time is 10 days-8 weeks, and / or the fourth period of time is 4-10 weeks, and / or the fifth period of time is 12-18 weeks.

[0285] Embodiment 12. The method of any one of embodiments 1 to 7, wherein the first period of time is from about 3 days to about 120 days, or from about 5 days to about 15 days, or about 3 days.

[0286] Embodiment 13. The method of embodiment 7, wherein the second period of time is from about 1 day to about 120 days, or from about 5 days to about 15 days, or about 11 days.

[0287] Embodiment 14. The method of embodiment 7, wherein the cells of step (ii) are cryopreserved from day 14 to day 120.

[0288] Embodiment 15 The method of embodiment 14, wherein cryopreservation is performed in a freezing solution.

[0289] Embodiment 16 The method of embodiment 7, wherein the freezing solution comprises CryoStem.

[0290] Embodiment 17 The method of embodiment 7, further comprising cryopreserving the cells from step (ii) at or near the completion of the first period of time.

[0291] Embodiment 18 The method of embodiment 16 or 17, wherein the cryopreserved cells are thawed and the thawed cells are cultured.

[0292] Embodiment 19 The method of embodiment 18, wherein the thawed cells are cultured according to step (iii) of embodiment 7.

[0293] Embodiment 20 The method of embodiment 7, wherein the cells from step (iii) are cryopreserved after the third period of time.

[0294] Embodiment 21 The method of embodiment 20, wherein the cryopreserved cells are thawed and cultured in any remaining steps of the method.

[0295] Embodiment 22. A method for preparing a photoreceptor neuronal cell (PNC) composition for administration to a subject immediately after thawing, comprising the steps of: (a) suspending the PNCs prepared according to any one of embodiments 1-21 in a cryopreservation medium to form a cell suspension; (b) storing the cell suspension at cryopreservation temperatures; and (c) thawing the cryopreserved suspension.

[0296] Embodiment 23. The method of embodiment 22, wherein the cryopreservation medium comprises one or more of adenosine, dextran-40, lactobionic acid, HEPES (N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid)), sodium hydroxide, L-glutathione, potassium chloride, potassium bicarbonate, potassium phosphate, dextrose, sucrose, mannitol, calcium chloride, magnesium chloride, potassium hydroxide, sodium hydroxide, dimethyl sulfoxide (DMSO), or water.

[0297] Embodiment 24. A pharmaceutical composition for administration to a subject, comprising the mature PNCs prepared according to any one of embodiments 7 to 23, and a cryopreservation medium.

[0298] Embodiment 25. The pharmaceutical composition of embodiment 24, wherein the cryopreservation medium comprises one or more of adenosine, dextran-40, lactobionic acid, HEPES (N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid)), sodium hydroxide, L-glutathione, potassium chloride, potassium bicarbonate, potassium phosphate, dextrose, sucrose, mannitol, calcium chloride, magnesium chloride, potassium hydroxide, sodium hydroxide, dimethyl sulfoxide (DMSO), and water.

[0299] Embodiment 26. The PNCs are about 1×10 5 cells / mL ~ approx. 100×10 6 26. The pharmaceutical composition of embodiment 24 or 25, which is in a concentration of cells / mL.

[0300] Embodiment 27. The pharmaceutical composition of any one of embodiments 24 to 26, wherein the pharmaceutical composition is stored in a volume of about 100 μL to about 1 mL, or about 250 μL, or about 600 μL.

[0301] Embodiment 28. The pharmaceutical composition of any one of embodiments 24 to 27, wherein the cryopreservation medium comprises a freezing solution.

[0302] Embodiment 29. The pharmaceutical composition of any one of embodiments 24 to 28, wherein the freezing solution comprises CRYOSTOR® CS2, CRYOSTOR® CS5, CRYOSTOR® CS10 or CRYOSTEM™.

[0303] Embodiment 30. The pharmaceutical composition of any one of embodiments 22 to 29, wherein the PNC expresses one or more markers selected from Crx, recoverin, cone arrestin (CAR), or a combination thereof.

