Compositions and methods for treating retinal diseases

Abstract

To provide compositions and methods for treating retinal diseases.SOLUTION: Disclosed herein are compositions and methods for treating, ameliorating or preventing a retinal disease or condition; for improving a photopic (day light) vision; for improving correcting visual acuity, for improving macular function, for improving a visual field, or for improving scotopic (night) vision by administration of retinal progenitor cells. The subject matter described herein also provides cell populations comprising retinal progenitor cells and methods of isolation thereof.SELECTED DRAWING: None

Description

[Technical field]

[0001] Technical Field The subject matter described herein generally relates to the field of stem cell biology and regenerative medicine.In particular, the subject matter disclosed herein provides compositions and methods for treating, alleviating or preventing retinal disease or retinal condition, improving photopic (daytime) vision, improving or correcting visual acurity, improving macular function, improving visual field, or improving scotopic (nighttime) vision by administering retinal progenitor cells.The subject matter described herein also provides cell populations comprising retinal progenitor cells and methods for isolating the same. In alternative embodiments, the present invention provides compositions and methods for treating, alleviating, or preventing a retinal disease or condition, such as Usher's disease, retinitis pigmentosa (RP), degenerative retinal disease, age-related macular degeneration (AMD), wet or dry AMD, geographic atrophy, retinal photoreceptor disease, diabetic retinopathy, cystoid macular edema, uveitis, retinal detachment, retinal damage, macular hole, macular telangiectasia, traumatic or iatrogenic retinal damage, ganglion cell or optic nerve cell disease, glaucoma, or optic neuropathy, ischemic retinal disease, such as retinopathy of prematurity, retinal vascular occlusion, or ischemic optic neuropathy, or improving photopic (daytime) vision, or improving correcting visual acurity, or improving macular function, or improving visual field, or improving scotopic (nighttime) vision. In alternative embodiments, the invention provides heterogeneous mixtures of fetal neural retinal cells, as well as methods and compositions (eg, kits, formulations, etc.) for making and using them. [Background technology]

[0002] background Retinal degenerations are a heterogeneous group of ocular diseases that result in permanent loss of vision and affect millions of individuals worldwide. Although the molecular mechanisms underlying these conditions are diverse, they share a common endpoint: irreversible death of photoreceptor cells. Effective treatments to restore lost photoreceptors and visual function are not currently available, and most therapeutic interventions at best only slow the progression of the disease.

[0003] Previous clinical trials in patients with retinal degeneration have involved the use of fetal retinal sheet grafts. This transplantation strategy utilizes immature retinal sheets that extend cellular processes and form synaptic connections with the degenerated host retina. The rationale behind this is that the host inner retinal neurons remain intact and therefore only require synaptic connections with photoreceptors to restore visual function. Trials investigating retinal sheet transplantation in patients have shown some subjective improvement in vision, but graft rejection, tissue availability, and unreliable clinical efficacy have prevented this approach from becoming a viable treatment option. Summary of the Invention [Problem to be solved by the invention]

[0004] Stem cells and other pluripotent cells are also intended for use in treating patients with retinal degeneration and can be isolated from several sources, including embryonic tissue, adult brain, genetically engineered skin fibroblasts, and even the retina. However, embryonic or stem cells have so far been shown to have little ability to differentiate into retinal phenotypes when transplanted into adult retina unless they are first predifferentiated into a fetal-like retinal progenitor cell population. Furthermore, their yield and engraftment efficiency are low, and contamination with residual tumorigenic pluripotent cells is problematic. The current challenge in the field of photoreceptor cell placement involves understanding the developmental process that directs cells toward photoreceptor differentiation so that large numbers of these cells can be transplanted at the optimal stage with the least risk of immune response or transformation of transplanted cells into tumorigenic phenotypes. [Means for solving the problem]

[0005] Abstract In alternative embodiments, the present invention provides compositions and methods for treating, alleviating, or preventing retinal diseases or conditions, such as Usher's disease, retinitis pigmentosa (RP), retinal degenerative diseases, age-related macular degeneration (AMD), wet or dry AMD, geographic atrophy, retinal photoreceptor diseases, diabetic retinopathy, cystoid macular edema, uveitis, retinal detachment, retinal damage, macular hole, macular telangiectasia, traumatic or iatrogenic retinal damage, ganglion cell or optic nerve cell disease, glaucoma or optic neuropathy, ischemic retinal diseases, such as retinopathy of prematurity, retinal vascular occlusion, or ischemic optic neuropathy, or improving photopic (daytime) vision, or improving corrected visual acuity, or improving macular function, or improving visual field, or improving scotopic (nighttime) vision.In alternative embodiments, the present invention provides a heterogeneous mixture of fetal neural retinal cells, as well as methods and compositions (e.g., kits, formulations, etc.) for making and using them.

[0006] In an alternative embodiment, the present invention provides a method for producing a medicament for use in a pharmaceutical composition comprising: (a)(i) for humans, at about 17-18 weeks gestational age, or at about 16-19 weeks gestational age, or at 15-20 weeks gestational age, or at 14-26 weeks gestational age, or at about 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 weeks gestational age, or at about 3, 4, 5, 6, 7, 8, 9, 10, or 11 weeks gestation for non-human animal, optionally feline, canine, or porcine cells, or at 6-7 weeks for felines, or at 6-9 weeks for porcine cells, or (ii) recovering a sample of cells comprising a plurality of mammalian fetal neural retinal cells from a mammalian fetus at a stage after the mammalian retina is clearly formed but before photoreceptor outer segments are fully formed and vascularization of the retina is substantially complete or complete, or at a similar mammalian fetal staging, optionally wherein the sample of cells comprises human or feline or canine cells; (b) enzymatically dissociating the sample of harvested cells to produce a dissociated suspension of cells and / or small and medium sized cell clusters; Optionally, the collected cell and / or small cell cluster sample is enzymatically dissociated using trypsin or equivalent, or TRYP-LE EXPRESS™ (TrypLE™ Express, Invitrogen-Life Technologies, Carlsbad Calif.) or equivalent; (c) culturing the cells and / or small populations of cells in a sterile environment comprising serum-free medium or medium containing serum, and containing antibiotics and antimycotics or containing no antibiotics or antimycotics, for no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more passages; Including, Optionally, the cells and / or small clusters of cells are cultured in a medium containing Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12™ (DMEM / F12™) medium, or ADVANCED DMEM / F12™ medium (Gibco-Invitrogen-Life Technologies, Carlsbad Calif.), or ULTRACULTURE™ medium (BioWhittaker-Lonza Walkersville, Inc., Walkersville, Md.) supplemented with N2 supplement (Invitrogen), or B27 or B27 Xeno Free (Invitrogen), L-glutamine or GlutaMax (Invitrogen), and human recombinant growth factors consisting of EGF and bFGF (Invitrogen), or other growth factors, as appropriate; Optionally, DMEM / F12™ medium is used for human cells and ULTRACULTURE™ medium is used for feline or canine cells; Cultivating or growing the cells under hypoxic conditions or oxygen conditions that approximate or closely mimic the oxygen levels of the developing fetal retina during gestation or at about 2%, 2.5%, 3%, 3.5% oxygen as appropriate; Optionally, the medium is supplemented with Vitamin C, optionally Vitamin C is added every 1 or 2 days, optionally Vitamin C is added in an amount having an initial concentration of about 0.1 mg / ml or 0.05 mg / ml, or between about 0.01 mg / ml and about 0.5 mg / ml; Optionally, the medium is supplemented with albumin, or human or feline or canine albumin, or recombinant albumin, or the albumin is added in an amount having an initial concentration of about 1.0 mg / ml; Optionally, the cell sample is screened for the presence of pathogens, bacteria, endotoxins, fungi, mycoplasma, viruses, hepatitis viruses, or HIV viruses; Optionally, the cell sample is screened for the presence of a normal karyotype; Optionally, the sample of cells does not exhibit elevated telomerase activity, Optionally, a sample of the cells is screened for viability; Optionally, the cell sample is screened for tumorigenicity. A formulation, product of manufacture, or composition is provided that comprises the heterogeneous mixture of mammalian fetal neural retinal cells produced by the method.

[0007] In an alternative embodiment of the formulation, product of manufacture, or composition, a method for producing a heterogeneous mixture of fetal neural retinal cells comprises: (a) selecting fetal neural retinal cells based on cell surface or genetic markers, optionally selecting the cells either prior to the culturing step (prospectively) or after the culturing step, or both; Optionally, the cell surface or genetic markers include CD15 / LeX / SSEA1 and / or GD2 ganglioside, optionally CD9, CD81, CD133, or AQP4, CXCR4; or (b) further comprising selecting a fetal neural retinal cell based on a fetal neural retinal cell transcriptomic, proteomic, and / or genomic profile. In an alternative embodiment, the method for producing a heterogeneous mixture of fetal neural retinal cells of the invention comprises: Culturing the cells under conditions that allow the cells to proliferate at an optimal rate (or near an optimal rate) and / or express a fetal neural retina cell phenotype or transcriptomic profile, Optionally, the fetal neural retinal cell phenotype profile comprises (optionally moderate) levels of Ki67, p21, and / or telomerase, and / or high levels of expression of one or more stem cell markers associated with multipotent but not pluripotent cells; Alternatively, optionally, the fetal neural retinal cell phenotype profile comprises (gene or message) expression of nestin, vimentin, Ki-67, a differentiation marker, β3-tubulin, glial fibrillary acidic protein (GFAP), and / or rhodopsin, or any combination thereof; Optionally, the method further comprises the steps whereby Dach1, Lhx2, and / or Pax6 message is measured.

[0008] In alternative embodiments, the formulation, product of manufacture, or composition is formulated for injection, or for injection into the vitreous or subretinal space. In an alternative embodiment, the present invention provides a method for treating a retinal disease or condition, comprising: (a) providing a formulation, product of manufacture, or composition of the invention; (b) injecting the formulation, product of manufacture, or composition of (a) into the vitreous or subretinal space; Including, Optionally, the vitreous or subretinal space is a human, feline or canine vitreous or subretinal space; Standard intraocular injection procedures are used as needed, Optionally, the method further comprises anterior chamber paracentesis; Thereby improving the safety of the procedure, Thereby, a method of treating a retinal disease or condition is provided.

[0009] In alternative embodiments of the method, the retinal disease or condition comprises Usher's disease, retinitis pigmentosa (RP), a retinal degenerative disease, age-related macular degeneration (AMD), wet AMD or dry AMD, retinal photoreceptor disease, diabetic retinopathy, retinal detachment, retinal injury, traumatic or iatrogenic retinal injury, ganglion cell or optic nerve cell disease, glaucoma, or optic neuropathy.

[0010] In an alternative embodiment, the present invention provides a method for improving photopic (daytime) vision, comprising: (a) providing a formulation, product of manufacture, or composition of the invention; (b) injecting the formulation, product of manufacture, or composition of (a) into the vitreous or subretinal space; Including, Optionally, the vitreous or subretinal space is a human, feline or canine vitreous or subretinal space; Standard intraocular injection procedures are used as needed, Optionally, the method further comprises anterior chamber paracentesis; Thereby, a method is provided for improving photopic (daytime) vision.

[0011] In an alternative embodiment, the present invention provides a method for improving corrected visual acuity, or improving macular function, or improving visual field, or improving scotopic (night) vision, comprising: (a) providing a formulation, product of manufacture, or composition of the invention; (b) injecting the formulation, product of manufacture, or composition of (a) into the vitreous or subretinal space; Including, Optionally, the vitreous or subretinal space is a human, feline or canine vitreous or subretinal space; Standard intraocular injection procedures are used as needed, Optionally, the method further comprises anterior chamber paracentesis; Thereby, a method is provided for correcting vision, or improving macular function, or improving visual field, or improving scotopic (nighttime) vision.

[0012] In an alternative embodiment, the present invention provides a method for producing a medicament for use in a pharmaceutical composition comprising: (a) for humans, at about 17-18 weeks gestational age, or at about 16-19 weeks gestational age, or at 15-20 weeks gestational age, or at 14-26 weeks gestational age, or at about 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 weeks gestational age, or at about 3, 4, 5, 6, 7, 8, 9, 10, or 11 weeks gestation for non-human animal, optionally feline, canine, or porcine cells, or at 6-7 weeks for feline cells, or at 6-9 weeks for porcine cells; Optionally, the sample of cells comprises human, feline or canine cells; (b) enzymatically dissociating the sample of harvested cells to produce a dissociated suspension of cells and / or small and medium sized cell clusters; Optionally, the collected cell sample is enzymatically dissociated using trypsin or equivalent, or TRYP-LE EXPRESS™ (TrypLE™ Express, Invitrogen-Life Technologies, Carlsbad Calif.) or equivalent; (c) culturing the cells in a sterile environment containing serum-free or serum-containing medium, free of antibiotics and antimycotics, for no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 passages; Including, Optionally, cells were cultured in a medium containing Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12™ (DMEM / F12™) medium (Gibco-Invitrogen-Life Technologies, Carlsbad CA) or in ULTRACULTURE™ medium (BioWhittaker-Lonza). Walkersville, Inc., Walkersville, MD) Optionally, DMEM / F12™ medium is used for human cells and ULTRACULTURE™ medium is used for feline or canine cells; Optionally, the medium is supplemented with Vitamin C, optionally Vitamin C is added every 1 or 2 days, optionally Vitamin C is added in an amount having an initial concentration of about 0.1 mg / ml or 0.05 mg / ml, or between about 0.01 mg / ml and about 0.5 mg / ml; Optionally, the medium is supplemented with albumin, or human or feline or canine albumin, or recombinant albumin, or the albumin is added in an amount having an initial concentration of about 1.0 mg / ml; Optionally, the cell sample is screened for the presence of pathogens, bacteria, endotoxins, fungi, mycoplasma, viruses, hepatitis viruses, or HIV viruses; Optionally, the cell sample is screened for the presence of a normal karyotype; Optionally, the sample of cells does not exhibit elevated telomerase activity, Optionally, a sample of the cells is screened for viability; Optionally, the cell sample is screened for tumorigenicity. Methods are provided for producing a heterogeneous mixture of fetal neural retinal cells produced by the methods.

[0013] In an alternative embodiment, the present invention provides a kit for treating a retinal disease or condition or for practicing a method of the present invention, comprising: (a) a formulation, product of manufacture, or composition of the invention, and / or (b) A heterogeneous mixture of fetal neural retinal cells produced by the methods of the invention. A kit is provided, comprising:

[0014] In alternative embodiments, the invention provides cell populations comprising mammalian retinal progenitor cells expressing one or more markers selected from the group consisting of nestin, Sox2, Ki67, MHC class I, and Fas / CD95, where nestin is expressed by greater than about 90%, or about 95-99%, of the cells in the population, Sox2 is expressed by greater than about 80%, or about 90-99%, of the cells in the population, Ki-67 is expressed by greater than about 30%, or about 40-60%, of the cells in the population, MHC class I is expressed by greater than about 70%, or about 90%, of the cells in the population, and Fas / CD95 is expressed by greater than about 30%, or about 40-70%, of the cells in the population. In some embodiments, the cells are derived from a human or non-human mammal.

[0015] In certain embodiments, the mammalian retinal progenitor cells in the population further express one or more markers selected from the group consisting of vimentin, CD9, CD81, AQP4, CXCR4, CD15 / LeX / SSEA1, GD2 ganglioside, CD133, β3-tubulin, MAP2, GFAP, OPN / SPP1, PTN, KDR, and TEK.

[0016] In another aspect, a method for isolating a population of mammalian retinal progenitor cells includes the steps of harvesting mammalian fetal retinal tissue at a stage at which the retina will later form, but before photoreceptor outer segments are fully formed throughout the retina and before vascularization of the retina is substantially complete or complete; dissociating the harvested tissue to produce a dissociated suspension of cells and cell populations; and culturing the dissociated suspension for about 10-30 passages, wherein the mammalian retinal progenitor cells express a number of receptors that are specific for Nestin, Sox2, Ki-67, β3-tubulin, MAP2, MHC class I, and IL-16. and Fas / CD95, nestin is expressed by greater than about 90% or about 95-99% of the cells in the population, Sox2 is expressed by greater than about 80% or about 90-99% of the cells in the population, Ki-67 is expressed by greater than about 30% or about 40-60% of the cells in the population, MHC class I is expressed by greater than about 70% or about 90% of the cells in the population, and Fas / CD95 is expressed by greater than about 30% or about 40-70% of the cells in the population. In some embodiments, the tissue is harvested from a human or non-human mammal. In some embodiments, the tissue is harvested from human fetal retina between about 12 weeks and about 28 weeks gestational age, or from postnatal or neonatal retinal tissue. In other embodiments, the tissue is harvested from non-human fetal retina between about 3 weeks and about 11 weeks gestational age. In other embodiments, tissue is harvested from a non-human mammal between about 3 and 11 weeks of gestation, or from postnatal or neonatal retinal tissue. The cells can be cultured at atmospheric oxygen levels or at low oxygen levels that approximate the oxygen levels of the developing fetal retina during gestation, such as between about 0.5% and about 7%, and can also be cultured in serum-free or low-serum cell culture medium, which can contain additional supplements, such as N2, B27, vitamin C, and albumin, as needed.

[0017] In another aspect, a method for treating a retinal disease or condition in a subject in need thereof comprises administering to the subject an effective amount of a composition comprising mammalian retinal progenitor cells, wherein the mammalian retinal progenitor cells express one or more markers selected from the group consisting of nestin, Sox2, Ki-67, β3-tubulin, MAP2, MHC class I, and Fas / CD95, wherein nestin is expressed by greater than about 90%, or about 95-99% of the cells in the population. wherein Sox2 is expressed by greater than about 80%, or about 90-99%, of the cells in the population, Ki-67 is expressed by greater than about 30%, or about 40-60%, of the cells in the population, MHC class I is expressed by greater than about 70%, or about 90%, of the cells in the population, and Fas / CD95 is expressed by greater than about 30%, or about 40-70% of the cells in the population; and optionally measuring a change in the subject's vision, thereby treating a retinal disease or condition.

