Photoreceptors and photoreceptor progenitors produced from pluripotent stem cells

Abstract

Methods are provided for the production of photoreceptor cells and photoreceptor progenitor cells from pluripotent stem cells. Additionally provided are compositions of photoreceptor cells and photoreceptor cells, as well as methods for the therapeutic use thereof. Exemplary methods may produce substantially pure cultures of photoreceptor cells and/or photoreceptor cells.

1 . A method of producing eye field progenitor cells, comprising (a) culturing pluripotent stem cells in a retinal induction culture medium. 
     
     
         2 . The method of  claim 1 , wherein said retinal induction culture medium comprises at least one component selected from the group consisting of insulin, DMEM/F12, DMEM/high glucose, DMEM/knock-out, D-glucose, antibiotics, N2 supplement, B27 supplement, non-essential amino acids, MEM non-essential amino acids, glutamine, GlutaMAX™, Noggin, BMP signaling inhibitor, and any combination thereof. 
     
     
         3 - 5 . (canceled) 
     
     
         6 . The method of  claim 2 , wherein said retinal induction culture medium comprises about 450 mg/ml D-glucose or between about 400 and about 500 mg/ml D-glucose, one or both of streptomycin and penicillin, optionally in concentrations of about 100 unit/ml of penicillin and 100 μg/ml of streptomycin, N2 supplement in a concentration of about 0.1 to 5% or about 1%, B27 supplement in a concentration of about 0.05-2.0% or about 0.2%, non-essential amino acids or MEM non-essential amino acids in a concentration of about 0.1 mM, or Noggin at a concentration of between about 5-100 ng/ml or about 10-100 ng/ml or about 50 ng/ml. 
     
     
         7 - 18 . (canceled) 
     
     
         19 . The method of  claim 1 , wherein said pluripotent stem cells comprise human ES cells or human iPS cells. 
     
     
         20 . The method of  claim 1 , wherein said pluripotent stem cells are cultured under feeder-free and/or xeno-free conditions prior to being cultured in said retinal induction culture medium comprising insulin, or cultured on a substrate comprising Matrigel™ and optionally in mTESR1 medium. 
     
     
         21 - 22 . (canceled) 
     
     
         23 . The method of  claim 1 , further comprising (b) culturing the cells in a neural differentiation medium. 
     
     
         24 . The method of  claim 23 , wherein said neural differentiation medium comprises at least one component selected from the group consisting of Neurobasal medium, D-glucose, antibiotics, N2 supplement, B27 supplement, non-essential amino acids, MEM non-essential amino acids, glutamine, GlutaMAX™, Noggin, BMP signaling inhibitor, and any combination thereof. 
     
     
         25 . The method of  claim 24 , wherein said neural differentiation medium comprises about 450 mg/ml D-glucose or between about 400 and about 500 mg/ml D-glucose, one or both of streptomycin and penicillin, optionally in concentrations of about 100 unit/ml of penicillin and 100 μg/ml of streptomycin, N2 supplement in a concentration of about 0.1 to 5% or about 2%, B27 supplement in a concentration of about 0.05-5.0%, about 0.05-2.0% or about 2%, non-essential amino acids or MEM non-essential amino acids in a concentration of about 0.1 mM, or Noggin at a concentration of between about 10-100 ng/ml or about 50 ng/ml. 
     
     
         26 - 37 . (canceled) 
     
     
         38 . The method of  claim 23 , wherein said cells are cultured in said neural differentiation medium for about 10-60 days or about 15-35 days or about 24 days. 
     
     
         39 - 43 . (canceled) 
     
     
         44 . A composition comprising eye field progenitor cells produced according to the method of  claim 1 . 
     
     
         45 . A composition comprising eye field progenitor cells, wherein said eye field progenitor cells comprise at least 50%, at least 75%, at least 85%, at least 95%, at least 99% or about 100% of the cells in said composition. 
     
     
         46 - 49 . (canceled) 
     
     
         50 . The composition of  claim 45 , wherein said eye field progenitor cells are cryopreserved. 
     
     
         51 - 52 . (canceled) 
     
     
         53 . A method of producing retinal neural progenitor cells or photoreceptor progenitor cells, comprising (a) culturing eye field progenitor cells in a neural differentiation medium. 
     
     
         54 .- 88 . (canceled) 
     
     
         89 . A composition comprising retinal neural progenitor cells produced according to the method of  claim 53 . 
     
     
         90 . A composition comprising retinal neural progenitor cells, wherein said retinal neural progenitor cells comprise at least 50%, at least 75%, at least 85%, at least 95%, at least 99% or about 100% of the cells in said composition. 
     
     
         91 - 94 . (canceled) 
     
     
         95 . The composition of  claim 89  or  90 , wherein said retinal neural progenitor cells are cryopreserved. 
     
     
         96 - 97 . (canceled) 
     
     
         98 . A composition comprising photoreceptor progenitor cells produced according to the method of  claim 53 . 
     
     
         99 . A composition comprising photoreceptor progenitor cells, wherein said photoreceptor progenitor cells comprise at least 50%, at least 75%, at least 85%, at least 95%, at least 99% or about 100% of the cells in said composition. 
     
     
         100 - 102 . (canceled) 
     
     
         103 . The composition of  claim 98 , wherein said photoreceptor progenitor cells are cryopreserved. 
     
     
         104 . A method of treatment of an individual in need thereof, comprising administering a composition of  claim 98  to said individual. 
     
     
         105 . (canceled) 
     
     
         106 . A method of producing photoreceptor cells, comprising (a) culturing photoreceptor progenitor cells in a photoreceptor differentiation medium. 
     
     
         107 . The method of  claim 106 , wherein said photoreceptor differentiation medium comprises at least one component selected from the group consisting of Neurobasal medium, D-glucose, antibiotics, N2 supplement, B27 supplement, non-essential amino acids, MEM non-essential amino acids, glutamine, GlutaMAX™, forskolin, BDNF, CNTF, LIF, DATP, and any combination thereof. 
     
     
         108 . The method of  claim 106 , wherein said photoreceptor differentiation medium comprises about 450 mg/ml D-glucose or between about 400 and about 500 mg/ml D-glucose, one or both of penicillin and streptomycin, optionally in concentrations of about 100 unit/ml of penicillin and optionally about 100 μg/ml of streptomycin, N2 supplement in a concentration of about 0.1 to 5% or about 2%, B27 supplement in a concentration of about 0.05-5.0%, about 0.05-2.0% or about 2%, non-essential amino acids or MEM non-essential amino acids are present in a concentration of about 0.1 mM, forskolin in a concentration between about 1-100 uM or about 5 μM, BDNF in a concentration between about 1-100 ng/ml or about 10 ng/ml, CNTF in a concentration between about 1-100 ng/ml or about 10 ng/ml, LIF in a concentration between about 5-50 ng/ml or about 10 ng/ml, or DATP in a concentration between about 1-100 μM or about 10 μM. 
     
     
         109 - 126 . (canceled) 
     
     
         127 . The method of  claim 106 , wherein said photoreceptor progenitor cells are differentiated from retinal neural progenitor cells, which are optionally human. 
     
     
         128 . A composition comprising photoreceptor cells produced according to the method of  claim 106 . 
     
     
         129 - 131 . (canceled) 
     
     
         132 . A method of treatment of an individual in need thereof, comprising administering a composition of  claim 128  to said individual. 
     
     
         133 . (canceled)