[0304] Embodiment 31. The method according to any one of embodiments 7 to 21, wherein the cells cultured after step (iii) comprise about 1% to 30% retinal epithelial cells.

[0305] Embodiment 32. A method of treating vision loss in a subject, comprising administering to the subject a therapeutically effective amount of a composition of any one of embodiments 24-30.

[0306] Embodiment 33 The method of embodiment 32, wherein the administering comprises administering the composition to the retina or in close proximity to the retina of the subject.

[0307] Embodiment 34 The method of embodiment 32 or 33, wherein the administration is by injection, implantation, or implantation. EXAMPLES

[0308] The following examples illustrate certain specific embodiments of the invention and are not intended to limit the scope of the invention.

[0309] The following examples and detailed protocols further illustrate the embodiments of the present specification.However, the examples are merely intended to illustrate the embodiments, and should not be interpreted as limiting the scope of the present specification.The contents of all references and published patents and patent applications cited throughout this application are hereby incorporated by reference.

[0310] Abbreviations and definitions: RI:Rock inhibitor NIC: Nicotinamide IWRe1: Wnt pathway inhibitor IGF-1: Insulin-like growth factor 1 DAPT: Notch inhibitors BDNF: Brain-derived neurotrophic factor NT4: Neurotrophin-4

[0311] Example 1 Dynamic Differentiation Protocol: Generation Run 44 (PR-44) Differentiation of human embryonic stem cells (hESCs) into retinal cells is based on a stepwise differentiation process in which cells are cultured in dynamic suspension and supplemented with relevant growth factors at each stage (Figure 1). The process began with seeding hESCs (HADC102) as single cell suspensions at two concentrations in a PBS Wheel™ small-scale vertical wheel bioreactor.

[0312] Cells were grown in dynamic culture at four different rotation speeds (A-D) according to Figure 2A-2D. The initial PBS Wheel rotation speed at seeding was 35 rpm or 40 rpm. The PBS Wheel rotation speed was increased by 15 rpm or 20 rpm every 1-3 weeks according to Figure 2A-2D.

[0313] The size of the aggregates was kept relatively small, 50-100 microns, 50-150 microns, 100-250 microns, 150-350 microns, or 250-450 microns. As the rotation speed increased, the aggregates became smaller (Figure 3A-B). To limit the size of the aggregates, the speed was increased throughout differentiation. This is because larger size may correlate with the development of a necrotic core and aggregate center. Variation in accessibility of factors to the aggregates may lead to altered cell populations and reduced control of induced differentiation.

[0314] Cells were fed twice a week by removing 75% of the medium and replacing it with fresh medium containing factors according to Table 1 below.

[0315] [Table 1]

[0316] Differentiation process of hESCs into eye field cells First, cells were cultured in a medium containing growth factors including a Rock inhibitor (RI), nicotinamide (NIC) and a Wnt inhibitor (IWRe1). This combination of growth factors was used from the start of differentiation until day 3, which was called the first period.

[0317] The cells were then supplemented with a medium containing growth factors including NIC and IWRe1. This combination of growth factors was used from day 3 to day 14, which was referred to as the second period.

[0318] Differentiation into embryonic retinal cells Differentiation into embryonic retinal cells was initiated by culturing the cells in medium containing the growth factors NIC and insulin growth factor 1 (IGF-1). This combination of growth factors was used from week 2 to week 5, a period called the third period.

[0319] At the fourth week of differentiation, the cells were tested for pluripotency using an established hESC residual FCM (flow cytometry) based assay. The results showed that the hESC residual population was less than 0.1% (Table 2 below). Cell differentiation continued for an additional 12 weeks. [Table 2]

[0320] Differentiation into PNCs Differentiation into PNCs was initiated by culturing the cells in medium containing the growth factors NIC (1–50 mM), IGF-1 (0.5–20 ng / mL), and a Notch inhibitor (DAPT 1–50 μM). This combination of growth factors was used from week 5 to week 6, referred to as the fourth period.