[0018] In alternative embodiments, the subject is a human or non-human mammal. In some embodiments, the composition is formulated for injection into the vitreous or subretinal space of the subject. Retinal diseases or conditions include retinitis pigmentosa (RP), Leber's congenital amaurosis (LCA), Stargardt syndrome, Usher syndrome, choroideremia, rod-cone or cone-rod dystrophies, ciliopathy, mitochondrial disorders, progressive retinal atrophy, retinal degenerative diseases, age-related macular degeneration (AMD), wet AMD, dry AMD, geographic atrophy, familial or acquired maculopathy, retinal photoreceptor disease, retinal pigment epithelial-based disease, diabetic retinopathy, cystoid macular edema, uveitis, retinal detachment, traumatic retinal injury, iatrogenic retinal injury, macular hole, macular telangiectasia, ganglion cell disease, optic nerve cell disease, glaucoma, optic neuropathy, ischemic retinal disease, retinopathy of prematurity, retinal vascular occlusion, familial microaneurysm, retinal vascular occlusion ... macroaneurysm), retinal vascular disease, ocular vascular disease, vascular disease, or ischemic optic neuropathy. In one embodiment of the present invention, for example, the following items are provided: (Item 1) A cell population comprising mammalian retinal progenitor cells, the mammalian retinal progenitor cells expressing one or more markers selected from the group consisting of nestin, Sox2, Ki-67, MHC class I, and Fas / CD95, wherein nestin is expressed by more than 90% of the cells in the population, Sox2 is expressed by more than 80% of the cells in the population, Ki-67 is expressed by more than 30% of the cells in the population, MHC class I is expressed by more than 70% of the cells in the population, and Fas / CD95 is expressed by more than 30% of the cells in the population. (Item 2) 2. The cell population of item 1, wherein the cells are derived from a human or non-human mammal. (Item 3) 2. The cell population of item 1, wherein the cells in the population further express one or more markers selected from the group consisting of vimentin, CD9, CD81, AQP4, CXCR4, CD15 / LeX / SSEA1, GD2 ganglioside, CD133, β3-tubulin, MAP2, GFAP, OPN / SPP1, PTN, KDR, and TEK. (Item 4) 1. A method for isolating a population of mammalian retinal progenitor cells, comprising: harvesting mammalian fetal retinal tissue at a stage at which a retina will subsequently form, but before photoreceptor outer segments are fully formed throughout said retina, and before vascularization of the retina is substantially complete or complete; dissociating the harvested tissue to produce a dissociated suspension of cells and cell populations; and culturing the dissociated suspension for about 10 to 30 passages, wherein the mammalian retinal progenitor cells express one or more markers selected from the group consisting of nestin, Sox2, Ki-67, MHC class I, and Fas / CD95, wherein nestin is expressed by more than 90% of the cells in the population, Sox2 is expressed by more than 80% of the cells in the population, Ki-67 is expressed by more than 30% of the cells in the population, MHC class I is expressed by more than 70% of the cells in the population, and Fas / CD95 is expressed by more than 30% of the cells in the population. (Item 5) 5. The method of claim 4, wherein the tissue is harvested from a human or non-human mammal. (Item 6) 5. The method according to item 4, wherein the tissue is harvested from a human fetal retina at about 12 weeks to about 28 weeks gestational age, or from a postnatal or neonatal retina. (Item 7) 5. The method of claim 4, wherein the tissue is harvested from a non-human fetal retina between about 3 weeks and about 11 weeks gestational age, or from a postnatal or neonatal retina. (Item 8) 5. The method of claim 4, wherein the cells are cultured at atmospheric oxygen levels. (Item 9) 5. The method of claim 4, wherein the cells are cultured at an oxygen level of between about 0.5% and about 7%. (Item 10) 5. The method of claim 4, wherein the cells are cultured in a serum-free or low-serum cell culture medium optionally containing vitamin C and albumin. (Item 11) 5. The method of claim 4, wherein the cells in the population further express one or more markers selected from the group consisting of vimentin, CD9, CD81, AQP4, CXCR4, CD15 / LeX / SSEA1, GD2 ganglioside, CD133, β3-tubulin, MAP2, GFAP, OPN / SPP1, PTN, KDR, and TEK. (Item 12) 1. A method for treating a retinal disease or condition in a subject in need thereof, comprising the steps of administering to the subject an effective amount of a composition comprising mammalian retinal progenitor cells, wherein the mammalian retinal progenitor cells express one or more markers selected from the group consisting of nestin, Sox2, Ki-67, MHC class I, and Fas / CD95, wherein nestin is expressed by more than 90% of the cells in the population, Sox2 is expressed by more than 80% of the cells in the population, Ki-67 is expressed by more than 30% of the cells in the population, MHC class I is expressed by more than 70% of the cells in the population, and Fas / CD95 is expressed by more than 30% of the cells in the population, and optionally measuring a change in the subject's vision, thereby treating the retinal disease or condition. (Item 13) 13. The method of claim 12, wherein the subject is a human or non-human mammal. (Item 14) 13. The method of claim 12, wherein the composition is formulated for injection into the vitreous or subretinal space of the subject. (Item 15) 13. The method of item 12, wherein the retinal disease or condition comprises retinitis pigmentosa (RP), Leber's congenital amaurosis (LCA), Stargardt's syndrome, Usher syndrome, choroideremia, rod-cone or cone-rod dystrophies, ciliopathy, mitochondrial disorders, progressive retinal atrophy, retinal degenerative diseases, age-related macular degeneration (AMD), wet AMD, dry AMD, geographic atrophy, familial or acquired maculopathy, retinal photoreceptor diseases, retinal pigment epithelium-based diseases, diabetic retinopathy, cystoid macular edema, uveitis, retinal detachment, traumatic retinal damage, iatrogenic retinal damage, macular hole, macular telangiectasia, ganglion cell disease, optic nerve cell disease, glaucoma, optic neuropathy, ischemic retinal disease, retinopathy of prematurity, retinal vascular occlusion, familial microaneurysm, retinal vascular disease, ocular vascular disease, vascular disease, or ischemic optic neuropathy. (Item 16) 13. The method of claim 12, wherein the cells in the composition further express one or more markers selected from the group consisting of vimentin, CD9, CD81, AQP4, CXCR4, CD15 / LeX / SSEA1, GD2 ganglioside, CD133, β3-tubulin, MAP2, GFAP, OPN / SPP1, PTN, KDR, and TEK. (Item 17) (a)(i) for humans, at about 17-18 weeks gestational age, or at about 16-19 weeks gestational age, or at 15-20 weeks gestational age, or at 14-26 weeks gestational age, or at about 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 weeks gestational age, or at about 3, 4, 5, 6, 7, 8, 9, 10, or 11 weeks for non-human animal, optionally feline, canine, or porcine cells, or at 6-7 weeks for feline cells, or at 6-9 weeks for porcine cells; (ii) recovering a sample of cells comprising a plurality of mammalian fetal neural retinal cells from a mammalian fetus at a stage after the mammalian retina is clearly formed but before photoreceptor outer segments are fully formed and vascularization of the retina is substantially complete or complete, or at a similar mammalian fetal stage; Optionally, said sample of cells comprises human or feline or canine cells; (b) enzymatically dissociating said sample of harvested cells to produce a dissociated suspension of cells and / or small and medium sized cell clusters, optionally said sample of harvested cells and / or small cell clusters being enzymatically dissociated with trypsin or equivalent, or TRYP-LE enzymatically dissociating the endothelial cell using EXPRESS™ (TrypLE™ Express, Invitrogen-Life Technologies, Carlsbad Calif.) or equivalent; (c) culturing the cells and / or small populations of cells in a sterile environment comprising serum-free medium or medium containing serum, and containing antibiotics and antimycotics or containing no antibiotics or antimycotics, for no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 passages; Including, Optionally, the cells and / or small clusters of cells are cultured in a medium containing Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12™ (DMEM / F12™) medium, or ADVANCED DMEM / F12™ medium (Gibco-Invitrogen-Life Technologies, Carlsbad CA) or ULTRACULTURE™ medium (BioWhittaker-Lonza). Walkersville, Inc., Walkersville, MD) with N2 supplement (Invitrogen), or B27 or B27 Xeno Free (Invitrogen), L-glutamine or GlutaMax (Invitrogen), and human recombinant growth factors consisting of EGF and bFGF (Invitrogen), or other growth factors as needed; Optionally, the DMEM / F12™ medium is used for human cells and the ULTRACULTURE™ medium is used for feline or canine cells; Cultivating or growing the cells under hypoxic conditions or oxygen conditions that approximate or closely mimic the oxygen levels of the developing fetal retina during gestation, or at about 2%, 2.5%, 3%, 3.5% oxygen as required; Optionally, the medium is supplemented with Vitamin C, optionally the Vitamin C is added every 1 or 2 days, optionally the Vitamin C is added in an amount having an initial concentration of about 0.1 mg / ml or 0.05 mg / ml, or between about 0.01 mg / ml and about 0.5 mg / ml; Optionally, the medium is supplemented with albumin, or human or feline or canine albumin, or recombinant albumin, or the albumin is added in an amount having an initial concentration of about 1.0 mg / ml, Optionally, the sample of cells is screened for the presence of a pathogen, bacteria, endotoxin, fungus, mycoplasma, virus, hepatitis virus, or HIV virus; Optionally, the sample of cells is screened for the presence of a normal karyotype; Optionally, the sample of cells does not exhibit elevated telomerase activity; Optionally, said sample of cells is screened for viability; Optionally, the sample of cells is screened for tumorigenicity. A formulation, product of manufacture, or composition comprising a heterogeneous mixture of fetal neural retinal cells produced by the method. (Item 18) The method for producing the heterogeneous mixture of fetal neural retinal cells comprises: (a) selecting fetal neural retinal cells based on cell surface or genetic markers, optionally selecting the cells before (prospectively) or after culture, or both; Optionally, the cell surface or genetic markers comprise CD15 / LeX / SSEA1 and / or GD2 ganglioside, optionally CD9, CD81, CD133, or AQP4, CXCR4; or (b) The formulation, product of manufacture, or composition of claim 17, further comprising the step of selecting fetal neural retinal cells based on a fetal neural retinal cell transcriptomic, proteomic, and / or genomic profile. (Item 19) 1. A method for treating a retinal disease or condition, comprising: (a) providing a formulation, product of manufacture, or composition according to any one of items 17-18; (b) injecting the formulation, product of manufacture, or composition of (a) into the vitreous or subretinal space; Optionally, the vitreous or subretinal space is human or feline or canine, and optionally standard intraocular injection procedures are used, and optionally the method further comprises anterior chamber paracentesis, thereby treating said retinal disease or condition, and optionally the retinal disease or condition comprises Usher's disease, retinitis pigmentosa (RP), retinal degenerative disease, age-related macular degeneration (AMD), wet AMD, or dry AMD, geographic atrophy, retinal photoreceptor disease, diabetic retinopathy, cystoid macular edema, uveitis, retinal detachment, retinal damage, macular hole, macular telangiectasia, traumatic or iatrogenic retinal damage, ganglion cell or optic nerve cell disease, glaucoma, or optic neuropathy, ischemic retinal disease, e.g., retinopathy of prematurity, retinal vascular occlusion, or ischemic optic neuropathy. (Item 20) 1. A method for improving photopic (daytime) vision, or for improving corrected visual acuity, or for improving macular function, or for improving visual field, or for improving scotopic (nighttime) vision, comprising: (a) providing a formulation, product of manufacture, or composition according to any one of items 17-18; (b) injecting the formulation, product of manufacture, or composition of (a) into the vitreous or subretinal space; Optionally, the vitreous or subretinal space is human or feline or canine, and optionally a standard intraocular injection procedure is used, and optionally the method further comprises anterior chamber paracentesis, thereby improving the safety of the procedure; thereby improving the photopic (daytime) vision, or improving corrected visual acuity, or improving macular function, or improving visual field.

[0019] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0020] All publications, patents, and patent applications cited herein are hereby expressly incorporated by reference for all purposes.

[0021] The following detailed description is provided by way of example and is not intended to limit the subject matter described herein to the specific embodiments described and can be understood in conjunction with the accompanying drawings, which are incorporated herein by reference. [Brief description of the drawings]

[0022] [Figure 1] Figure 1 illustrates the morphology of feline retina-derived progenitor cells, or RPCs, which was maintained at various time points over the course of sustained culture.

[0023] [Diagram 2] FIG. 2 is a graph depicting growth curves showing the differential proliferation of feline RPCs in SM medium versus UL medium.

[0024] [Diagram 3] FIG. 3 shows the morphology of human RPCs (hRPCs) over time during culture (days 0 to 56).

[0025] [Figure 4] 4 and 5 are graphs showing the growth kinetics of hRPCs over time.

[0026] [Diagram 5] 4 and 5 are graphs showing the growth kinetics of hRPCs over time.

[0027] [Figure 6] Figure 6A shows a comparison of growth conditions between Advanced DMEM / F12 and Basal DMEM / F12 cell culture media supplemented with N2. Figure 6B illustrates an experiment comparing the growth of hRPCs in cell culture media supplemented with N2 or B27.

[0028] [Figure 7]Figure 7A summarizes an experiment testing the effect of vitamin C supplementation in hRPC culture. Addition of vitamin C improved hRPC viability. Figures 7B and 7C depict an experiment testing the effect of albumin supplementation in hRPC culture.

[0029] [Figure 8] FIG. 8 reflects a comparative experiment testing different osmolarities of cell culture media on hRPC proliferation and viability.

[0030] [Figure 9] Figures 9-11 depict the results of experiments in which hRPCs were cultured under atmospheric oxygen conditions and compared to the same cells grown under hypoxic conditions. Growth kinetics, morphology, and gene expression were assessed. Figure 11 shows the reproducibility of growth characteristics of the three different cell samples used to generate the working cell bank.

[0031] [Figure 10] Figures 9-11 depict the results of experiments in which hRPCs were cultured under atmospheric oxygen conditions and compared to the same cells grown under hypoxic conditions. Growth kinetics, morphology, and gene expression were assessed. Figure 11 shows the reproducibility of growth characteristics of the three different cell samples used to generate the working cell bank.

[0032] [Figure 11] Figures 9-11 depict the results of experiments in which hRPCs were cultured under atmospheric oxygen conditions and compared to the same cells grown under hypoxic conditions. Growth kinetics, morphology, and gene expression were assessed. Figure 11 shows the reproducibility of growth characteristics of the three different cell samples used to generate the working cell bank.

[0033] [Figure 12] FIG. 12 shows the results of a steroid toxicity test performed on hRPCs.

[0034] [Figure 13]13A-13D are graphs showing the viability and stability of previously frozen hRPCs.

[0035] [Figure 14] FIG. 14 shows feline RPCs stained with ImmunoCytoChemistry (ICC) markers nestin, β3-tubulin, vimentin, rhodopsin, Ki-67, and GFAP.

[0036] [Figure 15] FIG. 15 is a graph showing feline RPC gene profile over time using qPCR of nestin, vimentin, GFAP, and PKC-α marker transcripts.

[0037] [Figure 16] Figure 16A is a graphical illustration of the differences in gene expression as measured by qPCR between RPCs grown under UL and SM media conditions. Figure 16B is a graph comparing feline RPCs vs. BPCs.

[0038] [Figure 17] FIG. 17 shows cell staining for RFP, vimentin, or opsin, ezrin, or PKC following in vivo transplantation of feline RPCs into the subretinal space of dystrophic Abyssinian cats.

[0039] [Figure 18] Figure 18 illustrates human cell morphology using marker expression by ICC, including (A) nestin, (B) vimentin, (C) Sox2, (D) SSEA-1 (also known as CD15, LeX), (E) GD2-ganglioside, and (F) Ki-67.

[0040] [Figure 19] Figure 19 illustrates human cell morphology using marker expression by ICC (A) shows β3-tubulin staining, (B) shows GFAP staining, and (C) shows GDNF staining.

[0041] [Figure 20] FIG. 20 depicts marker expression at the RNA level as detected by quantitative PCR heat mapping.

[0042] [Figure 21] Figure 21A shows the results of a qPCR analysis comparing the expression levels of genes in hRPCs versus human fibroblasts (hFBs). Figures 21B and 21C represent qPCR (gene expression) data from additional experiments.

[0043] [Figure 22] FIG. 22 is a summary table showing a list of 11 genes (from approximately 26 profiles used) that exhibit consistent behavior (in hRPCs vs. hFBs) across different donations.

[0044] [Figure 23] FIG. 23 shows marker expression at the RNA level as detected by qPCR ("real-time" PCR) at different time points in the cultures.

[0045] [Figure 24] FIG. 24 depicts changes in expression levels of various markers (eg, GDNF, Annexin V, β3-tubulin, Notch1, Six6, Ki-67, and CD133) in early versus late passage cells.

[0046] [Diagram 25] FIG. 25 is a summary of microarray data distinguishing hRPCs from BPCs.

[0047] [Figure 26] FIG. 26 shows marker expression after differentiation with retinoic acid (RA).

[0048] [Figure 27] FIG. 27 is a summary of culture conditions for hRPCs grown from different tissue donors.

[0049] [Figure 28] FIG. 28A illustrates gene expression by qPCR at different time points in cultures grown under SM conditions, and FIG. 28B illustrates gene expression by qPCR at two different time points in cultures grown under SM-UL (first UL, then SM) conditions.

[0050] [Figure 29] Figure 29A illustrates gene expression by qPCR under SM-FBS (initial plating conditions SM+5% FBS, then changed to SM alone) conditions at different time points. Figure 29B illustrates gene expression by qPCR in SM alone at two different time points. Figure 29C illustrates gene expression by qPCR in SM-hS (initial SM+human serum followed by SM) at the same two time points. As before, only GDNF is elevated.

[0051] [Diagram 30] Figure 30 is a table summary of the experimental results of the time point comparisons shown in Figures 28 and 29. Genes showing strong and consistent trends across treatment conditions are highlighted in yellow.

[0052] [Diagram 31] FIG. 31 depicts a heatmap analysis of qPCR data from hRPCs.

[0053] [Diagram 32] FIG. 32 depicts a histogram of qPCR data from hRPCs.

[0054] [Diagram 33]Figure 33A depicts the levels of angiogenesis-related genes in hRPCs, and Figure 33B depicts the levels of WNT pathway-related genes in hRPCs.

[0055] [Diagram 34] Figure 34A is a graph summarizing the results of a principal component analysis (PCA) showing gene expression differences between hRPCs versus tissue and fibroblasts. Figure 34B is another PCA graph showing gene expression differences between hRPCs versus tissue obtained from fetal retina on day 0. Figure 34C is another PCA graph depicting the overall similarities and differences between cell sample populations.

[0056] [Diagram 35] FIG. 35 shows cluster analysis of three hRPC populations (hypoxic MCB, normoxic MCB, and hypoxic WCB) versus fetal retinal tissue.

[0057] [Diagram 36] FIG. 36 is a volcano plot comparing differences in gene expression in hypoxic MCB versus tissue.

[0058] [Figure 37] Figure 37A is a Venn diagram showing the number of differentially expressed genes between hRPC groups as a function of treatment condition. Figure 37B is a Venn diagram showing the number of differentially expressed genes between hRPC groups normalized to tissue as a function of passage number and treatment condition.

[0059] [Figure 38] Figures 38A and 38B are volcano plots comparing different hRPCs versus tissue of origin.

[0060] [Figure 39] Figures 39A and 39B are volcano plots comparing hypoxic vs. normoxic hRPCs.

[0061] [Diagram 40]Figure 40 is a table summarizing the time point design for feline RPC. Donations and treatment conditions are compared (red underline for UL-d76 corresponds to a clear upward inflection in the cell growth curve, i.e., likely spontaneous immortalization).

[0062] [Diagram 41] Figure 41A illustrates gene expression by qPCR of feline cRPCs grown in UL medium at the indicated time points. Figure 41B includes additional markers not shown in Figure 41A.

[0063] [Diagram 42] 42-44 collectively represent additional data from the same experiment shown in FIG. [Diagram 43] 42-44 collectively represent additional data from the same experiment shown in FIG. [Diagram 44] 42-44 collectively represent additional data from the same experiment shown in FIG.

[0064] [Diagram 45] FIG. 45 is a radial graph showing the change in expression pattern of feline cRPCs according to time points and culture conditions.

[0065] [Figure 46] FIG. 46 is a chart summarizing qPCR data obtained from feline RPCs across different donations and culture conditions.

[0066] [Figure 47] Figure 47 shows the results of ELISA studies characterizing hRPCs cultured under normoxic and hypoxic conditions. Markers detected include OPN, VEGF, SDF-1, BDNF, and GDNF.

[0067] [Figure 48]Figure 48 depicts the results of FACS analysis of hRPCs cultured under normoxic and hypoxic conditions. Markers of interest include nestin, Sox2, Ki-67, GFAP, MHC class I and II, Fas / CD95, CXCR4, CD15, and GD2 ganglioside.

[0068] [Figure 49] FIG. 49 illustrates the proof-of-concept of the methods of the invention using in vivo transplantation of hRPCs into the eyes of dystrophic RCS rats in a model of inherited photoreceptor degeneration.

[0069] [Figure 50] Figures 50 and 51 are photographs showing staining of rat retina following subretinal injection of hRPCs. [Figure 51] Figures 50 and 51 are photographs showing staining of rat retina following subretinal injection of hRPCs.

[0070] [Figure 52] Figures 52 and 53 are photographs showing staining of rat retina following intravitreal injection of hRPCs. [Figure 53] Figures 52 and 53 are photographs showing staining of rat retina following intravitreal injection of hRPCs.

[0071] [Figure 54] Figure 54 is a photograph showing immunocytochemical staining of RCS whole mounts after injection of hRPCs into rat retina. A and B: Sham, C and D: Treated.

[0072] [Figure 55] FIG. 55 is a graph showing improvement in vision in patients treated with hRPCs. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0073] Detailed Description The features, structures, or characteristics described throughout this specification may be combined in any suitable manner in one or more embodiments. For example, the use of the phrases "exemplary embodiment", "exemplary embodiment", "some embodiments", or other similar phrases throughout this specification refers to the fact that a particular feature, structure, or characteristic described in connection with one embodiment may be included in at least one embodiment described herein. Thus, the appearance of the phrases "exemplary embodiment", "exemplary embodiment", "some embodiments", "other embodiments", or other similar phrases throughout this specification does not necessarily refer to the same group of embodiments, but rather the described features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0074] To facilitate understanding of this disclosure, certain terms are defined below. Terms defined herein have the meanings commonly understood by those skilled in the art in the field related to the subject matter described herein. Terms such as "a", "an", and "the" are not intended to refer to a singular entity only, but include general classes of which specific examples can be used for illustration. The terminology herein is used to describe specific embodiments of the subject matter described herein, but their use does not define the scope of the subject matter, except as outlined in the claims.

[0075] The present invention provides compositions and methods that include or use a heterogeneous mixture of fetal neural retinal cells to treat, reduce, or prevent retinal diseases or conditions, such as Usher's disease, retinitis pigmentosa (RP), retinal degenerative diseases, age-related macular degeneration (AMD), wet AMD or dry AMD, retinal photoreceptor diseases, diabetic retinopathy, retinal detachment, retinal damage, traumatic or iatrogenic retinal damage, ganglion cell or optic nerve cell disease, glaucoma, or optic neuropathy, or to improve photopic (daytime) vision, or to improve corrected visual acuity, or to improve macular function, or to improve visual field, or to improve scotopic (nighttime) vision. In alternative embodiments, the present invention provides a heterogeneous mixture of fetal neural retinal cells, as well as methods and compositions (e.g., kits, formulations, etc.) for making and using them.

[0076] In an alternative embodiment, the invention provides methods and uses of culturing retinal progenitor cells prepared as a cell suspension and used as an allogeneic graft injected into the vitreous cavity of patients with retinal disease. In an alternative embodiment, the invention provides cell-based therapies that comprise or consist of the use of a cultured heterogeneous cell population derived from an immature mammalian, e.g., human, retina.

[0077] In an alternative embodiment, proliferating mammalian, e.g., human, retinal cells are grown from donor tissue, characterized, and the cells are injected directly into the vitreous cavity under local anesthesia without the need for systemic immunosuppression of the recipient, e.g., patient.

[0078] In an alternative embodiment, the compositions and methods of the present invention are used to treat, prevent, or alleviate retinal diseases, such as retinal degeneration, such as retinitis pigmentosa (RP).

[0079] Although the present invention is not limited by any particular mechanism of action, in one embodiment, the donor fetal retinal cells provide a trophic effect to the host retina, particularly including the host cones. This trophic effect is not only neuroprotective, but also has a rapid revitalizing effect on the remaining host retinal cells, as determined by improved visual function. In one embodiment, the donor cells can incorporate into the retina and replace photoreceptors (which may be in limited numbers) through cell differentiation. The overall effect is to rapidly and persistently restore and maintain a clinically significant degree of visual function in a retina that would otherwise be heading for complete failure leaving the patient completely blind. Thus, in one embodiment, the compositions and methods of the present invention can rapidly and persistently restore and maintain a clinically significant degree of visual function in a mammalian, e.g., human, retina.

[0080] In an alternative embodiment, the method of the present invention includes making and using a dissociated suspension of fetal retinal cells, e.g., human retinal progenitor cells (hRPCs), optionally without tissue or scaffold. In an alternative embodiment, the cells are injected into the vitreous cavity, optionally without the need for vitrectomy or subretinal surgery. In one embodiment, the cells can be effectively implanted (e.g., injected) into the subretinal space, or they can be effectively implanted (e.g., injected) into the eye using any standard intraocular injection procedure, e.g., using a subcutaneous or oblique insertion route. In an alternative embodiment, neither retinotomy nor intraocular gas nor silicone oil is required.

[0081] In alternative embodiments, anterior chamber paracentesis may or may not be performed depending on the circumstances, as determined by one of skill in the art. In alternative embodiments, the eye does not need to be sutured during and / or after the procedure. In alternative embodiments, only topical anesthesia is used, e.g., no local, regional, or general anesthesia is used.

[0082] In an alternative embodiment, anti-inflammatory drugs and / or immunosuppression are not used, but anti-inflammatory and / or immunosuppressant therapy as post-operative drops can be included as needed. In an alternative embodiment, tissue typing of the graft and patient matching is not required.

[0083] In an alternative embodiment, there is no need for mandatory post-operative bed rest and / or "prone" positioning. In an alternative embodiment, the methods of the present invention are performed as an outpatient procedure, optionally without any overnight hospitalization.

[0084] In alternative embodiments, the compositions and methods of the present invention are used to prevent, alleviate and / or treat retinitis pigmentosa (RP), Usher's disease, or any retinal degenerative disease, such as AMD, or retinal photoreceptor diseases such as retinal detachment, or retinal diseases such as diabetic retinopathy, or ganglion cell / optic nerve diseases such as glaucoma or optic neuropathy.

[0085] In alternative embodiments, the use or practice of the compositions and methods of the invention provide, over time, sometimes rapid effects to improve photopic (daytime) vision, increase best corrected visual acuity, improve macular function resulting in the possibility of maintaining or regaining central fixation, improve visual fields, and improve scotopic (nighttime) vision; where hearing loss is concomitant in Usher syndrome, increased sensitivity to sound (improvement) has been reported, and / or for patients with LP vision (light only), where improvement in the treated eye is limited, vision in the contralateral eye is significantly improved.

[0086] In alternative embodiments, the use or practice of the compositions and methods of the present invention results in a variety of systemic benefits, such as changes in the appearance of the treated individual, improved sense of visual ability, improved independent walking, improved sense of health, and / or improved activities of daily living, likely due to physical improvements that may be related to the effects of light on circadian rhythms, pituitary function, hormone release, vascular tone, etc.

[0087] In alternative embodiments, the use or practice of the compositions and methods of the present invention does not result in unwanted cell proliferation, e.g., tumors, does not result in infection, e.g., endopthalmitis, which is a risk to any intraocular medical treatment, does not transmit disease (although prion or mad cow disease can be difficult to rule out), does not result in uveitis and / or acute graft rejection, does not increase intraocular pressure, does not result in angle closure, does not result in low pressure, does not result in retinal detachment and / or neovascularization.

[0088] In an alternative embodiment, the compositions and methods of the present invention use retinal progenitor cell cultures that include or consist of heterogeneous cultures of immature retinal cells obtained from fetal mammalian retina, optionally not clonally selected, but optionally mixed. In an alternative embodiment, the cells express progenitor cell markers and retinal markers. In an alternative embodiment, the cells are grown under xeno-free conditions for clinical use, since xeno-contamination can be a safety issue. In an alternative embodiment, the cells are grown under completely serum-free conditions, and can be grown under serum-containing conditions if desired.

[0089] In alternative embodiments, the cells are not immortal, are not allowed to become immortal, or are not forced to become immortal. In alternative embodiments, the cells do not grow indefinitely. Exemplary cell culture methods of the present invention can improve the speed and duration, and can significantly improve donor cell yields for a given tissue donation.

[0090] In alternative embodiments, the cells are not stem cells per se, but rather are immature and / or plastic, and optionally do not meet the definition of a true stem cell.

[0091] In alternative embodiments, the cells used to practice the invention are obtained from mammalian fetal tissue and do not optionally persist for the life of the organism. In alternative embodiments, the cells used to practice the invention are incapable of generating one germ layer (without further manipulation), or incapable of generating all three (3) germ layers (without further manipulation), and, optionally, they are pre-characterized for generating retinal tissue or cells.

[0092] In an alternative embodiment, the cells used to carry out the present invention are a population of closely related cells, not an isolated single cell type. In an alternative embodiment, the cells used to carry out the present invention are not pluripotent, but can appear to be multipotent if necessary. In an alternative embodiment, the cells used to carry out the present invention have never been cultured in a pluripotent state, and therefore they are safer. In an alternative embodiment, the cells used to carry out the present invention are not genetically modified, or instead they are genetically modified (e.g., stably, or transiently, or inducibly transformed).

[0093] In alternative embodiments, the compositions and methods of the present invention provide a clinically significant trophic effect on diseased retina or a regenerative effect on macular and / or scotopic visual function.

[0094] In alternative embodiments, the cells used to practice the invention have low immunogenicity when placed in the eye, or in the vitreous or subretinal space of the eye, for example, as an allograft.

[0095] In an alternative embodiment, the cells used to practice the invention are retinal progenitor cells (RPCs) that are differentiated from neural progenitor cells and / or neural stem cells (NSCs). In an alternative embodiment, the mammalian fetal retinal cells or RPCs used to practice the invention are multipotent but are not equivalent to NSCs. In an alternative embodiment, the mammalian fetal retinal cells or RPCs used to practice the invention are not derived from the brain but from the retina. In an alternative embodiment, the mammalian fetal retinal cells or RPCs used to practice the invention generate photoreceptors (brain-derived progenitor cells are inferior in this respect). In an alternative embodiment, the mammalian fetal retinal cells or RPCs used to practice the invention are multipotent but do not generate oligodendrocytes (without additional manipulation) (unlike NSCs).

[0096] In alternative embodiments, the mammalian fetal retinal cells or RPC cells used to practice the invention express quantitatively distinct gene profiles, e.g., as described herein, or they express quantitatively distinct soluble factor profiles, or they express quantitatively distinct surface marker profiles.

[0097] In alternative embodiments, the mammalian fetal retinal or RPC cells used to practice the invention are derived from the mammalian fetal neural retina and are not derived from the ciliary margin, ciliary epithelium, or RPE. In alternative embodiments, the mammalian fetal retinal or RPC cells used to practice the invention are not the progeny of differentiated Müller glia, are not themselves postmitotic precursors, are not themselves stem cells, and / or are not themselves a single isolated cell type.

[0098] In alternative embodiments, the mammalian fetal retinal cells or RPC cells used to practice the present invention have gene profiles that are not fixed, constant or invariant, or that change quantitatively and dynamically over time in culture.

[0099] In an alternative embodiment, the mammalian fetal retinal cells or RPC cells used to practice the invention are not found in early embryos, e.g., blastocysts. In an alternative embodiment, the mammalian fetal retinal cells or RPC cells used to practice the invention are not found in any useful abundance in normal mature mammals, e.g., humans. In an alternative embodiment, the mammalian fetal retinal cells or RPC cells used to practice the invention are found in their natural abundance in the developing (fetal) mammalian, e.g., human, retina. In an alternative embodiment, the mammalian fetal retinal cells or RPC cells used to practice the invention are not normally present in or derived from bone marrow.

[0100] In an alternative embodiment, the mammalian fetal retinal or RPC cells used to practice the invention are mostly mitotic, optionally with a minor mixture of post-mitotic cells, when grown under growth conditions. In an alternative embodiment, the mammalian fetal retinal or RPC cells used to practice the invention are artificially derived from a pluripotent cell line, but optionally do not contain a residual population of pluripotent cell types.

[0101] In an alternative embodiment, the mammalian fetal retinal cells or RPC cells used to practice the invention generate (differentiate into) retinal cells, including photoreceptors, but do not generate oligodendrocytes.

[0102] In an alternative embodiment, the mammalian fetal retinal cells or RPC cells used to practice the invention are immunologically tolerated as ocular allografts in an unrelated mammal, such as a human. In an alternative embodiment, the mammalian fetal retinal cells or RPC cells used to practice the invention are transplanted into the vitreous cavity for therapeutic or prophylactic therapy of mammalian or human visual or retinal diseases.

[0103] In an alternative embodiment, the mammalian fetal retinal cells or RPC cells used in practicing the present invention are free of any or substantial risk of tumor formation or other unwanted cell proliferation.

[0104] In alternative embodiments, the mammalian fetal retinal cells or RPC cells used to carry out the present invention are cultured as spheres or adherent monolayers, or as spheres and then monolayers, or as a combination of spheres and monolayers.In alternative embodiments, spheres are not required, or the cells are transplanted not as spheres, but as dissociated cells, or as a mixture of both.In alternative embodiments, the mammalian fetal retinal cells or RPC cells used to carry out the present invention include transplanted cells that can coalesce in the vitreous and become spheres as needed.

[0105] The present invention is not limited by any particular mechanism of action, but exemplary mechanisms of action are diffusive and / or trophic. Evidence is consistent with the concept of trophic reprogramming of dying host cones, switching from an apoptotic trajectory to regenerating light processing capacity. This may be direct or indirect. The involvement of other ocular tissues is not excluded. This mechanism allows for the placement of a heterogeneous mixture of fetal neural retinal cells of the present invention in the vitreous or subretinal space. In one aspect, placement in the vitreous enhances the diffusion-based therapeutic effect. This mechanism can allow for the placement of a heterogeneous mixture of fetal neural retinal cells of the present invention out of the line of sight, yet still treat the patient's macula.

[0106] In an alternative embodiment, vitreous placement is used to greatly simplify the procedure. This exemplary procedure can increase availability to needy patients worldwide, and vitreous placement can aid in immune tolerance by being away from the vasculature. The present invention is not limited by any particular mechanism of action, but in a relatively cell-free space with few native antigen-presenting cells, the exemplary vitreous placement of the present invention avoids the potential complications of subretinal surgery, avoids the risks of general anesthesia, does not require making a hole in the retina (retinotomy) (which increases the risk of retinal detachment, bleeding), and / or does not require performing a local detachment on the patient's retina (local detachment can result in tears, bleeding, total detachment, and in RP, detachment of the pathological retina is a difficult / dangerous procedure).

[0107] In alternative embodiments, visual benefits are rapid and may occur within the first week after treatment, hi alternative embodiments, incremental benefits occur over a longer period of time.

[0108] In alternative embodiments, retinal cell replacement is possible but not required for clinical efficacy, donor cell transfer to the retina is possible but not required for clinical efficacy, donor cell integration into the retinal circuitry is possible but not required for efficacy, donor cell integration into the outer nuclear layer / macula of the host retina is possible but not required for efficacy, donor cell integration into the retina is possible but not required for efficacy, donor cell integration into the retina is possible but not required for sustained graft survival.

[0109] In an alternative embodiment, the donor cells are cultured without antibiotics, which avoids cellular alteration and allows the use of very low passage cells, since the absence of antibiotics eliminates subclinical microbial contamination. In an alternative embodiment, the use of low passage cells reduces the risk of transformation and / or tumor formation, and the use of low passage cells is closest to the natural cells present in the developing retina.

[0110] In an alternative embodiment, a DMEM / F12 based medium or equivalent is preferred for the growth of human RPCs and an Ultraculture based medium or equivalent is preferred for the growth of feline progenitor cells.

[0111] The subject matter disclosed herein relates to a cell population comprising mammalian retinal progenitor cells isolated according to a defined cell culture method and expressing characteristic markers. The cell population can be a culture of cells isolated from a mammal and grown in vitro. For example, the culture can include a suspension of cells or adherent cells cultured in a culture plate, dish, flask, or bioreactor. The sample can be homogenous or heterogeneous, which can be determined by the expression of one or more markers as defined herein. In some embodiments, the cell population disclosed herein is a mixed cell population and can contain a mixture of undifferentiated and differentiated cells. The relative expression levels of markers characteristic of retinal progenitor cells as defined herein can vary between cells within the population.

[0112] In alternative embodiments, the terms "purified" or "enriched" indicate that a cell or cell population has been removed from its normal tissue environment and is present in a higher concentration compared to the normal tissue environment. Thus, a "purified" or "enriched" cell population can include further cell types in addition to retinal progenitor cells and can include additional tissue components, and the terms "purified" or "enriched" do not necessarily indicate the presence of only progenitor cells or exclude the presence of other cell types.

[0113] In alternative embodiments, the retinal progenitor cell populations disclosed herein can be at least 5% pure, at least 10% pure, at least 15% pure, at least 20% pure, at least 25% pure, at least 30% pure, at least 35% pure, at least 40% pure, at least 45% pure, at least 50% pure, at least 55% pure, at least 60% pure, at least 65% pure, at least 70% pure, at least 75% pure, at least 80% pure, at least 85% pure, at least 90% pure, at least 95% pure, at least 96% pure, at least 97% pure, at least 98% pure, at least 99% pure, or any increment between 5% and 99% pure.

[0114] In alternative embodiments, "marker" refers to any molecule that can be observed or detected. For example, markers can include, but are not limited to, nucleic acids such as transcripts of specific genes, polypeptide products of genes, non-gene product polypeptides, glycoproteins, carbohydrates, glycolipids, lipids, lipoproteins, or small molecules (e.g., molecules with molecular weights of less than 10,000 Daltons). In alternative embodiments, retinal progenitor cells can be characterized by the presence of one or more markers that can be expressed on the surface of cells in the cell population ("cell surface markers"), inside cells in the cell population (i.e., in the nucleus or cytoplasm of cells), and / or at the RNA or protein level as "genetic" markers.

[0115] In an alternative embodiment, the terms "express" and "expression" as used herein refer to the transcription and / or translation of a nucleic acid sequence in a host cell. The expression level of a desired product / protein of interest in a host cell can be determined or "screened" based on either the amount of corresponding mRNA present in the cell, or the amount of the desired polypeptide / protein of interest encoded by the selected sequence, as in this example. For example, the mRNA transcribed from the selected sequence can be quantified or detected by Northern blot hybridization, ribonuclease RNA protection, in situ hybridization to cellular RNA, microarray analysis, or by reverse transcription polymerase chain reaction (RT-PCR). The protein encoded by the selected sequence can be detected or quantified by a variety of antibody-based methods, e.g., by ELISA, by Western blotting, by radioimmunoassay, by immunoprecipitation, by assaying the biological activity of the protein, by immunostaining the protein (including, e.g., immunohistochemistry and immunocytochemistry), by flow cytometry or fluorescence activated cell sorting ("FACS") analysis, or by homogeneous time-resolved fluorescence (HTRF) assays.

[0116] Retinal progenitor cells can be characterized by their expression of molecular markers, including cell surface markers and non-surface ("genetic") markers. Although it is common in the art to say that cells are "positive" or "negative" for a particular marker, the actual expression level is determined quantitatively. The number of molecules on the cell surface (or located elsewhere) can vary by several logs, but still be characterized as "positive". It is also understood by those skilled in the art that cells that are negative for staining, i.e., whose binding level of a marker-specific reagent is not detectably different from a control, e.g., an isotype-matched control, may express minor amounts of the marker. Characterizing the level of labeling ("staining") allows fine distinction between cell populations. The staining intensity of cells can be monitored by flow cytometry, where a laser detects a fluorescent dye (which is proportional to the amount of cell surface marker bound by a specific reagent, e.g., an antibody) at a quantitative level. Flow cytometry, or FACS, can also be used to separate cell populations based on the strength of binding to specific reagents, as well as other parameters, such as cell size and light scattering. Absolute levels of staining may vary with particular fluorochromes and reagent preparations, but data can be normalized to controls.

[0117] To normalize the distribution to a control, each cell is recorded as a data point with a particular staining intensity. These data points can be displayed by a logarithmic scale, where the unit of measurement is the arbitrary staining intensity. As an example, the most brightly stained cells in a sample can be as much as 4 logs more intense than unstained cells. When displayed in this way, cells that fall within the highest logs of staining intensity are bright, while cells within the lowest are negative. "Low" positively stained cells have a staining level above the brightness of the isotype-matched control, but are not as intense as the most brightly staining cells typically found in a population. Low positive cells may have unique properties that are different from the negative and brightly stained positive cells of the sample. Alternative controls can utilize substrates, such as manufactured beads or cell lines, that have a defined density of markers on their surface, resulting in an intense positive control.

[0118] Marker expression may be variable during the culture of retinal tissue from which retinal progenitor cells and cell populations are obtained.For example, differences in marker expression may be affected by the culture conditions of oxygen level (i.e., atmospheric oxygen conditions, or "normoxic" conditions, or low oxygen conditions, also known as "hypoxic" conditions).Those skilled in the art will recognize that marker expression of retinal progenitor cells and cell populations is not static, but may vary as a function of one or more culture conditions, i.e., medium, oxygen level, number of passages, time of culture, etc.

[0119] Retinal progenitor cells and cell populations can express 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 25 or more, 30 or more, or any increment therebetween up to 50 or more of the markers defined herein.

[0120] In alternative embodiments, retinal progenitor cells and cell populations comprising retinal progenitor cells are characterized or screened by expression of one or more markers, such as nestin, vimentin, Sox2, Ki67, MHC class I, Fas / CD95, MAP2, CD9, CD81, AQP4, CXCR4, CD15 / LeX / SSEA1, GD2 ganglioside, CD133, β3-tubulin, GFAP, OPN / SPP1, PTN, KDR, and TEK. In certain embodiments, the retinal progenitor cells and cell populations express one or more markers selected from the group consisting of nestin, Sox2, Ki-67, MHC class I, and Fas / CD95, where nestin is expressed by greater than 90% or between 95-99% of the cells in the population, Sox2 is expressed by greater than 80% or between 90-99% of the cells in the population, Ki-67 is expressed by greater than 30% or between 40-60% of the cells in the population (i.e., 60-85% of cells grown under normoxic / atmospheric oxygen conditions, 80-90% of cells grown under hypoxic conditions), MHC class I is expressed by greater than 70% or between 90% of the cells in the population, and Fas / CD95 is expressed by greater than 30% or between 40-70% of the cells in the population. In some embodiments, the retinal progenitor cells and cell populations further express one or more markers selected from the group consisting of vimentin, CD9, CD81, AQP4, CXCR4, CD15 / LexA / SSEA1, GD2 ganglioside, CD133, β3-tubulin, MAP2, GFAP, OPN / SPP1, PTN, KDR, and TEK. GFAP may be expressed by 5-10% of the cells in the population. MHC class II may be expressed by 1-3% of the cells in the population. CXCR4 may be expressed by 5-30% of the cells grown under atmospheric oxygen conditions, but 90% of the cells grown under hypoxic conditions. CD15 may be expressed by 4-35% of the cells in the population, i.e., 4-8% of the cells grown under atmospheric oxygen conditions and 15-35% of the cells grown under hypoxic conditions. GD2 can be expressed by 2-15% of cells in a population, i.e., 2-4% of cells grown under atmospheric oxygen conditions and 15% of cells grown under hypoxic conditions.

[0121] In alternative embodiments, the retinal progenitor cells and cell populations are selected from the group consisting of ABCA4, AIPL1, AKT3, APC2, BSN, CCNG2, CDHR1, CRX, CD24, claudin 11, CNTF, CNTFR, DACH1, DAPL1, DCX, DLG2 and 4, DLL4, EPHA7, EYS, FLT1, FSTL5, FZD5, FGF9, 10, and 14, GADD45G, GRIA2, HES5 and 6, HEY2, HEYL, HGF, HIF3A, IMPG1 and 2, JAK2 and 3, KLF4, MAP6, myelin basic protein (MBP), MYCN, Nanog, NBL1, NEFL, NEFM, NEUROD1, NEUROD2, NEUROD3, NEUROD4, NEUROD5, NEUROD6, NEUROD7, NEUROD8, NEUROD9, NEUROD10, NEUROD11, NEUROD12, NEUROD13, NEUROD14, NEUROD15, NEUROD16, NEUROD17, NEUROD18, NEUROD19, NEUROD20, NEUROD21, NEUROD22, NEUROD23, NEUROD24, NEUROD25, NEUROD26, NEUROD27, NEUROD28, NEUROD29, NEUROD30, NEUROD31, NEUROD32, NEUROD33, NEUROD34, NEUROD35, NEUROD36, NEUROD37, NEUROD38, NEUROD39, NEUROD40, NEUROD41, NEUROD42, NEUROD4 The markers are characterized or screened for low, low, negative, or reduced expression of one or more of OD4, NEUROG1, NEUROG2, NOTCH1, 2, and 3, NRL, NRCAM, NRSN, NRXN1, 2, and 3, OCT4, OLIG2, OPN1MW1 and 2, OPN1SW, OTX2, PAR4, PAX6, PRPH2, RAX1 and 2, RBP3, RCVRN, RELN, RGR, rhodopsin, RICTOR, RP1, RRH, RXRG, SIX3 and 6, SOX8, SLC25A27, STAT1, STAT3, SYP, SYT4, WIF1, VSX2, and VSX1 and 2. The expression patterns of these markers can be used to distinguish retinal progenitor cells or cell populations derived from tissue of origin, for example, freshly isolated retinal tissue.

[0122] In alternative embodiments, other markers whose expression may be increased relative to the tissue of origin include, but are not limited to, ADM, ANGPT1, ANGPTL2 and 4, ATP5D, BHLHE40, CCL2, CCNB1, CCND2, and CCNDE1, CD44, CDKN2A, claudins 1, 4, and 6, CPA4, CTGF, CXCL12, DKK2, EMP1, FOXC2, FZD6, GADD45B, HES1, HIF1A, HOXB4, IGF1, IGFBP3, 5, and 7, IL1A, IL1R, IL1RAP, IL4R, IL7R, IL11, IL18, , JAG1, LIF, LOX, BDNF, EGF, EGFR, FGF1, 2, 5, and 9, KLF4, 5, and 12, MITF, MMP1, MYC, NCAN, NEFH, NOG, NTF3, NTRK2, NRP1 and 2, OSMR (IL31RA), OTX1, PAX8, PDGFA, B, and C, PLAU, PRRX1, RPE65, SDF-1, SFRP1 and 4, SIX1 and 4, SLC25A1, 19, and 20, TEK / TIE1, THBS1 and 2, TLR4, VEGFA, VEGFC, WNT5A, and WNT7B.

[0123] In alternative embodiments, retinal progenitor cells and cell populations are distinguished from other central nervous system progenitor cell types, such as brain progenitor cells and neural stem cells, as well as others that may be derived from fetal CNS tissues, such as the brain and spinal cord. Markers that may be increased relative to other CNS progenitor cells include, without limitation, ARR3 (arrestin C), CDF10, CDKN2A, CTGF, CXCL12 / SDF1, BHLHE41, BMP2, DKK1, EGFR, EPHB2, FN1, FOSL1 and 2, FOXD1, GABBR1, GAS1 and 6, GBX2, HHEX, HOXB2, IGFBP5 and 7, INHBA, JAG1, KDR, KLF10LHX9, LHX2, LIF, MET, NEUROD1, NTF3, NTRK2, OPTN (optineurin), RCVRN, SAG (S-arrestin), SERPINF1 (PEDF), SFRP1, SOX3, TBX3, TGFB, WIF, WNT5A, and WNT5B. Markers that may be decreased relative to other CNS progenitor cells include, without limitation, AQP4, ASLL1, CLDN11, CDKN1B, CCL2, CCNG2, CXCR4 (SDF1 receptor), DCX, DLX2 and 5, EMX2, EPHA3, 4, and 7, FABP7, FOXG1, GRIA1, 2, and 3, HGF, IL2, KLF4, LIFR, MNX1, NGF, NKX2-2, NOTCH1, NPY, NPY2R, OLIG2, OMG, PBX1, PDGFRA, RTN1, SCGN, SOX11, TFAP2B, TNFRSF21, and WNT7A.

[0124] In alternative embodiments, the cell populations can be harvested from healthy subjects (i.e., individuals not having retinal disease), diseased subjects, and can include fresh as well as frozen retinal cell populations. Sources include, but are not limited to, whole eye, or retinal tissue, or other sources obtained from embryonic, fetal, pediatric, or adult tissue. The method can include steps for cell isolation by further enrichment or purification procedures, or positive selection for other retinal progenitor cell specific markers. Retinal progenitor cells and cell populations can be obtained or harvested from any mammalian species or subject, such as humans, primates, horses, cows, pigs, dogs, cats, ferrets, rabbits, rodents, such as mice, rats, hamsters, etc.

[0125] During vertebrate embryonic development, the retina and optic nerve arise as outgrowths of the developing brain, and thus the retina is considered part of the central nervous system (CNS) and is in fact brain tissue. The retina is a layered structure with several layers of nerve cells interconnected by synapses. From closest to furthest from the vitreous, i.e., from closest to the front lateral side of the head toward the inside and back of the head, the retinal layers include the inner limiting membrane, which is made up of the Muller cell footplate, the nerve fiber layer, which contains the axons of the ganglion cell nuclei, the ganglion cell layer, which contains the nuclei of the ganglion cells whose axons become the optic nerve fibers, the inner plexiform layer, which contains the synapses between the bipolar cell axons and the dendrites of the ganglion cells and amacrine cells, the inner nuclear layer, which contains the nuclei and perikarya of the bipolar cells, the outer plexiform layer, which contains the projections of rods and cones that terminate in the rod globules and cone pedicles, respectively, the outer nuclear layer, which contains the cell bodies of the rods and cones, the outer limiting membrane, which separates the inner segment portions of the photoreceptors from their nuclei, the photoreceptor layer, and the retinal pigment epithelium (RPE), which is a single layer of cuboidal cells. The nerve cells that are directly sensitive to light are photoreceptor cells, which are primarily composed of two types: rods and cones. Rods function primarily in dim light and provide black-and-white vision, while cones support daytime vision and color perception. A third type of photoreceptor, the photosensitive ganglion cells, are important for reflex responses to bright daylight.