[0321] The cells were then cultured in medium containing growth factors such as IGF-1 (0.5–20 ng / mL), retinoic acid (RA, 0.1–2 μM), taurine (TA, 10–400 μM), BDNF (2–200 ng / mL), and NT4 (2–200 ng / mL). This combination of growth factors was used from week 7 to either week 12 or week 16, which was referred to as the fifth period.

[0322] At week 7, the amount of cells was estimated by visual inspection. The 0.1 L bioreactor was removed from the magnetic drive and the aggregates settled to the bottom of the vessel. The area covered by the aggregates was evaluated, indicating the relative amount of cells in each vessel (Figure 4). The results show that the initial seeding density is the main factor determining the cell amount 7 weeks after the start of the differentiation process (approximately double the cell amount at high seeding density versus low seeding density). A further 35% increase in aggregate amount can be attributed to the higher rotation speed of the dynamic culture (Group C vs. Group A in Figure 4).

[0323] At week 8, cells from groups A and C were roughly split into two. IGF-1 was added either at a basal concentration of 5 ng / mL (Fig. 5A1, A2) or at a two-fold higher concentration of 10 ng / mL (Fig. 5C1, C2). At week 16, cell mass was estimated again. Significant numbers of cells were sampled at weeks 4, 8 and 12, mainly from groups A1, A2, C1 and C2, for flow cytometry, immunostaining and QPCR, as shown in Fig. 5, but these conditions yielded higher cell numbers compared to the corresponding groups B and D, and in total more than 10-fold increase in final product yield compared to static non-adherent cultures.

[0324] Example 2 Dynamic Differentiation Protocol: Generation Run 45 (PR-45) In a repeat of the differentiation process, PR-45 cells were cultured as in Example 1. However, all cells were seeded at a high cell concentration of 250,000 / ml and a high rotation speed of 40-80 rpm (similar to PR-44 group C1) was applied over the entire 16 weeks of differentiation in some PBS Wheel vessels. At the end of 8, 12 and 16 weeks of differentiation, some vessels were sampled and examined for expression of various cell markers using flow cytometry and compared to control static non-adherent cultures grown in parallel (Figures 10-12). Quantitative monitoring of PR marker expression (Figure 10) allows for process control, crucial for realizing a viable industrial process, with a go / no-go point at only 16 weeks of differentiation.

[0325] Example 3 Quality Measurement: Production Work 46 (PR-46) Besides PR (production run) markers (e.g., CAR, Crx, recoverin, and rhodopsin), crucial monitoring of non-target cell populations was established for in-process monitoring of product safety specifications (Figure 9, Figures 11-12). For example, production run PR-46 was tested for expression of undifferentiated cell markers TRA-1-60 and SSEA5 at week 8, and high levels (>1%, Figure 11) of positive cells were detected, leading to the halting of the production run. Additionally, PR-44 and PR-45 week 8 and week 15 cells were dispersed with TrypLE Select and seeded in LN521-coated TC flasks. These flattened cultures were harvested after 2 weeks and aliquots were frozen for examination by flow cytometry. Significant differences were shown between control static and dynamic cultures, specifically, increased expression of photoreceptor (PR) markers such as CRX and recoverin, and termination of differentiation at week 16 in dynamic conditions versus static controls. Interestingly, transitioning from non-adherent dynamic to adherent static culture increased cone arrestin (CAR) expression by almost 10-fold, once again enhancing the prospects for industrial-grade process development in terms of product quality.