[0126] In some embodiments, the cells are harvested from a mammalian fetal retina at a stage after the retina is formed, but before the photoreceptor outer segments are fully formed throughout the retina, and before retinal vascularization is substantially complete or complete. This stage is generally between about 12 weeks and about 28 weeks of fetal gestation in human fetuses. For non-human cells derived from larger mammals, such as feline or porcine retinal progenitor cells, this stage is generally between about 3 weeks and about 11 weeks of fetal gestation. See, for example, Anand-Apte, B. and Hollyfield, JG, "Developmental Anatomy of the Retinal and Choroidal Vasculature." in The Retina and Its Disorders, Besharse, J. and Bok, D., Academic Press, (2001). However, the subject matter disclosed herein also includes the harvesting of cells from postnatal or neonatal mammalian tissues.

[0127] In an alternative embodiment, the retinal progenitor cells are purified from other tissue components after or simultaneously with the processing of the tissue sample. In one embodiment, the progenitor cells are purified from other cells and tissue components after the tissue sample is cultured under conditions suitable for cell growth and for a time sufficient to allow the cells to adhere to the culture dish. In certain embodiments, the purification of cells includes obtaining cells that migrate from the tissue sample during culture and are present in the culture medium or loosely attached to fibronectin or other substrate or feeder cell layer. These cells can be obtained by conventional methods, such as removing the culture medium and centrifuging to pellet the cells therein, and washing the cells remaining in the culture dish with a solution such as phosphate buffered saline (PBS) or Hank's balanced salt solution to remove the loosely attached cells as an adherent cell layer. This washing solution can then also be centrifuged to obtain the cells. In some embodiments, the purification of retinal progenitor cells and cell populations may further include separating the cells from certain insoluble tissue components, including residual tissue materials such as lipids. Cells can be separated from other tissue components by any means known and available in the art, including, for example, using density gradient, centrifugation, flow cytometry or magnetic cell sorting (MACS), and filtration, or a combination thereof.Examples of specific methods for purifying cells are known and described in the art, for example, in U.S. Patent No. 6,777,231.In certain embodiments, negative separation methods are used to remove one or more specific types of cells.

[0128] In certain embodiments, tissue is processed or "dissociated". Dissociation can be performed by physical dissociation and / or exposure to an enzyme preparation that promotes the release of cells from other tissue components to create a "dissociated suspension" of cells and / or cell groups. Examples of such enzymes include matrix metalloproteinases, clostripain, papain, trypsin, trypsin-like, pepsin, pepsin-like, neutral protease types, and collagenase. Suitable proteolytic enzymes are described in U.S. Patent Nos. 5,079,160, 6,589,728, 5,422,261, 5,424,208, and 5,322,790. In some embodiments, the enzyme preparation includes trypsin alone or in combination with one or more additional enzymes. Enzymatic dissociation can be performed in conjunction with physical dissociation, e.g., by mincing, pipetting, chopping, homogenization, grinding, freeze-thawing, osmotic shock, to remove unwanted cells or connective tissue, and can ultimately result in single cell cultures or contain cell clusters that can be defined by size, i.e., "small," "medium," and "large." Cell cluster size is subjective and can vary in the practice of the subject matter disclosed herein.

[0129] Cell culture describes the process by which cells are grown under controlled conditions, generally outside their natural environment. In alternative embodiments, cell populations are grown or cultured in any cell culture medium known in the art. In alternative embodiments, the "basal medium" used in the present invention refers to any medium capable of supporting cell growth. The basal medium provides standard inorganic salts, such as zinc, iron, magnesium, calcium, and potassium, vitamins, glucose, a buffer system, and important amino acids. Basal media that can be used in the present invention include, but are not limited to, Minimum Essential Medium Eagle, ADC-1, LPM (without bovine serum albumin), F10 (Ham), F12 (Ham), DCCM1, DCCM2, RPMI1640, BGJ medium (with and without Fitton-Jackson modification), Basal Medium Eagle (Earle's salt base), and other media. BME supplemented with Earle's salts base), Dulbecco's modified Eagle's medium (DMEM without serum), Yamane, IMEM-20, Glasgow modified Eagle's medium (GMEM), Leibovitz's L-15 medium, McCoy's 5A medium, medium M199 (M199E with Earle's salts base), medium M199 (M199H with Hank's salts base), minimum essential medium Eagle (MEM-E with Earle's salts base), minimum essential medium Eagle (MEM-H with Hank's salts base), and minimal essential medium Eagle (MEM-H with Hank's salts base). Examples include Minimal Essential Medium Eagle (MEM-NAA with non-essential amino acids), and among many others, Medium 199, CMRL1415, CMRL1969, CMRL1066, NCTC135, MB75261, MAB8713, DM145, Williams'G, Neuman&Tytell, Higuchi, MCDB301, MCDB202, MCDB501, MCDB401, MCDB411, MDBC153, and Ultraculture. Media suitable for use in culturing the retinal progenitor cells disclosed herein are Advanced DMEM / F12 and Ultraculture. Some of these media are summarized in Methods in Enzymology, Vol. LVIII, "Cell Culture", pp. 62-72.

[0130] In alternative embodiments, "conditioned medium" refers to a medium that has been altered compared to a basal or base medium. For example, conditioning of a medium can result in the addition or removal of molecules such as nutrients and / or growth factors from the original levels found in the basal medium. In some embodiments, a medium is conditioned by allowing a certain type of cell to grow or be maintained in the medium under certain conditions for a certain period of time. For example, a medium can be conditioned by allowing retinal progenitor cells to grow, differentiate, or be maintained in a medium of a defined composition at a defined temperature for a defined number of hours. As will be appreciated by those skilled in the art, numerous combinations of cells, medium types, durations, and environmental conditions can be used to generate an almost infinite array of conditioned media.

[0131] Examples of cell culture supplements or additives include, without limitation, components to partially or completely replace the role of serum in supporting cell survival or growth. In alternative embodiments, this includes insulin, transmetalloproteins, trace elements, vitamins, or other factors that are generally not included in basal media, but are provided by serum commonly used in the culture of cells. The supplement or additive may include at least one or more of the following components that support cell growth: one or more insulins or substitutes thereof, one or more transmetalloproteins or substitutes thereof, one or more trace elements (e.g., selenium, iron, zinc, copper, cobalt, chromium, iodine, fluoride, manganese, molybdenum, vanadium, nickel, tin), one or more vitamins (e.g., vitamin C, vitamin E, vitamin A, B-complex vitamins), one or more salts (e.g., sodium salts, magnesium salts, calcium salts, or phosphate salts), one or more buffers (e.g., phosphate buffered saline, HEPES buffer), one or more amino acids (e.g., L-glutamine), one or more riboflavins (e.g., riboflavin ... The serum replacement additives include a plurality of hormones, hormone-like compounds or growth factors (e.g., transferrin, EGF, NGF, ECGF, PDGF, FGF, IGF, LIF, interleukins, interferons, TGF, and / or VEGF, glucagon, corticosteroids, vasopressin, prostaglandins, etc.), serum albumin or substitutes thereof, one or more carbohydrates (glucose, galactose, fructose, mannose, ribose, glycolytic metabolites), one or more antibiotics and / or antimycotics (e.g., penicillin, streptomycin, fungizone), and one or more lipids (e.g., free and protein-bound fatty acids, triglycerides, phospholipids, cholesterol, ethanolamine). Many commercialized serum replacement additives, such as KnockOut serum replacement (KOSR), N2, B27, StemPro, insulin-transferrin-selenium supplement (ITS), and G5, are well known to those skilled in the art and are readily available.These additives are characterized by clearly defined components, so that the concentration of the component can be determined based on its ratio in the medium.

[0132] In alternative embodiments, cultures of mammalian retinal progenitor cells can be generated in medium containing low serum or in medium containing no serum. Examples of serum include fetal bovine serum, calf serum, newborn calf serum, goat serum, horse serum, human serum, rabbit serum, rat serum, mouse serum, among others. Under certain culture conditions, serum concentrations can range from about 0.05% v / v to about 20% v / v. For example, in some differentiation processes, serum concentrations in the medium can be less than about 0.05% (v / v), less than about 0.1% (v / v), less than about 0.2% (v / v), less than about 0.3% (v / v), less than about 0.4% (v / v), less than about 0.5% (v / v), less than about 0.6% (v / v), less than about 0.7% (v / v), less than about 0.8% (v / v), less than about 0.9% (v / v), or less than about 0.1% (v / v). The serum concentration of retinal progenitor cells may be less than about 1% (v / v), less than about 2% (v / v), less than about 3% (v / v), less than about 4% (v / v), less than about 5% (v / v), less than about 6% (v / v), less than about 7% (v / v), less than about 8% (v / v), less than about 9% (v / v), less than about 10% (v / v), less than about 15% (v / v), or less than about 20% (v / v). In some embodiments, retinal progenitor cells and cell populations comprising retinal progenitor cells are grown without serum ("serum-free"), without serum replacements, and / or without any supplements.

[0133] In some embodiments, the retinal progenitor cells or cell populations comprising retinal progenitor cells are cultured under "xeno-free" conditions. "Xeno-free" or "xenogen-free" refers to conditions in which cells of a particular species (e.g., human cells) are grown or cultured in the absence of products derived from other species, but only in the presence of human products or supplements (e.g., human serum albumin, human serum). This is particularly important for cells used for transplantation into humans. Cells exposed to various undefined animal-derived products make them undesirable for clinical use due to increased risk of graft rejection, immune responses, and viral or bacterial infections, prions, and as yet unidentified zoonotic diseases. Additionally, for all mammalian use, including human or non-human mammalian use (e.g., livestock use), the cells are screened for normal karyotype or the presence of infection or contamination, e.g., by mycoplasma, gram-negative bacteria (e.g., endotoxin testing), fungi, and the like. Cells can also be screened for tumorigenicity or transformation to a cancer phenotype by telomerase activity assays, hTERT gene expression, and growth in soft agar or tumor formation in nude mice. Such assays are known in the art and well within the purview of the skilled artisan.

[0134] In alternative embodiments, retinal progenitor cells or cell populations containing retinal progenitor cells can be cultured on a feeder cell layer (e.g., embryonic or adult fibroblasts) or in the presence of an extracellular matrix scaffold or substrate, such as collagen, entactin, heparin sulfate proteoglycan, fibronectin, laminin, gelatin, or matrigel. For example, PURECOL® collagen is known as the standard of all collagens in terms of purity (>99.9% collagen content), functionality, and the most natural-like collagen available. PURECOL® collagen is approximately 97% type I collagen with the remainder composed of type III collagen, making it ideal for coating surfaces and preparing thin layers to culture cells or use as a solid gel. Another example of a scaffold or substrate known in the art is CELLstart (Invitrogen).

[0135] In alternative embodiments, cell culture conditions can include growing cells in an incubator set at 37° C. with 5% CO2. Retinal progenitor cells or cell populations comprising retinal progenitor cells can be cultured under normoxia or ambient air (20%) and grown under conditions that approximate the oxygen levels of the developing fetal retina during pregnancy, i.e., "low" or "hypoxic" conditions, e.g., 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, or 7% oxygen, or any increment therebetween.

[0136] Plating density is the number of cells per volume of medium, or per cm in adherent cultures. 2Confluency refers to the number of cells per cell. A similar term in this context is "confluence", which is commonly used as a measure of the number of cells in a cell culture dish or flask, and refers to the coverage of the dish or flask by the cells. For example, 100 percent confluency means that the dish is completely covered by cells, and therefore there is no room for the cells to grow, while 50 percent confluency means that roughly half of the dish is covered, and there is still room for the cells to grow.

[0137] Passaging (also known as subculturing or splitting cells) involves the transfer of a small number of cells into a new container. In an alternative embodiment, cells are cultured for longer periods of time, as if they are split periodically, this avoids the senescence associated with long-term high cell density. Suspension cultures are easily passaged with small amounts of culture containing fewer cells diluted in a larger volume of fresh medium. For adherent cultures, cells first need to be detached. This is commonly done with a mixture of enzymes such as trypsin-EDTA, or a non-enzymatic solution such as Cell Dissociation Buffer, although a variety of enzymatic or non-enzymatic mixtures or preparations are available for this purpose. A small number of detached cells can then be used to seed a new culture.

[0138] Most early cell cultures have a limited life span and do not proliferate indefinitely. After a certain number of population doublings (called the Hayflick limit), the cells undergo a process of senescence and stop dividing, while generally retaining viability. In alternative embodiments, the retinal progenitor cells and cell populations can be cultured for 10 passages or less, e.g., passaged for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 passages. In alternative embodiments, the cells can be passaged more than 10 times, e.g., passages 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more. In certain embodiments, the retinal progenitor cells and cell populations are cultured for about 10-30 passages. In alternative embodiments, the retinal progenitor cells and cell populations comprising them are preferably not immortal, and are not allowed to immortalize or forced to immortalize. Although the cells described herein are considered to be immature progenitor cells, cell populations including retinal progenitor cells may contain cells that can themselves be considered to be "multipotent" stem cells, where such cells can generally differentiate under certain culture conditions to become retinal tissues or cells, including retinal progenitor cells. In alternative embodiments, "multipotent" refers to immature, undifferentiated and / or unspecialized cells that have the ability to self-renew but have limited ability to differentiate and are intrinsically committed to generate a specific cell type. In certain embodiments, the cells and cell populations described herein include closely related cells, but do not necessarily represent a single cell type.

[0139] In an alternative embodiment, the retinal progenitor cells are genetically modified to express one or more heterologous or exogenous nucleic acid sequences of interest. The nucleic acid sequence can be "exogenous", meaning that the nucleic acid sequence is independent of the cell into which the vector is introduced, or that the sequence is homologous to the sequence in the cell, but is in a location within the host cell nucleic acid where the sequence is not normally found. Nucleic acid sequences include, but are not limited to, plasmids, amplicons, cDNA, mRNA, antisense RNA, siRNA. The term "gene" refers to a functional protein, polypeptide, or peptide-encoding nucleic acid unit. As will be understood by those skilled in the art, this functional term includes genomic sequences, cDNA sequences, and smaller engineered gene segments that express or can be adapted to express proteins, polypeptides, domains, peptides, fusion proteins, and mutants.

[0140] Any method known in the art can be used to genetically modify tissue. One exemplary method is to insert a gene into the cells of the tissue using a recombinant viral vector. Any one of several different vectors, such as viral vectors, plasmid vectors, linear DNA, etc., known in the art, can be used to introduce exogenous nucleic acid fragments encoding therapeutic agents into target cells and / or tissues. For example, these vectors can be inserted using any one of infection, transduction, transfection, calcium phosphate-mediated transfection, DEAE-dextran-mediated transfection, electroporation, liposome-mediated transfection, biolistic gene delivery, liposomal gene delivery using fusogenic and anionic liposomes (which are alternatives to the use of cationic liposomes), direct injection, receptor-mediated uptake, magnetoporation, ultrasound, and others known in the art.

[0141] In an alternative embodiment, "vector" is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted to introduce it into a cell where it can be replicated. Vectors include plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). Those skilled in the art should be well equipped to construct vectors by standard recombinant techniques as described in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY; Ausubel et al. (1987) Current Protocols in Molecular Biology, Greene Publ. Assoc. & Wiley-Intersciences. In addition to encoding modified polypeptides, vectors can encode unmodified polypeptide sequences, such as tags or targeting molecules. Useful vectors encoding such fusion proteins include pIN vectors, vectors encoding stretches of histidines, and pGEX vectors for use in generating glutathione S-transferase (GST) soluble fusion proteins for subsequent purification and separation or cleavage.

[0142] In alternative embodiments, the vectors of the present invention are designed to introduce heterologous nucleic acid molecules, such as genes, into cells that are primarily "operably linked" or under the control of one or more control sequences. "Promoter" refers to one or more transcriptional control modules clustered around the initiation site for RNA polymerase II and other transcriptional activator proteins. Any promoter / enhancer combination (according to the Eukaryotic Promoter Database EPDB) can be used to drive the expression of a nucleic acid molecule of interest (i.e., constitutive, inducible, repressible, tissue-specific). Vectors can also contain selectable markers to facilitate their manipulation in vitro or ex vivo. Vectors can also contain polyadenylation signals, which can be obtained from the human growth hormone (hGH) gene, the bovine growth hormone (BGH) gene, or SV40. In addition, vectors can also contain internal ribosome binding site (IRES) elements used to create multigene or polycistronic messages. IRES elements can bypass the ribosome scanning model of 5-methylatd cap-dependent translation and initiate translation from internal sites (Pelletier, J. and Sonenberg, N. (1988) Nature 334(6180):320-325). IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating a polycistronic message. The IRES element ensures that each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter / enhancer to transcribe a single message.

[0143] In alternative embodiments, vector is viral vector.The viral vector known in the art includes, but is not limited to, adenovirus vector, retrovirus vector, vaccinia virus vector, adeno-associated virus vector, polyomavirus vector, alphavirus vector, rhabdovirus vector, lentivirus vector, Epstein-Barr virus vector, picornavirus vector or herpes virus vector.

[0144] In other embodiments, the nucleic acid sequence can be encapsulated in a liposome or lipid formulation. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before forming a closed structure, encapsulating water and dissolved solutes between the lipid bilayers (Ghosh, PC and Bachhawat, BK (1991) Targeted Diagn. Ther. 4:87-103). One example of a commercially available liposome or lipid formulation is Lipofectamine (Invitrogen). Others include FuGENE (Promega), PromoFectin (PromoKine), Affectene (Qiagen), Polyfect (Qiagen), Superfect (Qiagen), and TransMessenger (Qiagen).

[0145] The subject matter disclosed herein also provides a method of treating a retinal disease or condition in a subject in need of treatment for a retinal disease or condition by administering to the subject a composition comprising a cell population comprising mammalian retinal progenitor cells into the vitreous or subretinal space of the subject, and optionally measuring a change or improvement in the subject's vision.

[0146] In an alternative embodiment, the term "treating" in its various grammatical forms in the context of the present invention refers to curing, reversing, attenuating, mitigating, minimizing, inhibiting, or stopping the deleterious effects of a disease state, disease progression, disease causative agent, or other abnormal condition. For example, treatment can include alleviating a symptom (i.e., not necessarily all symptoms) of a disease or attenuating the progression of a disease. In an alternative embodiment, the term "preventing" means that the effects of a disease state or disease causative agent are forestalled due to administration of an agent such as those disclosed herein. A similar term in this context is "prophylaxis."

[0147] "Patient" or "subject" are used interchangeably herein and refer to the recipient of treatment. Mammalian and non-mammalian subjects are included. In some embodiments, the subject is a mammal, such as a human, non-human primate, dog, mouse, cat, cow, sheep, pig, or goat. In some embodiments, the subject is a human.

[0148] The cell populations and related compositions described herein can be administered to a subject or patient by a variety of different means. In certain embodiments, they are administered locally, e.g., at the site of actual or potential injury or disease. In some embodiments, they are administered using a syringe or needle to inject the composition at the site of potential or actual injury or disease. In other embodiments, they are administered systemically, i.e., administered into the bloodstream intravenously or intraarterially. The particular route of administration will depend primarily on the location and nature of the disease or injury to be treated or prevented. Thus, the subject matter described herein includes administering the cell populations or compositions of the invention via any known and available method or route, including, but not limited to, oral, parenteral, intravenous, intraarterial, intranasal, and intramuscular administration. Determination of appropriate dosages and treatment regimens can be readily accomplished and obtained by a physician based on information generally known in the art. Treatment may include a single treatment or multiple treatments. In particular, it is contemplated in certain embodiments that the purified cell populations of the invention are administered after stress that can potentially cause retinal damage, for preventative purposes. In other embodiments, the cell populations and compositions may be administered locally as a single injection into the vitreous or subretinal space of a subject. Alternatively, the compositions or cell populations may be administered two, three, four, or any number of times in the practice of the methods provided herein.

[0149] In alternative embodiments, the methods of the present invention include the isolation and characterization of mammalian retinal progenitor cells and compositions comprising such cells that are harvested from donor tissue, expanded in culture, and formulated for administration to a subject or patient. In some embodiments, the methods include administering the composition directly to the vitreous cavity under topical anesthesia, without the need for systemic immunosuppression of the subject. In alternative embodiments, tissue typing and matching of the graft to the patient or subject is not required, but may be performed if desired. Tissue typing and matching techniques are well known to those skilled in the art.

[0150] The retinal progenitor cells and cell populations used to practice the present invention can be formulated as compositions for administration by any or a variety of means, including orally, parenterally, by inhalation spray, nasally, topically, intrathecally, intrathecally, intracerebrally, epidurally, intracranially, or rectally. The compositions and formulations disclosed herein can include pharma- ceutical or veterinarily acceptable liquids, carriers, adjuvants, and vehicles, and can be in the form of liquids, tablets, capsules, implants, aerosols, gels, liposomes, nanoparticles, and the like.

[0151] In some embodiments, the retinal progenitor cells and cell populations can be administered to a subject in the form of a pharmaceutical or veterinary composition. The phrase "pharmaceutical acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to animals or humans. As used herein, "pharmaceutical acceptable carriers" include any and all aqueous and non-aqueous carriers, including water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, and the like) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the presence of microorganisms can be ensured by sterilization procedures and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, sorbic acid, and the like. It may be desirable to include isotonic agents such as sugars, sodium chloride, etc. in the composition. In addition, the extended absorption of the injectable pharmaceutical form can be brought about by including agents that delay absorption, such as aluminum monostearate and gelatin. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The use of such media and agents for pharmaceutical active substances is well known in the art.

[0152] In certain embodiments, an effective amount of retinal progenitor cells or cell populations must be administered to a subject. An "effective amount" or "therapeutically effective amount" refers to the amount of a composition that produces a desired effect. The effective amount depends in part on, for example, the molecule or agent being delivered (here, retinal progenitor cells or cell populations), the indication for which the therapeutic is being used, the route of administration, and the size (body weight, body surface or organ size), and the condition (age and overall health) of the subject or patient. Thus, the clinician or physician can titer the dosage and modify the route of administration to obtain optimal therapeutic effects. The effective amount of a particular agent for a specific purpose can be determined using methods well known to those skilled in the art. For any composition provided herein, the effective amount can be estimated first either in cell culture assays or in animal models such as mice, rats, rabbits, dogs, pigs, or monkeys. Animal models can also be used to determine appropriate concentration ranges and routes of administration. Such information can then be used to determine useful doses and routes for administration to humans.

[0153] Examples of effective amounts of the compositions described herein include cell suspensions in volumes of 5 μl, 10 μl, 15 μl, 20 μl, 25 μl, 50 μl, 100 μl, 150 μl, 200 μl, 250 μl, 300 μl, 350 μl, 400 μl, 450 μl, 500 μl, or any increment therebetween up to 5000 μl (5 ml). The lower and upper volume limits are limited by the delivery system and / or method. See, for example, Kayikcuiglu, OR et al. (2006) Retina 26(9):1089-90. For example, the upper volume limit when administered without vitrectomy is about 200 μl due to elevated intraocular pressure. The upper volume limit when administered into the vitreous cavity following vitrectomy is limited by the volume of the vitreous cavity and can include up to 5 ml or more. The upper limit for subretinal injections can be up to 200 μl due to retinal detachment. In alternative embodiments, these volumes may range anywhere between 10-10 million cells per dose, or between 1000-2000 cells per dose, 2000-3000 cells per dose, 3000-4000 cells per dose, 400-5000 cells per dose, 5000-6000 cells per dose, 6000-7000 cells per dose, 7000-8000 cells per dose, 8000-9000 cells per dose, 9000-10,000 cells per dose, 10,000-15,000 cells per dose, 15,000-20,000 cells per dose, 20,000-25,000 cells per dose, 25,000-30,000 cells per dose, 30,000-35,000 cells per dose, 35,000-40,000 cells per dose, 40,000-45,000 cells per dose, 45,000-50,000 cells per dose, 50,000-55,000 cells per dose, 55,000-60,000 cells per dose, 60,000-65,000 cells per dose cells, 65,000-70,000 cells per dose, 70,000-75,000 cells per dose, 75,000-80,000 cells per dose, 80,000-85,000 cells per dose, 85,000-90,000 cells per dose, 90,000-95,000 cells per dose, 95,000-100,000 cells per dose000 cells, 100,000-125,000 cells per dose, 125,000-150,000 cells per dose, 150,000-200,000 cells per dose, 200,000-250,000 cells per dose, 250,000-300,000 cells per dose, 300,000-350,000 cells per dose, 350,000-400,000 cells per dose, 400,000-450,000 cells per dose, 450,000-500,000 cells per dose, 500,000-550,000 cells per dose, Examples of suitable infusion doses include 550,000-600,000 cells per dose, 600,000-650,000 cells per dose, 650,000-700,000 cells per dose, 700,000-750,000 cells per dose, 750,000-800,000 cells per dose, 800,000-850,000 cells per dose, 850,000-900,000 cells per dose, 900,000-950,000 cells per dose, 950,000-1,000,000 cells per dose, or any increment between 1,000 cells and up to 10 million cells per dose. Dosages may, of course, vary with frequency and duration of administration. In alternative embodiments, the dosage of cells in the compositions described herein contains a large number of cells in a small volume, such as, for example, 500,000 cells per 100 μl. The number of cells can be counted by any method known in the art, such as, for example, hemocytometer, spectrophotometry, Coulter counter, flow cytometry, etc. Dosing can be administered once or over the course of several treatments.

[0154] In alternative embodiments, the compositions of the present invention are delivered by transscleral delivery, or by using, for example, transscleral delivery as described in U.S. Pat. No. 7,585,517; a sustained delivery device for delivery to the inside of a patient's eye as described in U.S. Pat. No. 7,883,717; a device for insertion into the vitreous region of the eye as described in U.S. Pat. Nos. 5,378,475 or 5,466,233; or by using a subcutaneous syringe or oblique insertion route as described, for example, in U.S. Patent Application Publication Nos. 20110112470 or 20100256597 (which describe fine needles for targeted administration to a patient's eye); or by using, for example, puncta as described in U.S. Patent Application Publication No. 20100209478. The cells can be formulated for parenteral administration to the eye (particularly into the vitreous or subretinal space), the vitreous or subretinal space, the retina, the brain, the nerves, or the CNS, by any method or protocol known in the art, including through a hydrophilic polymer hydrogel with dimensions to pass through a vitreous membrane (lacrimali); or by a device that provides access to the subretinal space of the human eye, as described, for example, in US Patent Publication No. 20100191176. Anterior chamber paracentesis can also be performed as determined by those skilled in the art. In alternative embodiments, the methods do not require suturing of the eye during and / or after the procedure, especially for intravitreous placement. However, this may be necessary for methods that utilize a vitrectomy procedure, for example, when placing cells in the subretinal space.

[0155] The compositions disclosed herein can also be formulated for intrathecal, intracerebral, epidural, subcutaneous, intravenous, intramuscular and / or intraarterial administration by injection route, but including various infusion techniques, for example, as described in U.S. Patent Application Publication No. 200500480021. Administration can be performed by using a catheter, or a pump, for example, an intrathecal pump, or an implantable medical device. In an alternative embodiment, the methods of the present invention are directed to the use of the methods disclosed in U.S. Patent Nos. 7,388,042, 7,381,418, 7,379,765, 7,361,332, 7,351,423, 6,886,568, 5,270,192, and U.S. Patent Application Publication Nos. 20040127987, 20080119909, 20080118549, 2008 This may also include administering or transplanting implants comprising the retinal progenitor cells, cell populations, or compositions disclosed herein, such as those described in US Pat. Nos. 20020015, 20070254005, 20070059335, and 20060128015, as well as artificial organs, bioreactor systems, cell culture systems, plates, dishes, tubes, bottles, flasks, and the like.

[0156] In alternative embodiments, the methods provided herein are directed to the treatment of a retinal disease or condition, such as, but not limited to, retinitis pigmentosa (RP), Leber's congenital amaurosis (LCA), Stargardt's syndrome, Usher syndrome, choroideremia, rod-cone or cone-rod dystrophies, ciliopathy, mitochondrial disorders, progressive retinal atrophy, retinal degenerative diseases, age-related macular degeneration (AMD), wet AMD, dry AMD, geographic atrophy, familial or acquired maculopathy, retinal photoreceptor diseases, retinal pigment epithelium-based diseases, diabetic retinopathy, cystoid macular edema, uveitis, retinal detachment, traumatic retinal injury, iatrogenic retinal injury, macular hole, macular telangiectasia, ganglion cell disease, optic nerve cell disease, glaucoma, optic neuropathy, ischemic retinal disease, retinopathy of prematurity, retinal vascular occlusion, familial microaneurysm, retinal vascular occlusion ... macroaneurysm), retinal vascular disease, ocular vascular disease, vascular disease, or ischemic optic neuropathy, or to improve photopic (daytime) vision, or to improve corrected visual acuity, or to improve macular function, or to improve visual field, or to improve scotopic (nighttime) vision.

[0157] In alternative embodiments, the treatment methods of the present invention utilize topical anesthesia, although any local, regional or general anesthesia may be used during administration. Examples of local anesthetics suitable for use in the methods disclosed herein include, without limitation, mepricaine, proparacaine, prilocaine, ropivacaine, benzocaine, bupivacaine, butamben picrate, chlorprocaine, cocaine, dibucaine, dimethisoquine, dyclonine, etidocaine, hexylcaine, ketamine, lidocaine, mepivacaine, pramoxine, procaine, tetracaine, salicylates, and derivatives, esters, salts, and mixtures thereof.

[0158] The composition comprising the retinal progenitor cells or cell population can be optionally co-administered with one or more drugs. Examples of drugs include anti-angiogenic agents, such as angiostatin, anecortave acetate, thrombospondin, VEGF receptor tyrosine kinase inhibitors, and anti-vascular endothelial growth factor (anti-VEGF) drugs, such as ranibizumab, and bevacizumab, pegaptanib, sunitinib, and sorafenib, and any of a variety of known small molecules and transcription inhibitors with anti-angiogenic effects; known classes of ophthalmic drugs, including glaucoma drugs, such as acebutolol, atenolol, bisoprolol, carvedilol, esmolol, and glaucoma drugs. adrenergic antagonists, including beta-blockers such as asmolol, labetalol, nadolol, penbutolol, pindolol, propranolol, metipranolol, betaxolol, carteolol, levobetaxolol, levobunolol, and timolol; adrenergic agonists or sympathomimetics, such as epinephrine, dipivefrin, clonidine, aparclonidine, and brimonidine; parasympathomimetics or cholinergic agonists, such as serotonin, serotonin, serotonin, and serotonin; agonists, such as pilocarpine, carbachol, phospholine iodine, and physostigmine, salicylates, acetylcholine chloride, eserine, diisopropyl fluorophosphate, demecarium bromide; muscarinic agonists; carbonic anhydrase inhibitors, including local and / or systemic agents, such as acetozolamide, brinzolamide, dorzolamide, and methazolamide, ethoxzolamide, diamox, and dichlorphenamide; mydriatic-cycloplegic agents, such as atropine, cyclopentolate, succinylcholine, homatropine, phenylephrine, scopolamine, and tropicamide; prostaglandins, such as prostaglandin F2α, antiprostaglandins, prostaglandin precursors, or prostaglandin analog agents, such as bimatoprost, latanoprost, travoprost, and unoprostone.

[0159] Other examples of drugs include, for example, glucocorticoids and corticosteroids, such as betamethasone, cortisone, dexamethasone, dexamethasone 21-phosphate, methylprednisolone, prednisolone 21-phosphate, prednisolone acetate, prednisolone, fluroometholone, loteprednol, medrysone, fluocinolone acetonide, triamcinolone acetonide, triamcinolone, triamcinolone acetonide, beclomethasone, budesonide, flunisolide, fluorometholone, Anti-inflammatory agents including fluticasone, fludrocortisone, hydrocortisone, hydrocortisone acetate, loteprednol, rimexolone, as well as anti-inflammatory agents such as, for example, aspirin, diclofenac, flurbiprofen, ibuprofen, bromfenac, nepafenac, and ketorolac, salicylates, indomethacin, naxoprene, piroxicam, and nabumetone, diflunisal, etodolac, fenoprofen, flurbiprofen, indomethacin, ketoprofen, meclofenamate, mefenamic acid, meloxicam, nabumetone, oxapeptide, nabumetone ... nonsteroidal anti-inflammatory agents, including roxine, piroxicam, salsalate, sulindac, and tolmetin; COX-2 inhibitors, such as celecoxib, rofecoxib, and valdecoxib; for example, tetracycline, chlortetracycline, bacitracin, neomycin, polymyxin, gramicidin, cephalexin, oxytetracycline, chloramphenicol, rifampicin, ciprofloxacin, tobramycin, gentamicin, erythromycin, penicillin, sulfonamides, sulfadiazine, sulfacetamide, Anti-infective or anti-microbial agents such as antibiotics including sulfamethizole, sulfisoxazole, nitrofurazone, sodium propionate, aminoglycosides, e.g., gentamicin, tobramycin, amikacin, and streptomycin; fluoroquinolones, e.g., ciprofloxacin, gatifloxacin, levofloxacin, moxifloxacin, norfloxacin, ofloxacin; bacitracin, erythromycin, fusidic acid, neomycin, polymyxin B, gramicidin, trimethoprim, and sulfacetamide;Also included are antifungal agents such as amphotericin B, caspofungin, clotrimazole, fluconazole, itraconazole, ketoconazole, voriconazole, terbinafine, nystatin, and miconazole; antimalarials such as chloroquine, atovaquone, mefloquine, primaquine, quinidine, and quinine; antimycobacterial agents such as ethambutol, isoniazid, pyrazinamide, rifampin, and rifabutin; antiparasitic agents such as albendazole, mebendazole, thiobendazole, metronidazole, pyrantel, atovaquone, iodoquinaol, ivermectin, paromycin, praziquantel, and trimatrexate;

[0160] Other examples of drugs include antiviral agents such as idoxuridine, trifluorothymidine, acyclovir, cidofovir, famciclovir, ganciclovir, valacyclovir, valganciclovir, vidarabine, trifluridine, and foscarnet; protease inhibitors such as ritonavir, saquinavir, lopinavir, indinavir, atazanavir, amprenavir, and nelfinavir; nucleotide / nucleoside / non-nucleoside Reverse transcriptase inhibitors, such as abacavir, ddl, 3TC, d4T, ddC, tenofovir, and emtricitabine, delavirdine, efavirenz, and nevirapine; other antiviral agents, such as interferons, ribavirin, and trifluridiene; antibacterial agents, including carbapenems, such as ertapenem, imipenem, and meropenem; cephalosporins, such as cefadroxil, cephalosporins, and cephalosporins. Fazolin, cefdinir, cefditoren, cephalexin, cefaclor, cefepime, cefoperazone, cefotaxime, cefotetan, cefoxitin, cefpodoxime, cefprozil, ceftaxidime, ceftibuten, ceftizoxime, ceftriaxone, cefuroxime, and loracarbef; other macrolides and ketolides, such as azithromycin, clarithromycin, dirithromycin, and telithromycin; amoxicillin, Penicillins (with and without clavulanate), including ampicillin, pivampicillin, dicloxacillin, nafcillin, oxacillin, piperacillin, and ticarcillin; tetracyclines, such as doxycycline, minocycline, and tetracycline; other antibacterial agents, such as aztreonam, chloramphenicol, clindamycin, linezolid, nitrofurantoin, and vancomycin.

[0161] Other examples of drugs include immunomodulators such as antiallergic drugs including, for example, sodium chromoglycate, antazoline, methapyriline, chlorpheniramine, cetirizine, pyrilamine, and profenpyridamine; aldesleukin, adalimumab, azathioprine, basiliximab, daclizumab, etanercept, hydroxychloroquine, infliximab, leflunomide, methotrexate, mycophenolate mofetil, and sulfasalazine; antihistamines, for example, azetidin, cyclosporine ... cyproheptadine, emedastine, loratadine, desloratadine, cetirizine, diphenhydramine, chlorpheniramine, dexchlorpheniramine, clemastine, cyproheptadine, fexofenadine, hydroxyzine, promethazine, and levocabastine; immunological agents (such as vaccines and immunostimulants); MAST cell stabilizers, such as cromolyn sodium, ketotifen, lodoxamide, nedocrimil, olopatadine, and pemirolast; ciliary body ablative and other ophthalmic agents such as verteporfin, proparacaine, tetracaine, cyclosporine, and pilocarpine; inhibitors of cell surface glycoprotein receptors; decongestants such as phenylephrine, naphazoline, tetrahydrazoline; lipids or antihypertensive lipids; dopaminergic agonists and / or antagonists such as quinpirole, fenoldopam, and ibopamine; vasospasm inhibitors; vasodilators;antihypertensives;angiotensin-converting enzyme (ACE) inhibitors;angiotensin-1 receptor antagonists such as olmesartan;microtubule inhibitors;molecular motor (dynein and / or kinesin) inhibitors;actin cytoskeleton regulators, such as cyctchalasin, latrunculin, swinholide A, ethacrynic acid, H-7, and Rho-kinase (ROCK) inhibitors;remodeling inhibitors;modulators of the extracellular matrix, such as tert-butylhydroquinolones and AL-3037A;Adenosine receptor agonists and / or antagonists, such as N-6-cyclohexyl adenosine and (R)-phenylisopropyl adenosine; serotonin agonists; hormonal agents, such as estrogen, estradiol, progesterone, insulin, calcitonin, parathyroid hormone, peptides, and vasopressin hypothalamic releasing factor; growth factor antagonists or growth factors, including epidermal growth factor, fibroblast growth factor, platelet derived growth factor, transforming growth factor beta, somatotrapin, fibronectin, connective tissue growth factor, bone morphogenetic protein (BMP); cytokines, such as interleukins, serum amyloids, such as CD44, cochlin, and serum amyloid A;

[0162] Other therapeutic agents include neuroprotective agents, such as lubezole, nimodipine, and related compounds, including blood flow enhancers, sodium channel blockers, glutamate inhibitors such as memantine, neurotrophic factors, nitric oxide synthase inhibitors; free radical scavengers or antioxidants; chelating compounds; apoptosis-related protease inhibitors; compounds that reduce new protein synthesis; radiation therapy agents; photodynamic therapy agents; gene therapy agents; gene modulators; and dry eye medications, such as cyclosporine A, delmulcent, and sodium hyaluronate; alpha blockers, such as doxazosin, prazosin, and terazosin; calcium channel blockers, such as amlodipine, bepridil, diltiazem, felodipine, isradipine, nicardipine, anticoagulants, including heparins and heparinoids, such as heparin, dalteparin, enoxaparin, tinzaparin, and fondaparinux; other anticoagulants, such as hirudin, aprotinin, argatroban, bivalirudin, desirudin, lepirudin, warfarin, and ximelagatran; antiplatelet agents, such as abciximab, clopidogrel, dipyridamole, eptifibatide, ticlopidine, and tirofiban.

[0163] Other therapeutic agents include prostaglandin PDE-5 inhibitors and other prostaglandin agents, such as alprostadil, carboprost, sildenafil, tadalafil, and vardenafil; thrombin inhibitors; antithrombotic agents; antiplatelet aggregation agents; thrombolytic and / or fibrinolytic agents, such as alteplase, anistreplase, reteplase, streptokinase, tenecteplase, and urokinase; antiproliferative agents, such as sirolimus, tacrolimus, everolimus, zotarolimus, paclitaxel, and mycophenolic acid; levothyroxine, fluoxymesterone, and the like. ne), hormone-related agents, including methyltestosterone, nandrolone, oxandrolone, testosterone, estradiol, estrone, etropipate, clomiphene, gonadotropins, hydroxyprogesterone, levonorgestrel, medroxyprogesterone, megestrol, mifepristone, norethindrone, oxytocin, progesterone, raloxifene, and tamoxifen; antineoplastic agents, including alkylating agents, e.g., carmustine, lomustine, melphalan, cisplatin, fluorouracil 3, and procarbazine; antibiotic-like agents antiproliferative agents, such as 1,3-cis retinoic acid, 5-fluorouracil, taxol, rapamycin, mitomycin C, and cisplatin; antimetabolites, such as cytarabine, fludarabine, hydroxyurea, mercaptopurine, and 5-fluorouracil (5-FU); immunomodulators, such as aldesleukin, imatinib, rituximab, and tositumomab; mitotic inhibitors, docetaxel, etoposide, vinblastine, and vincristine; radioactive agents, such as strontium-89; and other antineoplastic agents, such as irinotecan, topotecan, and mitotane.

[0164] The uses and methods described herein can rapidly and persistently restore and / or preserve the visual function of the retina of a mammalian subject to a clinically significant degree, including, but not limited to, improved photopic (daytime) vision, increased best-corrected visual acuity, improved macular function, preserved or restored central fixation, improved visual field, improved scotopic (nighttime) vision, increased or improved sensitivity to sound, and improved visual acuity of the contralateral eye.Other changes can include various systemic benefits, such as changes in appearance due to physical improvements that may be related to the effects of light on circadian rhythm, pituitary function, hormone release, vascular tone, improved perception of visual ability, improved independent walking, improved perception of health, and / or improved activities of daily living.Such changes in vision can be measured by methods known in the art.Vision benefits can occur within the first week after treatment, but incremental benefits can also occur over a longer period of time. Retinal cell replacement and / or donor cell transfer to the retina, retinal circuitry, or outer nuclear layer or macula can be performed but is not required for clinical efficacy, and donor cell transfer within the retina is possible but is not required for clinical efficacy.

[0165] Measurement of visual changes, including visual improvement, resulting from treatment with the retinal progenitor cell compositions disclosed herein can be accomplished using standard ophthalmic examination techniques, including, but not limited to, fundus examination, best corrected visual acuity (BCVA), IOP, slit lamp examination, fluorescein angiography (FA), optical coherence tomography (OCT), stereo fundus photography, electroretinography (ERG), cone flicker electroretinography, perimetry (visual field), microperimetry, dark adaptation, maze negotiating skill, optokinetic / optokinetic response, pupillary reflex, visual evoked potential (VIP), and adaptive optics scanning laser ophthalmoscopy (AOSLO).

[0166] Kit and Instructions The invention provides kits comprising compositions (e.g., heterogeneous mixtures of fetal neural retinal cells) and methods of the invention (e.g., treating a retinal disease or condition or producing a heterogeneous mixture of fetal neural retinal cells), including instructions for their use. In alternative embodiments, the invention provides kits comprising a composition, product of manufacture, or mixture of cells or culture of cells of the invention (e.g., a heterogeneous mixture of fetal neural retinal cells), optionally further comprising instructions for carrying out the methods of the invention.

[0167] The present invention provides kits comprising cell populations comprising mammalian retinal progenitor cells as described herein, provided as fresh or frozen cells in culture, or formulated as compositions for administration to a subject. The kits may further comprise instructions for carrying out the methods of the present invention. Such kits may further comprise agents (e.g., antibodies or oligonucleotide primers) that bind to one or more markers of retinal progenitor cells as described herein, and basal or conditioned medium. For example, the kits may comprise a first container comprising an antibody specific to one or more markers, the antibody being suitable for isolation or detection, for example, by conjugation to a fluorescent marker or magnetic beads, and a second container comprising basal or conditioned medium. In various related embodiments, the kits may further comprise one or more additional reagents useful for the preparation of the cell populations of the present invention, such as cell culture medium, cell culture dishes coated with extracellular matrix, and enzymes suitable for tissue processing. The kits may also comprise instructions for their use to purify and expand retinal progenitor cells or cell populations obtained from tissue samples. In other embodiments, the kit may further comprise a means for obtaining a tissue sample from a patient or donor, and / or a container for containing the obtained tissue sample.

[0168] Veterinary Use In alternative embodiments, the compositions and methods of the invention may be used in veterinary applications, for example, the present invention demonstrates the first successful proliferation of feline RPCs and the first therapeutic use of the retina in dystrophic cats and other animals, such as any mammalian pet, common domesticated and rare wild mammal species, zoo animals, farm animals, sports (e.g., racing dogs or horses) animals, etc.

[0169] There are several domestic animals that carry genes that cause blindness as a result of extensive inbreeding. These included cats, dogs, and horses, and possibly other species. There are retinal diseases and injuries that occur in wild and domestic animals that would benefit from treatment using the compositions and methods of the present invention.

[0170] Manufactured Articles, Implants, and Artificial Organs The invention also provides implants comprising one or more formulations or medicaments of the invention, including a heterogeneous mixture of fetal neural retinal cells, as well as artificial organs, bioreactor systems, cell culture systems, plates, dishes, tubes, bottles, flasks, and the like.

[0171] In alternative embodiments, the present invention provides a bioreactor, implant, stent, artificial organ, or similar device containing a heterogeneous mixture of fetal neural retinal cells, e.g., as described in U.S. Patent Nos. 7,388,042; 7,381,418; 7,379,765; 7,361,332; 7,351,423; 6,886,568; 5,270,192; and U.S. Patent Application Publication Nos. 20040127987; 20040127987; The present invention provides implants similar to or described in US Pat. Nos. 80119909 (describing auricular implants); 20080118549 (describing ocular implants); 20080020015 (describing bioactive wound dressings); 20070254005 (describing heart valve bioprostheses, vascular grafts, and meniscus implants); 20070059335; and 20060128015 (describing liver implants).