[0326] Example 4 Monitoring viable cell mass by quantification of cell metabolism Before each medium change, glucose and lactate levels were measured. Metabolite levels correspond to the amount of viable cells, with glucose levels being reduced and lactate levels increasing when more viable cells are present. The graph shown in Figure 6 shows that the glucose consumption of PR-44 groups A and C was similar to the control static group throughout the study. Considering that PR-44 groups A and C were split at week 8 and more cells were sampled from the vessel throughout the differentiation, the difference in the cells initially seeded was maintained throughout the process. Increasing the agitation speed (PR-44 groups C1 and C2) also increased the metabolism (i.e., lower glucose and more lactate, then the speed was reduced). On the other hand, increasing the IGF-1 concentration did not seem to have a significant effect on the metabolism (e.g., PR-44 groups C1 vs. C2).

[0327] Cells were harvested from the bioreactor after 8 or 16 weeks, dispersed, and seeded on PDL / laminin-coated coverslips for 2 weeks. Immunostaining clearly identified various PR cell populations, including early photoreceptors. Double staining with CRX and recoverin antibodies showed a large population of cells expressing CRX and some cells expressing recoverin (Figures 7 and 9). Interestingly, in all conditions where IGF-1 was used at 5 ng / mL, recoverin staining illuminated relatively long neurites. In contrast, in conditions where IGF-1 was used at 10 ng / mL, positive cells had significantly shorter neurites, indicating that high IGF-1 may maintain the cells in a relatively immature state. Immunostaining with both anti-CAR and anti-rhodopsin antibodies identified two distinct mature photoreceptor populations, i.e., a large cone arrestin (CAR)-positive cell population displayed (i.e., cone photoreceptors) and rhodopsin-positive cells (Figure 8). Rhodopsin antibody labeling was mainly at the periphery of the cells.

[0328] References Transl Vis Sci Technol. 2019 May 30;8 (3):30. Reichman、S. et al.、Generation of Storable Retinal Organoids and Retinal Pigmented Epithelium from Adherent Human iPS Cells in Xeno-Free and Feeder-Free Conditions Stem Cells 2017(5):1176-1188. Gonzalez-Cordero、A. et al. Recapitulation of Human Retinal Development from Human Pluripotent Stem Cells Generates Transplantable Populations of Cone Photoreceptors. Stem Cell Reports 2017、9:820-837. EP3583204A1 EP3761998A1 EP2796545A1 Meyer、S. et al、Stem Cells 29 (2011) 1206-1218. Eiraku、M. et al.、Self-organizing optic-cup morphogenesis in three-dimensional culture、Nature 472 (2011) 51-56. Meyer、J. et al.、Modeling early retinal development with human embryonic and induced pluripotent stem cells、Proc. Natl. Acad. Sci. U. S. A. 106(2009) .16703-16698. Nakano、T. Self-formation of optic cups and storable stratified neural retina from human ESCs、Cell Stem Cell 10 (2012) 771-785. Osakada, F. et al. Nat. Biotechnol. 26 (2008): 224-215. Osakada, F. et al., Stepwise differentiation of pluripotent stem cells into retinal cells, Nat. Protoc. 4 (2009) 811-824. Phillips, M. et al., Blood-derived human iPS cells generate optic vesicle-like structures with the capacity to form retinal laminae and develop synapses, Invest. Ophthalmol. Vis. Sci. 53 (2012) 2007-2019. Reichman, S. et al., From confluent human iPS cells to self-forming neural retina and retinal pigmented epithelium, Proc. Natl. Acad. Sci. USA 111 (2014) 8518-8523. Zhong, X., et al. Generation of three-dimensional retinal tissue with functional photoreceptors from human iPSCs, Nat. Commun. 5 (2014) 4047.

[0329] Other embodiments Although the present invention has been described in conjunction with its detailed description, the foregoing description is intended to illustrate, but not to limit, the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0330] The patents and scientific literature referred to herein establish knowledge available to those skilled in the art. All references, including U.S. patents, U.S. patent application publications, PCT patent applications designating the U.S., published foreign patents, and patent applications cited herein, are incorporated herein by reference in their entirety. GenBank and NCBI submissions with accession numbers cited herein are incorporated herein by reference. All other published references, documents, manuscripts, and scientific literature cited herein are incorporated herein by reference. In case of conflict, the present specification, including definitions, will take precedence. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0331] While the present invention has been particularly shown and described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and detail can be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

1. A pharmaceutical composition containing a population of retinal cells, (a) More than 10% of the cells in the population express pyramidal-rod homeoboxes (Crx); (b) Three percent or more of the cells in the population express the recabin; (c) Three percent or more of the cells in the population express pyramidal arrestin (CAR); and (d) Less than 1% of the cells in the population express TRA-1-60 and / or SSEA5, The aforementioned pharmaceutical composition comprises a pharmaceutically acceptable carrier.