[0172] The present invention will be further described with reference to the following examples, but it should be understood that the present invention is not limited to such examples. EXAMPLES

[0173] Example 1 Isolation and culture of retinal progenitor cells Whole fetal eyes from fetuses of approximately 16-19 weeks gestational age (GA) were obtained from donors and placed in 15 ml tubes containing RPMI-1640 medium with L-glutamine (BioWhittaker). Eyes were shipped on ice for periods ranging from 4.5 hours to approximately 21.5 hours. At the same time, blood samples were drawn from the donors and sent for testing for exposure to adventitious agents. Upon arrival, each eye was inspected to ensure that the cornea was clear and of normal shape, and then placed under a laminar flow hood under sterile conditions. Whole fetal eyes were rinsed three times in 40 ml of cold phosphate buffered saline (PBS) containing antibiotics in separate 50 ml tubes. The optic nerve and remaining mesenchymal tissue were then removed by dissection. This procedure was adopted to avoid the possibility of contaminating the retinal isolate with unwanted cells of non-retinal origin. After dissection, the eyes were rinsed one more time in cold PBS containing antibiotics.

[0174] Under a binocular dissecting microscope, a hole was punctured in each eye at the surgical limbus using a 1 ml "TB" type syringe fitted with a 25-5 / 8 gauge needle. The globe was then opened circumferentially by cutting along the limbus with fine scissors. The anterior structures (cornea, lens) and any remaining vitreous were removed from the eyecup. The retina was then detached from the retinal pigment epithelium (RPE) by carefully scraping it and transferred into a small Petri dish containing approximately 2 ml of cold DMEM / F12 medium. The retinal tissue was manually broken into small pieces by gentle trituration with a 1 ml tip in the Petri dish. The retinal chunks were transferred in suspension into a 15 ml cold conical bottom tube and any remaining tissue was collected by rinsing the Petri dish 2-3 times in 1 ml of cold DMEM / F12 and adding this to the 15 ml tube. The tissue was pelleted by centrifugation at 1000 rpm (179×g) for 5 min and the supernatant was discarded.

[0175] The tissue was then subjected to enzymatic digestion by incubation in 0.8 ml of TrypLE Express stock solution (Invitrogen) for 40 seconds at room temperature. The trypsinized tissue was then pumped up and down with a 1 ml pipette tip. Trypsin was neutralized by subsequent addition of 10 ml of cold fresh serum-free cell culture medium, and the mixture was collected by centrifugation at 1000 rpm (179×g) for 4 minutes. The supernatant was removed, the pellet was resuspended in cold fresh cell culture medium, and cell viability and cell number were then determined by trypan blue (Invitrogen) dye exclusion and counted using Countess (Invitrogen) or manually. Approximately 10×10 6 Cell populations were obtained, where approximately 80% were small / medium clusters, approximately 9-18% were single cells, and approximately 1-2% were large clusters. This technique resulted in approximately 92% cell viability.

[0176] Cells were then seeded into two T75 culture flasks pre-coated with human (xeno-free) fibronectin. Human plasma fibronectin (Invitrogen) was used in some experiments. In other experiments, ornithine, polylysine, laminin, or matrigel were used. Cells were then incubated at 37°C under 5% CO2 and atmospheric oxygen, or alternatively in 3% O2, using a LowOx incubator. During culture, care was taken to further dissociate clusters by trypsinization and / or trituration to prevent premature differentiation when cells were subsequently passaged. Every 1 or 2 days, 90% of the cell culture medium was replaced and cells were passaged at 60–80% confluence, and 40–90% confluence as required, using TrypLE Express for 5–6 min at 37°C. Trypsinization was stopped by adding 10 ml of cold medium or cold PBS. Cell viability was determined by trypan blue staining, and cell numbers were counted. Dissociated cells were cultured at 1–6.7 × 10 4 / cm 2 The seeds were subsequently sown at a density of 1000×1000.

[0177] Cells were prepared for freezing by first harvesting them with TrypLE Express. Cells were harvested by centrifugation at 1000 rpm (179 x g) for 5 min. The supernatant was removed and the cell pellet was resuspended in fresh medium or cold PBS. Cell viability and cell number were determined. Cells were subsequently pelleted again at 1000 rpm for 5 min and 0.5-5 x 10 cells were harvested per cryovial. 6 Cells were aliquoted and resuspended in cell cryopreservation medium (90% fresh complete medium, 10% DMSO). Cryovials were placed in a freezer at 1° C. and then transferred to a −80° C. freezer, liquid nitrogen tank, or other permanent cryogenic storage.

[0178] To thaw the cells, the cryovial was removed from liquid nitrogen / storage and placed in a 37°C water bath for 2-3 minutes until the ice crystals had disappeared. The thawed cells were then immediately transferred to a 15 ml cold conical tube using a 1 ml pipette tip, and the vial was rinsed twice with cold fresh medium. Ten milliliters of cold fresh medium was added dropwise into the 15 ml tube with gentle shaking. The cells were then collected by centrifugation at 800 rpm (115 x g) for 3 minutes, the supernatant was discarded, and the resulting cell pellet was resuspended in fresh medium. Cell counts and viability were determined as described herein, and the cells were then seeded into new fibronectin-coated flasks and incubated under the conditions described above.

[0179] The morphology of feline retina-derived progenitor cells is shown in Figure 1. As can be seen, the morphology was maintained at different time points during the course of sustained culture. Figure 2 illustrates the proliferation curves of feline RPCs grown in two different types of cell culture media. Feline RPCs in SM senesced at passage day 10 (P10), while the same cells in UL continued to proliferate. After P14, there was an upward inflection of proliferation.

[0180] The morphology of the human cells in Figure 3 shows small clusters of cells initially observed, which were systematically converted into adherent single cell cultures by the end of the first week. This was achieved by dissociation during the passaging procedure described above. In general, the initial use of small or medium sized clusters promoted cell viability, which was advantageous in the complete absence of serum. Subsequent complete dissociation while maintaining a relatively high cell density can promote proliferation while avoiding differentiation.

[0181] In Figures 4 and 5 the growth kinetics of hRPCs can be seen. Cells derived from the donation were used clinically at P4. The growth observed under atmospheric oxygen was substantial and sustained for at least 10 passages (P10), but growth was not indefinite. Unlimited growth characteristics are contraindicated as they may indicate increased risk of pluripotency, immortalization, and tumor formation.

[0182] Various serum-free and xeno-free cell culture media, including DMEM / F12 (standard, Advanced and KnockOut; Invitrogen), Neurobasal (Invitrogen), Ultraculture (Lonza), and ReNcell (Chemicon), were tested to determine optimal conditions for RPC propagation. Cell culture media used in culturing RPCs were supplemented with N2 supplement (Invitrogen), B27 (Invitrogen or other brands), Stempro (Invitrogen), vitamin C, albumin, recombinant human epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), GlutaMAX I, L-glutamine, and / or penicillin-streptomycin (Invitrogen) at intervals over the first two weeks. As used herein, "SM" refers to standard growth medium based on DMEM / F12. "UL" refers herein to growth medium using Ultraculture as the basal medium. Serum-free medium was used in the absence or with antibiotics for the first 2 weeks, after which antibiotic-free medium was used for about 6 weeks. No antimycotic was used. Figure 5 shows the results of an experiment carried out to determine the optimal medium for RPCs. For the basal medium, standard DMEM / F12, media supplemented with N2 supplement, growth factors, glutamine or GLUTAMAX™ (GlutaMax™) were compared with a similarly supplemented "Advanced DMEM / F12". As can be seen in Figure 6A, the "Advanced" version, which also contains supplemental vitamin C and albumin, proved more effective, increasing the yield of hRPCs by 18-29%.

[0183] The effect of cell culture supplementation was also explored. To that end, N2 supplement was also compared with B27 xeno-free supplement. Figure 6B shows that supplementation with B27 xeno-free indeed increases the yield of RPCs. N2 increases the yield moderately, and these amounts are sufficient for therapeutic efficacy. The effect of additional vitamin C was also tested. Vitamin C supplement was added to the medium every 2 days. Figure 7A shows that vitamin C improves the yield of hRPCs by about 30%. Advanced DMEM / F12 contains added vitamin C, but it is clear that the higher levels provided by the additional supplementation (0.05mg / ml to 0.1mg / ml) are useful for hRPC growth. Adding fresh vitamin C every 2 days was sufficient, and daily addition was not found to be necessary. Finally, albumin supplementation was tested. When xeno-free human recombinant albumin was added to the medium at 1.0 mg / ml, enhanced proliferation was observed (up to 27%) when added to standard DMEM / F12-based medium, but no detectable improvement was observed when additional albumin was added to Advanced DMEM / F12-based medium, which is the preferred basal medium in this context (and already contains added albumin), see Figures 7B and 7C.

[0184] The osmolarity of the cell culture media was also examined by using media of different osmolarities (OSM) in combination with different commercially available supplements. See Figure 8. Low OSM media (KnockOut DMEM / F12; 276 mOsm / kg) was not beneficial for the commonly used neural supplements N2 or B27, and in some cases resulted in significantly lower yields compared to regular OSM media (DMEM / F12; 300-318 mOsm / kg). When less common supplements were used in combination (STEMPRO™ kit, Invitrogen, data not shown), there were indications of benefit to low OSM.

[0185] In addition to % viability and proliferation rate, an additional metric was developed that predicts optimal (e.g., in vitro) outcome of the first harvest, referred to herein as "time to drop." "Time to drop" specifically refers to the time it takes for dissociated cells to settle to the bottom of the flask in the growth medium in which they are incubated. Retention of suspension (lack of drop) may be associated with injury or non-viability of the cells resulting from damage from the isolation process, while successful self-repair by the cells is associated with the observation of their ability to regain membrane homeostasis, normal osmolarity, etc., thereby restoring negative buoyancy and thus "dropping." Delayed self-repair is stressful for the cells, resulting in less active / healthy cultures. Using approximately 90% drop (based on % of population) as the reporting criterion, a drop time of approximately 6 hours was observed for cells from tissues where the transport time was 21.5 hours, shortening to approximately 1.5 hours for cells from tissues where the transport time was 4.5 hours. Immediately plated feline RPCs and brain progenitor cells resulted in a drop time of 1 hour. Fall times of approximately 1 hour or less were achievable in human cells.

[0186] The cells used to demonstrate improved vision in humans with RP were cultured under conditions of atmospheric oxygen. Ramification of proliferating RPCs under low oxygen conditions that more closely mimic the oxygen levels of the developing fetal retina during pregnancy, in this case 3% oxygen ("lowOx"), was explored. As Figure 9 shows, 3% oxygen ("lowOx") significantly improved hRPC proliferation and sustained proliferation longer (at day 56, P10), greatly increasing the overall cell yield from a given donation. Growth profiles under atmospheric oxygen (20%) are shown for comparison, revealing a significantly less vigorous proliferation rate and more premature senescence of proliferation (at day 47, P7), and poorer overall yield. Figure 9 also shows an inflection point corresponding to accelerated proliferation of hRPCs in lowOx, which occurs at a confluence level of approximately 40%, highlighting the importance of high-density culture conditions for optimal hRPC proliferation. Similar results are seen in cells grown under hypoxic conditions (Figure 10). The growth characteristics of WCB cells grown under hypoxic conditions were reproducible (Figure 11).

[0187] Since steroids are often used in patients at the time of transplantation, the cells were also subjected to steroid toxicity testing. Figure 12 shows the toxicity of triamcinolone acetonide, a steroid commonly used in ophthalmic applications, but only at levels above expected clinical use, to hRPCs. Clinical doses of triamcinolone acetonide did not appear to affect cell proliferation, but higher doses (approximately 10-fold greater than the expected clinical dose) may reduce donor cell viability.

[0188] Cells previously frozen in liquid nitrogen were thawed and tested for viability. Figure 13 shows the results of the freeze-thaw experiment. These previously frozen cells were viable under both hypoxic and normoxic cell culture conditions. Cell viability was found to be approximately 94% for both oxygen conditions. Post-thawing, cells exhibited similar proliferation kinetics as hRPCs maintained under continuous culture conditions.

[0189] Example 2 Characterizing RPC Immunocytochemistry Cells were dissociated and grown on 4- or 8-well chamber slides for 1-3 days, then fixed in 4% paraformaldehyde for 15 min and washed three times in PBS. Slides were blocked for 1 h in a solution containing 5% donkey serum and / or 0.3% Triton X-100, followed by another wash in PBS. A panel of antibodies was then incubated overnight at 4°C to detect antigens expressed by the progenitor cells. These included anti-nestin (Chemicon 1:200), anti-vimentin (Sigma 1:200), anti-Sox2 (Santa Cruz 1:400), anti-SSEA-1 (BD 1:200), anti-GD2 (Chemicon 1:100), anti-Ki-67 (BD 1:200), anti-β3-tubulin (Chemicon 1:400), anti-GFAP (Chemicon 1:400), and anti-GDNF (Santa Cruz 1:200). This was followed by incubation with anti-mouse Alexa546 (Invitrogen 1:400), anti-goat Alexa488 (Invitrogen 1:400), or anti-rabbit FITC (Chemicon 1:800) secondary antibodies. Fluorescence was detected using a Leica converse microscope and visualized by Metamorph software. Total cell numbers were determined using DAPI, and the positive profile rate was calculated by counting profiles expressing specific immunoreactivity within six randomly selected fields.

[0190] RNA extraction Total RNA was extracted by using the RNeasy mini kit (Qiagen, CA, USA) according to the manufacturer's instructions and treated with DNase I. RNA was quantified by measuring the optical density (OD) at 260 nm / 280 nm of 1.90–2.10 and the optical density (OD) at 260 nm / 230 nm of 1.90–2.20 by spectrophotometer (ND-1000; NanoDrop Technologies Inc., Wilmington, DE).

[0191] Microarray analysis After assessing the quality of all starting total RNA samples, the target preparation / processing step was initiated by running a small amount of each sample (typically 25–250 ng / well) on an RNA 6000 Nano LabChip and evaluated on an Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA). Double-stranded cDNA was synthesized from poly(A)+ mRNA present in the isolated total RNA using the GeneChip WT cDNA Synthesis Kit (Affymetrix, Inc., Santa Clara, CA) and random hexamers tagged with a T7 promoter sequence. Typically, 100 ng of total RNA starting material was used for each sample reaction. Multiple copies of antisense cRNA were then generated by using the double-stranded cDNA as a template in an in vitro transcription reaction in the presence of T7 RNA polymerase for 16 h using the Affymetrix Genechip WT cDNA Amplification Kit. Ten micrograms of cRNA was used in a second cycle cDNA reaction with random hexamers reverse transcribed to generate single-stranded DNA in the sense orientation.

[0192] Single-stranded DNA samples were fragmented (WT Terminal Labeling Kit, Affymetrix) to an average length of 60 bases (range 40–70 bp) according to the prescribed protocol (Affymetrix GeneChip WT Sense Target Labeling Assay Manual). The fragmented single-stranded DNA was subsequently labeled with recombinant terminal deoxynucleotidyl transferase and Affymetrix proprietary DNA Labeling Reagent covalently linked to biotin. Following the recommended procedure, 0.54 μg of this fragmented single-stranded target cDNA was hybridized to the probe sets present on the Affymetrix Human Gene 1.0 ST array at 45°C for 17 hours with rotation (Affymetrix GeneChip Hybridization Oven 640). GeneChip arrays were washed and then stained with streptavidin-phycoerythrin in an Affymetrix Fluidics Station 450 (Fluidics protocol FS450_007). CEL intensity files were generated by scanning the arrays using a GeneChip Scanner 3000 7G and GeneChip Operating Software v1.4.

[0193] Normalization was performed using the probe logarithmic intensity error (PLIER) estimation method, which includes a quantile normalization protocol within the accompanying software algorithm. Briefly, the probe cell intensity files (*.CEL) generated above were analyzed using Affymetrix Expression Console software v1.1, which uses the PLIER algorithm, to generate probe-level summary files (*.CHP). The algorithm used was from PLIER v2.0 (quantification scale: linear; quantification type: signal and detection p-value; background: PM-GCBG; normalization method: sketch-quantile). Microarray data were then evaluated using JMP Genomics 4.1 (SAS Americas). Data were analyzed by one-way ANOVA with post-hoc t-tests, and the resulting p-values ​​were corrected using an FDR α<0.05. The resulting data tables were annotated. In addition to volcano plots from the ANOVA results, JMP software was also used to generate principal component analyses, Venn diagrams, and hierarchical clusters and heat maps, using the default fast Ward method.

[0194] Real-time qPCR assays The selection of candidate markers was based on their potential suitability for the present study as well as the results of previous work with this type of cell. Particular emphasis was placed on markers associated with immature cells of the neural lineage, as well as selected markers for neuronal and glial differentiation. Two micrograms of total RNA from sample preparations were reverse transcribed using the Omniscriptase Reverse Transcriptase kit (Qiagen, CA, USA) and 10 μM random primers (Sigma, MO, USA) according to the manufacturer's instructions. Quantitative PCR was performed using the 7500 fast Real-Time PCR System (Applied Biosystems, Irvine, USA) using Power SYBR green (Applied Biosystems, Irvine, USA) or Taqman gene expression assays (Applied Biosystems).

[0195] Discrimination of products of interest from non-specific product amplification was achieved by melting curve analysis when using the SYBR Green method. β-actin or GDNPH was used as an endogenous control to normalize gene expression. The following general real-time PCR protocol was used: denaturation program (95°C for 10 min), quantification program (95°C for 15 s and 60°C for 130 min) repeated for 40 cycles, melting curve program (95°C for 15 s and 60°C for 1 min with continuous fluorescence measurement), and a final cooling program at 40°C. Each reaction was performed in triplicate. ΔΔC t Graphs were plotted and analyses were performed by the method (7500 Fast System Software 1.4 and DataAssist 2.0, Applied Biosystems, Irvine, USA) and JMP software 4.1 (SAS Americas). All data points are expressed as mean ± standard error (SE). Statistical differences were determined using t-test. Data were considered significant when p<0.05.

[0196] Cytotoxicity Testing The cytotoxicity of RPCs was determined using Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies Inc., Gaithersburg, MD). This kit uses WST-8, which upon biological reduction in the presence of the electron carrier 1-methoxy PMS produces a water-soluble colored formazan. 96-well plates containing 90 μl of cell suspension per well were inoculated with 10 μl of CCK-8 pre-packaged solution. Plates were incubated for 2 hours and the OD of the supernatant was measured. 450 Each experiment was performed in quadruplicate in at least three separate experiments.

[0197] Feline RPC cells stained with immunocytochemistry (ICC) markers showed high levels of expression of vimentin. See Figure 14. The same was observed with human RPCs. Lineage markers including nestin, vimentin, Ki-67, β3-tubulin, glial fibrillary protein (GFAP), and rhodopsin revealed the presence of neurons, photoreceptors, and glia, demonstrating the retention of multipotency and heterogeneity of RPC cultures. Feline RPC gene profile was observed over time using qPCR of marker transcripts. Dynamic changes in expression profile, as also observed with human RPCs: a general trend of downward quantitative changes in marker expression over time in culture. See for example Figure 15. Gene expression was also compared between UL and SM cell culture medium conditions by qPCR (Figure 16A). As proliferation of feline progenitor cells (both RPC and BPC) was less sustained in SM cultures, investigations were directed to comparative gene expression. Using day 13 SM as a baseline for comparison (set at "1.0"), feline RPCs grown in SM and UL were compared at day 31 in culture. Most markers examined showed a decrease in expression over time in culture, but SM cultures showed a marked increase in GFAP expression, consistent with a progressive loss of pluripotency and a trend towards restriction along the glial lineage. Cells in UL medium did not show this.

[0198] Feline RPCs versus brain progenitor cells (BPCs) were also compared. See FIG. 16B. Feline RPCs showed relatively increased expression of several markers compared to BPCs, including transcription factors Dach1, Lhx2, and Pax6, which are involved in retinal specification and development. Similarly, transcription factors Hes1 and Hes5 were involved in retinal development. CD133, nestin, Sox2, and vimentin are general CNS progenitor markers, while β3-tubulin, Map2, and PKCα are lineage markers.

[0199] Feline RPCs were transplanted into the subretinal space of dystrophic Abyssinian cats and isolated transplanted cells were subjected to staining. See FIG. 17. The cells survived transplantation into the subretinal space and further demonstrated the ability to migrate into the recipient retina. The transplanted cells demonstrated the ability to differentiate into what appeared to be Müller glia, both morphologically and by vimentin labeling. Müller cells are glia that are retina specific and spread throughout all layers of the retina providing structural stabilization. They are important for many retinal functions, including neuronal survival.

[0200] Morphology of human RPCs using marker expression by ICC was determined. Figures 18 and 19 show the percentage expression of certain key markers within the cultured population. Expression of nestin was confirmed, which is thought to be associated with neural stem / progenitor cells and RPCs. Vimentin was expressed in very large amounts by this population, but its expression was thought to be non-specific to RPCs. Sox2 is a transcription factor also associated with neural development and was also present in hRPCs. SSEA-1 (also known as CD15, LeX) has been associated with pluripotency in ES cells, and studies have shown expression of this marker by a subset of non-pluripotent, multipotent brain and retinal progenitor cells (RPCs). Expression of GD2-ganglioside was also observed in hRPCs. Ki-67 is a marker of active proliferation and was used to confirm that the cells of this embodiment were proliferative and mitotically active at the time of clinical use. The presence of this marker distinguishes hRPCs from postmitotic precursor populations (Surani, MA and McLaren A. (2006) Nature 443(7109):284-285). Ki-67 in the relative absence of OCT4 expression distinguishes RPCs from mitotically active pluripotent stem cells (ES, iPS) that are not safe for transplantation unless they undergo further differentiation. The level of Ki-67 activity also allowed monitoring of the quality (health and fitness) of the hRPC cultures being isolated. β3-tubulin is a marker of neuronal development. It was found to be expressed at moderate levels in the cultures, suggesting potential for neural lineage commitment and differentiation into neurons. As neuronal formation tends to be lost at high passages, expression of this marker can confirm the retention of pluripotency by the cultures. GFAP is a marker commonly associated with glial differentiation, particularly astrocytes, but is also expressed by retinal Müller cells after various perturbations or in culture. Again, the low percentage of GFAP expression observed suggests a proportion of spontaneous differentiation into glial cells and helps confirm the retention of multipotency by the cultures, although GFAP is known to be expressed by immature progenitor cells as well.GDNF is a neurotrophic factor associated with neuronal rescue, including photoreceptors, in several animal models, and although the hRPC populations isolated as described herein can express this factor by ICCs, negative ELISA data indicate that the factor is not necessarily secreted. Additional factors may be more important for RPC-mediated photoreceptor rescue.

[0201] Notably, hRPCs can be distinguished from fibroblasts (hFBs) as shown by marker expression at the RNA level. See Figure 20. This figure shows qPCR heatmaps demonstrating that vimentin is a marker very highly expressed by hRPCs but not fibroblasts, yet still reveals dynamic changes in expression of the relative profile over time in culture. Figures 21A-21C show the results of qPCR experiments comparing hRPCs vs hFBs and extending marker detection. Several genes have been identified that are more abundant in RPCs (AQP4, CD133, GFAP, MAP2, MASH1, nestin, Notch1, recoverin, SIX6, SOX2), and one that is less abundant (and therefore more abundant in hFBs) (KLF4). Expression levels change over time in culture, but the relative predominance between cell types appears to be fairly consistent. Figure 22 contains a list of 11 genes (from about 26 profiles used) that exhibit consistent behavior between different donations (in hRPCs vs. hFBs). Genes that are consistent across all three donations are highlighted in yellow and are as follows: KLF4 (RPCs<FB)、ならびにGFAP、MAP2、ネスチン、リカバリン、SIX6、およびSOX2(RPC> FB).

[0202] The expression levels of selected markers were tracked by real-time qPCR as a function of time in culture. As shown in Figure 23, highly expressed markers tend to moderate from peak expression levels over time. For example, Ki-67 tends to decline over time, which is consistent with the progenitor state and lack of immortalization. MHC and GDNF expression show a moderate increase over time in culture. The change in expression levels of markers in early versus late passage cells was examined. The results are shown in Figure 24. Cell cycle genes and Six6 (a transcription factor critical for retinal development) are most strongly downregulated in late passage cells. Conversely, the neuroprotective factor GDNF tends to be upregulated in late passage, possibly as a result of cell stress.

[0203] An overview of the microarray data distinguishing (human) RPCs from neural stem cells (BPCs) is shown in Figure 25. Principal component analysis shows a clear separation of the RPC dataset (3) from BPCs (3), demonstrating that RPCs as a cell population type can be distinguished from similar brain-derived cell types such as BPCs based on their transcriptome. Volcano plots show the basis of this difference in the form of close to 1000 transcripts that are significantly upregulated in RPCs compared to BPCs, and about 600 transcripts that are significantly downregulated. A dendrogram of transcripts by specific gene categories is used to further represent this comparison and identify specific genes. For example, the transcription factor BHLHE41 (Basic Helix-Loop-Helix Family, member e41) is highly expressed by RPCs and very low expressed by BPCs. This gene encodes a transcription factor that belongs to the Hairy / Enhancer of Split subfamily of basic helix-loop-helix factors. The encoded protein functions as a transcriptional repressor. Other transcription factors preferentially expressed by RPCs over BPCs include HHEX, SOX3 and SOX13, HOXB2, LHX2, KLF10, TLE4, MYCBP, TFAP2A, FOSL1 and 2, FOXD1, NHLH1, GBX2, NEUROD, MET, etc. In terms of signaling molecules, a number of transcripts are shown to be expressed at significantly higher levels by RPCs than in BPCs / neural stem cells, including WNT5A and B, KDR, LIF, CALB1, RGS4, CAV2, IL11, IL1R1, IL1RAP, IL4R, IL21R, CXCL6 and 12, CXCR7, DKK1, HBEGF, SMAD7, BMP2, etc. Similarly, the ECM matrix genes fibronectin, LUM, ALCAM, TGFBI, ECM1, PARVA, and collagens: 4A1, 4A2, 5A1, 5A2, 7A1, 9A2, 13A1, 18A1, and various other ECM genes are also preferentially expressed by RPCs.

[0204] Experiments were performed to test marker expression after differentiation with retinoic acid (RA) in hRPCs. Results are shown in Figure 26. Gene profiles of hRPCs grown in standard growth medium (SM) were compared to RA-based differentiation conditions (SM+RA without GFs). The proliferation marker Ki-67 is decreased, as is vimentin, while the tumor suppressor gene p21 is elevated along with lineage markers such as AIPL-I, MAP2, NRL, CRALBP, GFAP, and recoverin. Increases in both glial and neuronal markers are consistent with the multipotency of the cultured RPC population. Other markers that can be used in practicing the present invention to identify hRPC cells are described in U.S. Pat. No. 7,419,825.

[0205] Additionally, gene expression of hRPCs grown under lowOx conditions compared to ambient Ox was examined (Figure 27). The data show that there was a detectable change in gene expression, with surface markers CD9 and CD73 being upregulated, whereas downregulation of most genes was observed, including the tumor suppressor gene p21, and lineage markers CRALBP, GFAP, MAP2, NRL, and recoverin. These changes are most consistent with a decrease in expression of non-essential genes as cells grow in a sustained manner under these permissive conditions. GDNF is also downregulated, likely due to a decreased need for autocrine neuroprotection.

[0206] We tested the differences in gene expression between hRPCs obtained from different donors and cultured in various cell culture medium conditions and time points. The results are shown in Figures 28-30. Figure 28A shows the results of an experiment in which gene expression was detected by qPCR in SM conditions at different time points, Figure 28B illustrates gene expression by qPCR in SM-UL (first UL, then SM) conditions at different time points, and Figure 29A illustrates gene expression by qPCR in SM-FBS (first SM+FBS, then SM) conditions at different time points. Figure 29B illustrates gene expression by qPCR in SM alone at two different time points. Most of the markers tested showed a decrease in expression over time during culture, while some remained at approximate levels. Notably, of the markers tested, only GDNF expression increased over time during culture. Figure 29C illustrates gene expression by qPCR at the same two time points in SM (after first SM+FBS). As shown in Figure 29B, only GDNF is elevated. Figure 30 provides a summary comparison of these time points.

[0207] qPCR was used to further characterize hRPCs with respect to growth factor pathways and targeted the relative expression of secreted factors. Specific panels included angiogenesis and WNT signaling pathways. Cells used for these experiments were obtained from low passage hRPCs grown under normoxic (20% oxygen) and hypoxic (3% oxygen) conditions, from working cell banks containing higher passage hRPCs grown under hypoxic conditions, or from human fetal retinal tissue at day 0 (donation day), representing the baseline origin of hRPCs. Human fetal RPE (hRPE) cells and human fetal fibroblasts (hFB) were used for comparison.

[0208] Figure 31 is a heatmap analysis of qPCR data for growth factor pathway studies. Each vertical column is a different cell type or treatment condition. Human fetal RPE and fibroblasts were used as comparators (first two columns). The right three columns are hRPCs. One cytokine that was particularly highly expressed in two of the three hRPC banks and moderately expressed in the other bank, but not expressed by RPE or FBs, is SPP1 (osteopontin, OPN), which is currently the primary candidate for a trophic mechanism of action factor. Another candidate is PTN (pleiotrophin), which is also highly expressed, but this factor appears to be less specific than SPP1 (OPN). In terms of expression levels, other potential candidates from this data are MDK (midkine), TGFB1 (TGFβ1), JAG1, VEGFA (VEGF A), PGK1 (phosphoglycerate kinase 1), and depending on the criteria used, other candidates can be argued to various extents such as GDF11, DKK1, PPIA (peptidyl prolyl isomerase A), and LIF, etc. Also, B2M (β2-microglobulin, a component of MHC class I), although not a cytokine per se, is strongly expressed by hRPCs and is a component of MHC class I.

[0209] Where specificity is emphasized, dendrogram clustering indicates that all of the enumeration from HBEGF (heparin-binding EGF-like growth factor, HB-EGF), JAG1, to SPP1 (OPN) show general specificity for hRPC over fetal hRPE and fetal hFB, and thus may contribute to a heterogeneous "cocktail" effect. This includes PTN, and low levels of IL1B (interleukin 1β). 20% MCB may have maximum trophic efficacy, which is what the enumeration from MDK to LEFTY2 targets. UBC (ubiquitin C) was also detected. Several of these genes are known to play a role in retinal development, including GDF11, Lefty, Nodal, DKK1, LIF, etc. Many are known to be neurotrophic, including SPP1, NTF3, HB-EGF, or to be involved in neurodevelopment, for example midkine, neuregulin (NRG1,3), JAG, etc.

[0210] Another summary of qPCR data from the growth factor pathway study is shown in Figure 32, here presented as a histogram. Human fetal RPE cells were used for comparison. When examined quantitatively for expression levels, secreted genes that showed particularly high expression by hRPCs included FGF9, GDF10, IL-1A (Interleukin 1 alpha), PTN, and SPP1 (Osteopontin, OPN). All of these genes were found to group in what appears to be a relatively hRPC-specific cluster of the heatmap, as above, and are therefore likely candidates for mediating trophic mechanisms. Other genes showing lower relative expression, but still high expression, include BMP2, FGF7, 13, 14, Lefty 1, 2, Nodal, NTF3, thrombopoietin, and potentially VEGF A, C. Genes preferentially downregulated by hRPCs compared to hRPE were JAG2, NGF, inhibin beta B (INHBB), and IL-10.

[0211] RPCs are active before and during the retinal vascularization period, and therefore can be expected to play a role in angiogenesis, especially when they begin to differentiate.The factors and receptors involved in the molecular pathways that regulate angiogenesis can also potentially be neurotrophic in the context of degenerative retina.The activation or inhibition of these pathways can also be important in a range of retinal conditions, including AMD, either as beneficial or as undesirable side effects.

[0212] All genes shown in Figure 33A were upregulated at least 10-fold compared to RPE. Genes upregulated >100-fold included the VEGF receptor KDR, chondromodulin / LECT1 (hypoxic conditions only), transcription factor PROX1, and receptor TEK (TIE2). The factor with the highest expression compared to RPE was pleiotrophin (PTN), >10,000-fold for hypoxic hRPCs and close to 10,000-fold for normoxic cells.

[0213] Clear evidence of increased expression of angiogenesis-related genes, and additional confirmation of high levels of PTN expression, one of the top trophic factor candidate genes, was detected. Elevated expression of surface markers KDR and TEK, both identified in previous screens, was also confirmed here. The VEGF receptor KDR was consistently found to be elevated in RPCs versus other cell types. Microarray data also showed KDR expression 80-fold higher in hRPCs than hBPCs (brain progenitor cells) and 106-fold higher in porcine RPCs than BPCs. Thus, KDR is a potentially useful surface marker for the identification and enrichment of RPCs, a way to distinguish RPCs from BPCs (neural stem cells), and likely important for RPC function.

[0214] The WNT pathway is believed to be important for neural development throughout the CNS, including neuronal and glial differentiation, and including the retina. Genomic studies have previously identified substantial evidence of WNT pathway activity in hRPCs based on gene expression levels of WNT-related genes, including WNT, "frizzled" receptors, and WIF (WNT inhibitor). In Figure 33B, all genes shown were upregulated at least 10-fold compared to RPE. Genes that were upregulated >100-fold included FRZB, SFRP4 (normoxia), TLE2 (hypoxia only), and WNT7B (normoxia). Both SFRP4 (hypoxia) and WNT7B (hypoxia) were upregulated at levels >1000. Barely reaching a 10,000-fold change was WIF1 (hypoxia only), which showed an approximately 10,000-fold change in expression.

[0215] In summary, clear evidence of WNT pathway gene expression was observed by microarray analysis, including prominent expression of WNT inhibitor genes. Previous microarray data had already shown that WIF1 is preferentially expressed by hRPCs 45-fold over hBPCs (brain). The prominent upregulation of SFRP4 and especially WIF1 (both of which may result from FRZB activation) appears to be characteristic of hRPCs grown under hypoxic conditions. This relates to the ability of hRPCs to better maintain their immature state and grow for extended periods under hypoxic conditions, thereby greatly increasing the yield of these difficult-to-grow cells. The qPCR data presented above meaningfully intersect with previous results obtained from microarrays. These showed that LIF is preferentially expressed in hRPCs over hBPCs (brain) by 65-fold, HB-EGF by 30-fold, DKK1 (dickkopf 1) by 23-fold, osteopontin (SPP1, OPN) by 6-fold, TGF β1 and BMP2 by 5-fold, and JAG1 by 3-fold. Each of these genes contributes to the composition / identity of hRPCs compared to similar brain stem / progenitor cells (neural stem cells), with KDR contributing to an even greater extent (80-fold) and WIF (45-fold).

[0216] Principle component analysis (PCA) data from microarray data shows the ability to distinguish cell populations (treated, non-treated / control) based on overall gene expression patterns. This is also relevant to characterizing hRPCs by showing how closely related the samples are and the extent to which generation (time in culture) and culture conditions (normoxia vs. hypoxia) affect expression patterns.

[0217] Three pairs of fetal eyes (biological replicates) were obtained on the same day and were used to provide RNA (retinal tissue) on day 0 and also to grow RPCs (normoxic and hypoxic), as well as scleral fibroblasts. RNA was later extracted from the cultured cell populations and all were sent for simultaneous microarray analysis.

[0218] Figure 34A presents PCA data, which clearly shows that cultured RPCs differ in gene expression from the fetal retinal tissue from which they were derived, but also from scleral fibroblasts. RPCs are closer to the fetal retina than are fibroblasts. Furthermore, normoxic and hypoxic RPCs can be distinguished, but they appear to form a continuum with younger cells at one end and older cells at the other. In Figure 34B, hRPCs are clearly distinguished from the P0 retinal tissue of origin.

[0219] The effect of time in culture is evident in that the oldest cells (hypoxic WCB) can be differentiated from other younger samples. The difference between normoxic and hypoxic conditions does not show a strong separation.

[0220] Whole genome microarray studies were performed to characterize the composition of hRPCs compared to other cell types, including how different they are from their tissue of origin (fetal retina) and from their grossly damaged, dangerous tumorigenic analogue, retinoblastoma (RB). Also, studies were performed to delineate similarities and differences between normoxic and hypoxic hRPC cultures. Global gene expression of the cell populations was compared on Affymetrix human gene chips.

[0221] Principal component analysis (PCA) provides an immediate three-dimensional visualization of the overall similarities and differences between cell sample populations. See FIG. 34C. Classifying similar samples together (all the same color, all triplicates) demonstrates the reliability of the data. A clear separation between fetal retinal tissue ("retina") was observed in comparison to the four different hRPC samples. RPCs differed somewhat between each treatment condition, but differentiated from the other ocular cell types tested, i.e., fetal RPE, fetal FB, and RB. A line can be drawn to separate neural retina and neural retina-derived cells (left) from non-neural retinal cells (top right). Finally, normoxic and hypoxic hRPCs are relatively close to each other, but it may be possible to differentiate them to some extent, as they appear to be closely related. The data shown is consistent with previous data on the relative similarities / differences of these different cell types. FIG. 35 shows a cluster analysis of the three hRPC populations (in order: hypoxic MCB, normoxic MCB, hypoxic WCB) versus fetal retinal tissue (triplicates used for each population). Figure 35 is a volcano plot showing the results of a comparative experiment between hypoxic MCB and tissue. The data show that the hRPC population is distinguishable from the original tissue population. With few exceptions, genes seen as red in tissue (right column) are green in RPCs and vice versa.

[0222] Figure 37A shows the number of genes differentially expressed between hRPC groups as a function of treatment condition (fetal retinal tissue was used as a comparator). The majority of the differences are accounted for by changes to the tissue (11, 706, center, grey), which are shared among the RPC populations, while individual overlaps and differences are seen at the periphery of the figure. Each hRPC population expresses between about 1300-2100 distinct genes. The number of genes differentially expressed between hRPC groups was measured as a function of passage and treatment condition. See Figure 37B. Cells grown under normoxic conditions were also compared. The hypoxic MCBs are not as different as the normoxic MCBs (at the same passage number) and the hypoxic WCBs at a later passage number. Thus, while time in culture does have a demonstrable effect on gene expression in hRPCs, this is much lower than between cell types, or between hRPCs and the tissue of origin.

[0223] Figures 38A and 38B are volcano plots showing different hRPCs versus tissue of origin, while the volcano plots in Figures 39A and 39B show hypoxic versus normoxic hRPCs. "Volcano" refers to a style of plot showing each gene as a data point plotted against fold change (up or down, X-axis) and statistical significance (function of p-value, Y-axis), providing an overview of how many genes are changing, how much and in what direction (up vs. down). The results show that a greater number of genes change consistently going from tissue to hRPC than between hRPC conditions. In the former, more genes are downregulated than upregulated, presumably because more differentiated cell types present in the tissue are lost in culture or overgrow with more primitive proliferative types. However, when comparing hypoxia and normoxia, more genes are upregulated in hypoxic conditions versus normoxia.

[0224] Specific genes and pathways were similarly examined by microarray analysis. hRPCs, fetal retinal tissue (day 0), and human fetal fibroblasts (normoxia) were compared. Adrenomedullin was upregulated in hRPCs versus tissue. Pleiotrophin was found to be upregulated in hRPCs versus tissue and hFBs. Osteopontin was upregulated in hRPCs versus hFBs. Angiogenesis pathways were also found to be upregulated. Angiopoietin was generally elevated in hRPCs versus tissue and to a lesser extent in hFBs. ANGPTL4 is upregulated 30-60-fold in hRPCs versus tissue and approximately 20-fold upregulated versus hFBs. BAI3 (an inhibitor) is downregulated with an approximately 30-fold decrease in hRPCs versus tissue. Thrombospondin 1 is upregulated approximately 100-fold in hRPCs versus tissue, while thrombospondin 2 is upregulated approximately 40-fold in hRPCs versus tissue. Matrix metallopeptidase 1 is upregulated approximately 200-fold in hRPCs versus tissue. The adhesion molecule NCAN is downregulated 16-21-fold in hRPCs versus tissue. The oncogene / proliferation-related genes MYC, MYCN, and NBL1 were also measured. MYC is upregulated 8-9-fold in hRPCs versus tissue, consistent with hRPC proliferation. MYCN (v-myc-associated, neuroblastoma) is downregulated 25-40-fold in hRPCs versus tissue. NBL1 is downregulated 3-4-fold. ATP synthase H + A broad range of mitochondrial / metabolism-related genes, including transporter and solute carrier family 25, were all generally upregulated 2- to 4-fold in hRPCs versus tissue, whereas SLC25A27 (member 27) was downregulated approximately 20-fold. These data are consistent with the notion that cultured hRPCs are more metabolically active and proliferative than cells in the fetal retinal tissue of origin.

[0225] Growth factor expression was also examined. CTGF (connective tissue growth factor) is strongly upregulated (>70-fold) in hRPCs versus tissue. LIF (leukemia inhibitory factor) is similarly upregulated in hRPCs versus tissue. BDNF and EGF are moderately upregulated (2-14-fold) in hRPCs versus tissue. CNTF is downregulated approximately 25-fold in hRPCs versus tissue. FGF9, a candidate trophic factor based on previous studies, is confirmed to be upregulated (4-6-fold) in hRPCs versus hFBs, but is reduced 14-25-fold versus tissue. FGF5 is the most strongly upregulated (20-40-fold) family member in hRPCs compared to tissue, but expression is lower versus hFBs. FGF14 is the most downregulated, 16-38-fold versus tissue. HGF (hepatocyte growth factor) is downregulated 30-fold versus hFBs. Members of the insulin-like growth factor binding protein family, i.e., IGFBP3, -5, and -7, are generally upregulated in hRPCs versus tissues. Members 3, 5, and 7 are all strongly upregulated (15-110 fold). IGFBP3 and 5 are most strongly upregulated, especially in hypoxic cells (MCB, WCB). Candidate factor NTF3 was not elevated versus tissues, but was elevated 8-fold in normoxic hRPCs versus hFBs. NTRK2 (neurotrophic tyrosine kinase, receptor, type 2) was moderately upregulated in hypoxic hRPCs versus tissues, and strongly upregulated in all hRPCs versus hFBs (50-180 fold). Another candidate trophic factor, PDGFC (platelet-derived growth factor C), was upregulated 20-fold versus tissues. VEGFA (VEGF A) was moderately upregulated by hypoxic hRPCs. DACH1 was moderately higher in hFBs but strongly downregulated in hRPCs versus tissues. DLG2 (a synaptic marker) was also strongly downregulated in hRPCs versus tissues. KLF family members showed downregulation of KLF4 (3-4 fold) versus hFBs and upregulation of KLF5 (4-5 fold) versus tissues.

[0226] NeuroD1 (a differentiation-associated transcription factor) was very strongly downregulated in tissues (<300-fold), as was NeuroD4 and neurogenin 1 (and 2), and neuronatin (2- to 10-fold). NOG (noggin), a developmental fate specification-associated factor, is strongly expressed by hRPCs versus tissues (10- to 21-fold). OTX2 (an eye transcription factor) was very strongly downregulated in hRPCs versus tissues. PAX6, SIX3, SIX6, RAX, and RAX2 (eye transcription factors) were only moderately downregulated in hRPCs versus tissues. PAX6 is at least known to be expressed by at least some RPCs.

[0227] DCX (a postmitotic neuroblast marker) and the migratory neuroblast marker RELN were strongly downregulated relative to tissue (the latter in hypoxic hRCs). SOX2, a key neurogenesis transcription factor, was unchanged relative to tissue but very strongly upregulated (>100-fold) in hRPCs relative to hFBs. The adult retinal synaptic markers bassoon and recoverin (adult retinal cell markers) were strongly or very strongly downregulated relative to tissue (20-fold and 50-450-fold, respectively).

[0228] The surface glycoprotein CD44 was upregulated 6-fold in hRPCs versus tissue. The CCL2 chemokine (MCP-1) was strongly upregulated (68-105-fold) in hRPCs versus tissue. CXCL12 (SDF-1) is a ligand for the (non-specific) hRPC surface marker CXCR4. SDF-1 is downregulated compared to hFBs and therefore expression is low (as seen by ELISA), but this is elevated 4-12-fold in hRPCs versus tissue. CXCR4 is a surface marker for hRPCs and was clearly upregulated in hRPCs versus hFBs. The comparison with tissue was important in that normoxic MCB was lower than tissue, but hypoxic cells were similar. This confirms that CXCR4 is expressed by both hRPC conditions, more so in hypoxia, and retinal tissue is similar to hypoxic cells.

[0229] IL-11 and IL-18 were upregulated relative to tissue. IL-1A (IL-1α) was upregulated relative to tissue but not in the hypoxic MCB. Several IL receptors were upregulated relative to tissue, most notably IL-7R (60-105 fold), IL-31RA (28-80 fold), and IL-4R (18-40 fold). These all represent potential positive markers of hRPCs relative to the tissue of origin.

[0230] WNT pathway genes upregulated relative to tissue included several previously identified by qPCR, including WNT7B, and SFRP4. DKK2, FZD6, SFRP1, and WNT5A were also upregulated in hRPCs relative to tissue. WIF1 was strongly upregulated relative to hFBs (for hypoxia) but strongly downregulated relative to tissue (for normoxia). Notch pathway genes were downregulated or unchanged relative to tissue, with the exception of Jag1, a candidate hRPC trophic marker, which was upregulated (8-13 fold). DLL4 and HEYL were most strongly downregulated.