2. (i) 15% or more of the cells in the population express Crx; (ii) 5% or more of the cells in the population express the recabin; and / or (iii) The pharmaceutical composition according to claim 1, wherein 6% or more of the cells in the population express CAR.

3. (e) More than 30% of the cells in the population express Pax6; (f) Less than 40% of the cells in the population express β-tubulin 3; (g) Less than 30% of the cells in the population express PMEL; (h) Less than 30% of the cells in the population express PKCa; or (i) Less than 10% of the cells in the population express HuCD, The pharmaceutical composition according to claim 1.

4. (a) Approximately 10% to 70% of the cells in the population express Crx; (b) Approximately 3% to 90% of the cells in the population express the recabin; (c) Approximately 3% to 90% of the cells in the population express CAR; and (d) 0 to about 0.1% of the cells in the population express TRA-1-60 and / or SSEA5. The pharmaceutical composition according to claim 1.

5. The pharmaceutical composition according to claim 1, wherein the population of retinal cells comprises early ocular field cells, embryonic retinal cells, precursor cells of photoreceptor neurons (PNCs), mature photoreceptor neurons, or any combination thereof.

6. (i) The initial ophthalmic cells include cells expressing Six3, Six6, Rx1, Pax6, RXRy, LHX2 and / or RAX; (ii) The embryonic retinal cells include cells expressing Otx2, NeuroD, Blimp1, Transducin, Phosducin (PdC), RXRy and Tr-32, Atoh7 and / or Isl-1; or (iii) The PNC precursor cells and mature PNCs include cells expressing Crx, recoverin and / or pyramidal arrestin (CAR), The pharmaceutical composition according to claim 5.

7. The pharmaceutical composition according to claim 1, wherein the population of retinal cells comprises neuronal retinal cells (NRCs), Müller glial cells, retinal pigment epithelial (RPE) cells, or any combination thereof.

8. The pharmaceutical composition according to claim 1, comprising a cryopreservation medium.

9. The pharmaceutical composition according to claim 8, wherein the cryopreservation medium comprises a cryoprotective agent selected from the group consisting of glycerol, sucrose, dextran, and dimethyl sulfoxide (DMSO).

10. The pharmaceutical composition according to claim 9, comprising about 1% to about 10% of the cryoprotective agent.

11. The aforementioned population of retinal cells is 1 × 10 ５ cells / mL ~ approx. 100 x 10 ６ The pharmaceutical composition according to claim 1, having a concentration of cells / mL.

12. The pharmaceutical composition according to claim 1, wherein the population of cells is present in a suspension or in a scaffold.

13. A method for producing a composition containing a population of retinal cells, wherein the method is as follows: (a) A step of culturing a population of undifferentiated pluripotent stem cells for a first period in a first cell culture medium containing nicotinamide (NIC), rel-4-[(3aR,4S,7R,7aS)-1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindole-2-yl]-N-8-quinolinylbenzamide (IWRe) and a Rock inhibitor (RI), (b) A step of culturing the population of cells generated in step (a) in a second cell culture medium containing NIC and IWRe for a second period, (c) A step of culturing the population of cells generated in step (b) for a third period in a third cell culture medium containing NIC and insulin-like growth factor 1 (IGF-1), (d) a step of collecting the population of cells and thereby producing the composition containing the population of retinal cells, A method comprising culturing the population of cells in a culture vessel for at least 14 weeks under conditions sufficient to produce cell aggregates with a diameter of 100 μm to 800 μm, and culturing the cells under dynamic culture conditions.