[0231] JAK-STAT genes were moderately but uniformly downregulated for hFBs (2-5 fold) with the exception of STAT3, which was unchanged for hypoxia versus FBs. These were unchanged for tissues. Apoptosis genes were mixed. GADD45B was the most elevated for tissues. GADD45G and DAPL1 were more significantly downregulated (20-56 fold). BMP2 (candidate) was upregulated for hFBs but unchanged for tissues. Other BMPs were generally decreased for FBs and also unchanged for tissues. TGFβ genes were unchanged throughout.

[0232] HIF1A (HIF1α) was moderately decreased (4-fold) relative to tissue. Neuropilin 1 and especially 2 appear to be increased (except in hypoxic MCBs). RICTOR was moderately decreased (2-fold) relative to tissue. Toll-like receptors 3-7, and 9 were unchanged relative to tissue. DCX was strongly downregulated relative to tissue, with NRXN1 (neurexin 1, 25-fold less) being the next most downregulated, as were several other neural markers that were downregulated to a lesser extent. GFAP was clearly upregulated relative to hFBs and less obviously upregulated relative to fetal retinal tissue. A number of retina-related genes are strongly downregulated by hRPCs relative to the fetal retinal tissue of origin. These include CRX, EYS, IMPG1, 2; NRL, recoverin, RGR, RP1, and VSX1, 2. Of the 98 small non-coding RNAs (SNORDs) examined, almost all were downregulated in tissues, sometimes in large amounts, except for 114-6, 49B, 75, and 78, while 114-2, 44, 49A, 74, 79, and 96A were suggestive but somewhat inconsistent between conditions. In any case, the upregulation was not to a high level.

[0233] In summary, the data show that CTGF is a hRPC marker and can be useful for determining the trophic mechanism underlying therapeutic efficacy.Other such markers include SPP1 (OPN), PTN, LIF, FGF9, JAG1, IL-1A, IL-11, IL-18, and Noggin.NTRK2 neurotrophin receptor is a hRPC surface marker, as are interleukin receptors IL7R, IL-31RA, and IL-4R.In particular, HGF seems to have potential as a negative marker for hRPCs, or alternatively in methods for detecting contaminating cell types. Downregulation of ocular transcription factors DACH1, OTX2, CRX, NRL, VSX1, 2, as well as differentiation transcription factors NeuroD1 (and 4), postmitotic blastema marker doublecortin (DCX), synaptic marker DLG2, many notch pathway genes, neurexin 1, as well as adult retinal marker recoverin, and interphotoreceptor matrix genes IMPG1, 2 are also useful in distinguishing cultured hRPCs from the tissue of origin. Cells expressing these markers are more specified, mature, and less proliferative, and therefore much less abundant than those in proliferative hRPC cultures. CPA4 is highly upregulated, whereas PAR4 and many SNORDs are downregulated.

[0234] Similar data were obtained for feline RPCs. In Figure 40, a tabular summary of experiments testing cell culture conditions over time is shown. Figures 41-44 show the results of experiments measuring gene expression in feline RPCs by qPCR in UL medium at various time points. Notably, in UL medium, the markers tested were downregulated, and the progenitor markers nestin and vimentin showed elevated expression relative to the day 0 baseline. Cyclin D2 was elevated in UL at time points after the day 0 baseline. The pattern of change in expression within the profiles tested for feline cRPCs is presented as a radial graph in Figure 45. Genes with high copy number are toward the center of the graph, while genes with lower expression are at the periphery. Progenitor markers are listed clockwise (on the radial graph) from nestin at 12 o'clock to vimentin at approximately 6:30. Lineage markers are also shown. Note that expression is highest across the markers at day 0 (dark blue) and tends to decrease over time for some, but not all, markers. The greatest decrease in expression appears to occur from day 0 to the first time point in culture measured (day 31). The decrease in expression is seen in a subset of both lineage and progenitor markers. A chart summarizing the expression data for feline RPCs is presented in Figure 46, which depicts the qPCR data across different donors and culture conditions.

[0235] Enzyme-linked immunosorbent assays (ELISAs) were performed to characterize hRPCs and potential mechanisms of action (i.e., neuroprotection). These tests were performed by multiplex assays or sandwich ELISAs. For multiplex ELISA assays, assay buffer was added to a 96-well filter plate and the plate was placed on a shaker for 10 minutes. The plate was then cleaned by vacuum and 25 μl of assay buffer or other appropriate buffer was added to each well and 25 μl of standard / sample / control was added to the appropriate wells. 25 μl of a mixture containing the requested cytokine conjugated to beads (1:50 dilution) was then added. The plate was then placed on a shaker overnight at 4°C. The plate was then washed three times. Detection antibodies were added and the plate was placed on a shaker for 1 hour at room temperature. 25 μl of phycoerythrin (1:25 dilution) was then added to each well on the shaker for 30 minutes. The plate was then washed three times and 150 μl of Sheath Fluid was added to each well. Plates were then read using a Luminex 100 reader and Softmax Pro software. Data was calculated using Millipore's BeadLyte Software.

[0236] For sandwich ELISA, markers were measured by two-antibody ELISA using biotin-strepavidin-peroxidase detection. Capture antibody was coated onto polystyrene plates overnight at 25°C. Plates were washed four times with 50 mM Tris, 0.2% Tween-20, pH 7.0-7.5, then blocked with assay buffer for 90 min at 25°C. Plates were washed four times and 50 μl of assay buffer was added to each well along with 50 μl of samples or standards prepared in assay buffer. Plates were then incubated for 2 h at 37°C. Plates were washed four times and 100 μl of biotinylated detection antibody in assay buffer was added and incubated for 1 h at 25°C. Plates were washed four times followed by the addition of streptavidin-peroxidase polymer (RDI) in casein buffer and incubation for 30 min at 25°C. Plates were washed four times and 100 μl of commercially prepared substrate (TMB; Neogen) was added and incubated at 25 °C for approximately 10–30 min. The reaction was stopped with 100 μl of 2 N HCl and A450 (-A650) was read on a microplate reader (Molecular Dynamics). Curves were fitted against standards using a computer program (SoftPro; Molecular Dynamics) and cytokine concentrations in each sample were calculated from the standard curve equation. Data reflect protein secretion from the averaged samples. Expression of five candidate trophic factors GDNF, BDNF, VEGF, OPN, and SDF-1 was assessed under standard normoxic (20% O2) and hypoxic (3% O2) conditions.

[0237] In FIG. 47, GDNF expression was found to be undetectable (less than 0.1 picogram / ml) at the level of secreted protein. BDNF expression was out of range (OOR) on the low side, but barely detectable at an average concentration of 1.58 pg / ml for normoxic conditions and 0.55 pg / ml for hypoxic conditions. VEGF was on the low side OOR for normoxic (20% O2) conditions, but detectable for hypoxia (3% O2) at an average concentration of 92 pg / ml. OPN (osteopontin, SPP1) expression was strongly positive under both conditions (nanogram range instead of picogram) and was approximately twice as high for normoxic versus hypoxic (average = 72.2 ng / ml versus 31.3 ng / ml, respectively). These are notable levels in both cases, suggesting a role for this known neuroprotective / anti-apoptotic factor. SDF-1 (stromal cell-derived factor 1) was on the lower side of OOR but was detectable at a mean concentration of approximately 48 pg / ml for both normoxic and hypoxic cultures.

[0238] Osteopontin (OPN, SPP1) is highly expressed by hRPCs and secreted into the surrounding medium at concentrations predicted to be physiologically significant. Other expression data have shown differential expression of this gene relative to other cell types. Taken together, these data suggest that OPN may play a role in the neuroprotective / cone reactivation effects of hRPCs. Other factors tested are unlikely to play such a role (unless they happen to be massively upregulated after transplantation in response to the vitreous / degenerative retinal microenvironment, which is unlikely).

[0239] Fluorescence-activated cell sorting was performed to characterize hRPCs and reveal the extent of marker expression within the population. Cultured cells or dissociated retinal single cells were either stained with conjugated surface antibody markers / isotype control (BD) or fixed and permeabilized followed by conjugated intracellular antibody markers / isotype control (BD) staining for 30 min at room temperature. After three washes in staining buffer (BD), cells were run on a BD Aria II Sorter. Data were obtained from the same three simultaneously donated fetal eyes used for microarray studies.

[0240] Figure 48 shows the expression of 10 markers, either lineage-associated or potentially associated surface or genetic markers, in retinal tissue at day 0 compared to cultured hRPCs grown under normoxic (20%) or hypoxic (3%) conditions. The data show differences between the retinal tissue of origin and cultured RPCs, particularly the massive upregulation of MHC class I, but not MHC class II, with a large increase in Fas (CD95). GFAP is increased to a lower but lesser extent. Other markers are altered to various degrees.

[0241] Example 3 In vivo efficacy of RPC RPCs were prepared for transplantation by first harvesting with TrypLE Express and collecting by centrifugation at 1000 rpm for 5 min. Cells were washed once in HBSS and then resuspended in cold HBSS to determine cell viability and cell number. For transplantation into humans, 0.5 × 10 cells were collected in 100 μl of HBSS. 6 Cells were used in various doses ranging from 4000 to 75,000 cells in 2 μl of HBSS for transplantation into rats.

[0242] Human RPCs were transplanted as a suspension into the vitreous or subretinal space of hooded RCS rats. Rats were maintained on cyclosporine A and steroids to avoid xenograft rejection. Sham injections (subretinal, intravitreal) consisting of vehicle alone were administered to control eyes. Transplanted animals were functionally tested in an unrestrained awake state using a commercially available instrument designed to quantify the optokinetic response (OR). A subset of animals was tested for luminance thresholds across the entire visual field. This was done by electrophysiology via extracellular recordings in the contralateral superior colliculus. At the end of the study, eyes were collected, fixed, and histologically analyzed for evidence of host photoreceptor rescue and donor cell survival.

[0243] FIG. 49 illustrates the proof of concept of the method of the invention for use in in vivo transplantation. hRPCs (or vehicle alone, "sham") were injected into the eyes of dystrophic RCS rats (a model of inherited photoreceptor degeneration). Injections were made into either the vitreous or subretinal space. Animals with hRPC grafts in either location performed significantly better than sham or untreated controls at 60 days of age. Histology at that time point showed widespread rescue, which was located in the area of ​​the subretinal injection (FIGS. 50-51) or extended throughout the retina in the case of intravitreal transplants (FIGS. 52-53). Some cases were further examined for luminance thresholds at 90 days of age across the entire visual field using electrophysiological recordings in the contralateral superior colliculus. Animals with grafts exhibited significant improvement in sensitivity compared to sham or untreated controls. Histology at 90 days showed sustained high-level rescue of host photoreceptors. Figures 54-55 show the results of immunocytochemistry performed on all RCS specimens.

[0244] The in vivo data confirms and demonstrates that hRPC transplantation is successful at the functional and anatomical levels in rat models. The data also show that in certain circumstances, intravitreal injection may have advantages over subretinal transplantation in terms of the degree of host retina rescued. RPCs are also effective when placed subretinal, even if in a more restricted manner (such restrictions of rescue also apply to subretinal placements where various investigators have used most, if not all, non-malignant cells).

[0245] Example 4 Clinical trials of hRPC transplantation in patients A prospective, open-label, feasibility study of intraocular injection of RPC in patients with retinal disease to obtain preliminary safety data was conducted. After initial evaluation of cells and tissues for safety, clinical trials were initiated. Tissue was supplied by Advanced Bioscience Resources, Inc. (ABR). This company can also provide tissue samples with GTP levels that are expected to form the basis for establishing a CGMP master cell bank based on this technology. Pathology and blood testing of donor samples for exposure to adventitious agents (e.g., HIV, Hepatitis B and C viruses, cytomegalovirus) was performed, as well as tests for endotoxin, mycoplasma, and fungi, such as the Limulus amebocyte lysate (LAL) test (kinetic turbidimetric method) for endotoxin detection, and the Fungitell dynamic dye method (kinetic turbidimetric method) for detection of fungal contamination. Final container sterility tests by PCR chromogenic method or direct inobulation were performed on culture donor samples. Cell culture media were collected and measured in our laboratory or at the Mycoplasma Core Service using the LookOut Mycoplasma PCR Detection Kit (Sigma) or MycoAlert Mycoplasma Detection Kit (Lonza). Culture donor samples were also subjected to soft agar assay to test for tumorigenicity. 0.2 × 10 cells from different donations and different time points were cultured in a soft agar assay. 5 cells or 1.0 x 10 5 A cell suspension of cells was seeded in growth medium containing 0.35% agar and then placed on a 0.7% agar gel. After 28 days of incubation, colonies were stained with 0.005% crystal violet and scored for growth compared to positive and negative controls. Cells for transplantation were also tested for telomerase activity. One microgram of cell protein from different donations and different time points in culture was tested using a TRAPEZE RT Telomerase Detection Kit (Chemicon) with TITANIUM Taq DNA Polymerase (BD Clontech) according to the manufacturer's instructions. In addition, cells were karyotyped by a third-party company to detect the presence of any abnormalities, since the presence of these abnormalities may indicate spontaneous immortalization and cancerous behavior of cultured progenitor cells. Finally, cell viability and cell number were determined by trypan blue (Invitrogen) staining, by a Countess automated cell counter (Invitrogen), or by manual counting using a hemocytometer (Fisher Scientific).

[0246] Clinical laboratory results showed that blood samples from the donors were negative for lethal viruses and other adventitious agents. The transplantation cells were also negative for endotoxin, mycoplasma, and fungal infections. Furthermore, the cells prepared for transplantation did not form colonies in soft agar, indicating that they lacked tumorigenicity. Karyotyping was also performed to ensure the clinical safety of the cultured hRPCs (outsourced to an established FDA-reputed supplier, Cell Line Genetics). Karyotyping did not reveal any abnormalities. For cell viability and cell number, the harvested cells were left in transplantation medium on ice outside the incubator for various lengths of time. A subset was also expelled through a 27g hypodermic needle to assess its effect on survival. Expulsion through the subcutaneous tissue had no detectable effect. The expected actual procedure time is less than 1 hour. Survival outside the incubator began to appreciably decline at 2.5 hours but remained above 90% (acceptable) by 3.5 hours, suggesting that the clinical transplantation procedure had no significant effect on donor cell survival.

[0247] Cells prepared for transplantation exhibit low or moderate, but not high, levels of telomerase activity. Telomerase activity is generally downregulated in mammalian cells beyond early embryonic development, except in certain cells, including cells of the germ line, which should be effectively immortal and transmit indefinitely. The normal loss of telomerase activity is associated with a limited life span of the organism, but also suggests a low probability of tumorigenicity. Increased telomerase activity is found in mammalian cancer cells, and pluripotent stem cells, and immortalized human cells in culture. The strong association with malignant activity means that this is potentially dangerous for transplanted cells with elevated telomerase levels. Safety studies show that the conditions for preparing cells for transplantation do not induce elevated telomerase activity over a range of time points in culture up to 97 days (i.e., beyond the current usable limit). Indeed, telomerase activity tends to decrease over time, which is consistent with developmental "senescence." In other words, cells eventually enter a senescent state and lose the ability to proliferate.

[0248] Eligible patients received cells unilaterally. Eligibility was determined by selecting patients with one of the following: severe end-stage retinal or optic nerve disease, poor residual central vision of 20 / 200 or less with corrective lenses, and / or dismal visual prognosis. Patients must also have adequate pupil dilation and clear media to allow stereoscopic fundus photography, intraocular pressure of 21 mmHg or less, and wide anterior chamber angles. Patients were excluded if they presented with a history of narrow anterior chamber angles, anterior chamber synechiae or neovascularization, angle-closure glaucoma, or significant pre-existing media opacity that would interfere with observations during treatment, fundus examination, visual acuity measurements, or general evaluation of toxicity; any intraocular surgery in the same eye within 3 months prior to study entry; known severe allergy to the fluorescein dye used in angiography; or previous history or evidence of severe cardiac disease (NYHA functional class III or IV), myocardial infarction within 6 months, or ventricular tachyarrhythmia requiring continued treatment, or unstable angina.

[0249] Three legally blind patients with visual acuity of 20 / 200 or less with retinal disease (i.e., retinitis pigmentosa) and poor prognosis for visual improvement were selected. Enrolled patients ranged in age from 46 to 57, two were female and one was male, and all of them had a diagnosis of RP. Two had IOLs and one had cataracts.

[0250] Early passage fetal human retinal progenitor cells were characterized by transcript and protein expression profiles and tested for normal karyotype. Cells were also negative for fungi, bacteria (endotoxin), and mycoplasma. Tissues were also screened for adventitious viruses and were positive for HIV1 and HIV2 antibodies, Hepatitis B antigen, Hepatitis C antibody, Syphilis, Herpes Simplex Virus IgM antibody, West Nile Virus TMA: Singlet, and EBV IgM. It was found to be negative for VCA antibodies. Cells were prepared for intravitral bolus injection in a volume of 100 μl of cell suspension, which delivered approximately 500,000 cells to the eye.

[0251] For 3 days prior to day 0, patients self-treated at home with topical antibiotic drops. On day 0, patients underwent baseline clinical examination, including fundus photography. Eyes were washed and topically administered antibiotics, then pupils were dilated and topical anesthesic drops were administered. In awake, unsedated patients, under sterile surgical conditions and direct visualization by a surgical microscope, cells were injected once through the wall of the globe at the level of the "surgical limbus" into the vitreous cavity, using a standard oblique entry technique to create a self-sealing entrance passage and avoid graft reflux. Anterior chamber paracentesis was performed to prevent iatrogenic elevation of intraocular pressure. No immunosuppressive therapy, suturing or wound closure procedures were required. Post-injection antibiotics were administered thereafter and intraocular pressure was carefully monitored. All patients were released on the same day with an accompanying person and no hospitalization was required. Patients were advised to keep their head elevated at least 45 degrees for at least 2 hours after injection to avoid mass collection of cells on the macula. Prior to release from the hospital, patients were instructed to sleep at home with their heads slightly elevated for 2–3 days after the injection.

[0252] Clinical follow-up was scheduled at 1 day, 3 days, 1 week, 1 month, 2 months, 3 months, 6 months, 1 year, and annually for 5 years after treatment. The incidence and severity of ocular adverse events were determined by standard ophthalmologic examination techniques, including fundus examination, best corrected visual acuity (BCVA), IOP, slit-lamp examination, fluorescein angiography (FA), optical coherence tomography (OCT), stereo fundus photography, and cone flicker electroretinography (ERG) responses.

[0253] Follow-up slit lamp examinations in all patients revealed that the graft cells could be visualized within the vitreous, some of which had solidified into chain-like structures, confirming that the graft cells entered and remained in the eye, and that vitreous placement resulted in aggregation of the vitreous cells. Notably, this did not negate the improvement in vision due to line of sight occlusion (blocking the subject's view from the eye), as could theoretically occur.

[0254] Follow-up B-scans were performed and vitreous cells were tracked by ultrasound device at 4 months postoperatively. Results indicate no intraocular tumor formation and no tumor formation within the anterior orbit (behind the eye). The persistence of graft cells (and sustained visual improvement) suggests that patients receiving grafted cells do not require routine immunosuppression. No evidence of tumor, vascular complications, or retinal detachment was noted on examination or by fundus photography. Notably, no patient experienced a loss of vision due to the procedure, and all patients reported detectable improvement in vision. Improvement was related to disease severity, with worse initial vision resulting in more limited improvement and better initial vision resulting in greater and more rapid improvement. One patient (002) with hand motion vision was able to see the eye chart again at 4 months. Another patient (003) regained central fixation and some macular function (which is required to achieve vision better than 20 / 200). Patient 003 achieved an improvement in visual acuity of "20 letters" on the eye chart, a very large and significant improvement. Vision improvement was maintained and visual acuity gains were sustained for at least up to 1.5 years. The patient also reported better general visual function and improvement in activities of daily living.

[0255] The results of visual acuity tests used to compare trends between patients are shown in Figure 55. None of the scales are exactly linear. Evidence of rapid improvement in vision was observed within the first week in all three cases, mostly consistent with a clinically significant trophic effect on the host retina. A trend toward further improvement that may include engraftment and retinal cell replacement was also noted after one month. Several issues with the patients correspond to certain apparent biases in the trends of the data. Patient 002 (blue) was not compliant with postoperative drops and developed anterior uveitis, which resolved rapidly with standard postoperative medication. Notably, her long-term evolution shows improvement. Patient 001 (pink) had a pre-existing cataract that worsened over the first few months after transplantation, possibly due to the procedure, and may have contributed to a secondary decline in her vision (although vision improved from initial levels). No IOP abnormalities were found associated with transplantation of hRPCs (see Table 1). [Table 1]

[0256] On visual field testing, all three patients had lost central fixation prior to treatment due to poor visual acuity associated with loss of macular function resulting from end-stage retinitis pigmentosa. Patient 003 regained central fixation on day 3 and was able to perform automated visual field testing, which revealed a small 2-degree area (normal) with 20 DB sensitivity. This patient's visual sensitivity was also increased in another area nasal to the foveal fixation point. Now that this patient had regained central fixation, it was possible to test his visual field using automated perimetry (HVF). This testing showed that he had regained a small but highly sensitive island of vision within the foveal region. The data support the concept that this treatment appears to restore function to the remaining host cones, with rapid trophic action from transplanted cells distributed through the vitreous to the retina. Reports of improved night vision (in several patients) and improved ERG performance (in patient 003) also support a role for improved rod photoreceptor function. These improvements may also be due to functional cell integration into the host retina. Other retinal examinations, such as retinal topography, RNFL, and OCT, were also performed. No evidence of tumor formation or immunological tissue rejection was observed.

[0257] In summary, all patients in the clinical trial experienced improvement in vision with treatment. Visual benefits persisted for at least 20 months after injection. At least one patient had an improvement in visual field characterized by return of central fixation. No postoperative complications or evidence of immune rejection were observed despite the absence of immunosuppression or administration of immunosuppressive drugs. No significant donor cell proliferation was noted in vivo based on all clinical examinations, i.e., slit lamp, indirect ophthalmoscopy, ocular and orbital ultrasound (B-scan), and fundus photography. No tumor formation was noted for up to at least 20 months.

[0258] Although some variations have been described in detail above, other modifications or additions are possible. In particular, further functions and / or variations can be provided in addition to those shown herein. For example, the implementations described above may be directed to combinations and subcombinations of the features disclosed and / or combinations and subcombinations of some further features disclosed above. Furthermore, the logic flows depicted in the accompanying drawings and / or described herein do not require the particular order or sequential order shown to achieve desirable results. Other embodiments may be within the scope of the following claims.

Claims

[Claim 1] A method or composition as described in the specification.

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