14. The method according to claim 13, wherein the culture vessel includes a wheel bioreactor, and in each step (a) to (c), the rotational speed of the wheel bioreactor is increased, thereby generating a cell aggregate with a diameter of 100 μm to 800 μm suspended in the wheel bioreactor.

15. (i) The rotational speed is 35 to 80 revolutions per minute (rpm), and / or (ii) The method according to claim 14, comprising an initial rotational speed of about 30 to 50 rpm, which increases to about 60 to 80 rpm.

16. (i) The first period is 1 to 15 days, (ii) The second period is 1 to 30 days, and / or (iii) The method according to claim 13, wherein the third period is 5 days to 14 weeks.

17. The method according to claim 13, further comprising the step of culturing the population of cells generated in step (c) for a fourth period in a fourth cell culture medium containing IGF-1, NIC, and tert-butyl(S)-{(2S)-2-[2-(3,5-difluorophenyl)acetamide]propanamide}phenylacetate (DAPT) prior to step (d).

18. The method according to claim 17, wherein the fourth period is 1 to 18 weeks.

19. The method according to claim 17, comprising step (ii) following step (i), wherein step (ii) comprises culturing the population of cells from step (i) for a fifth period in a fifth cell culture medium containing IGF-1, retinoic acid (RA), taurine (TA), brain-derived neurotrophic factor (BDNF), and neurolofin-4 (NT4).

20. The method according to claim 19, wherein the fifth period is 4 weeks to 24 weeks.

21. (i) The RI is in the first cell culture medium at a concentration of about 1 to 20 μM; (ii) The NIC is present in the first, second, third and / or fourth cell culture medium at a concentration of about 1 to 50 mM; (iii) The IWRe is present in the first cell culture medium and / or the second cell culture medium at a concentration of about 0.01 to 20 μM; (iv) The IGF-1 is present in the third, fourth and / or fifth cell culture medium at a concentration of about 0.5 to 20 ng / mL; (v) The DAPT is present in the fourth cell culture medium at a concentration of approximately 1 to 50 μM; (vi) The BDNF is present in the fifth cell culture medium at a concentration of approximately 5 to 50 ng / mL; (vii) The NT4 is present in the fifth cell culture medium at a concentration of about 2 to 200 ng / mL; and / or (viiii) The method according to claim 19, wherein the TA is present in the fifth cell culture medium at a concentration of 10 to 400 μM.

22. The method according to claim 13, wherein the undifferentiated pluripotent stem cells include human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs).

23. The method according to claim 22, wherein the hESC comprises HADC102 cells.

24. This includes cryopreserving the aforementioned population of retinal cells, The method according to claim 13, wherein the cryopreservation includes a cryopreservation medium suitable for administration to the eye of the subject.

25. The method according to claim 24, wherein the cryopreservation medium comprises a cryoprotectant selected from the group consisting of glycerol, sucrose, dextran, and dimethyl sulfoxide (DMSO).

26. The method according to claim 25, wherein the composition comprising the population of retinal cells comprises about 1% to about 10% of the cryoprotective agent.

27. The method according to claim 25, wherein the cryopreservation medium comprises 2% DMSO, 5% DMSO, or 10% DMSO.

28. Before step (d), (i) A step of collecting the cell aggregate, (ii) A step of dispersing the cell aggregate to generate dispersed cells, (iii) The step of seeding the dispersed cells into a tissue culture flask, (iv) The method according to claim 13, comprising the step of culturing the dispersed cells under adhesive static conditions for at least one week, wherein optionally the dispersed cells are cultured for one to three weeks.

29. A method for treating a subject having a visual disorder, comprising administering a therapeutically effective amount of the pharmaceutical composition described in any one of claims 1 to 12.

30. The method according to claim 29, wherein the visual pathology includes a neurodegenerative disease of the retina or damage to the retina.

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