Method for maturation of retinal tissue containing continuous epithelium
The present invention provides a method for maintaining a continuous epithelial structure of a retinal tissue including culturing the retinal tissue in a medium comprising a methyl group donor or a substrate of the methyl group donor at a concentration at which cell differentiation of a neural retinal progenitor cell is suppressed, and a neurite extension inhibitor at a concentration at which neurite extension is suppressed.
1 . A method for maintaining a continuous epithelial structure of a human retinal tissue comprising culturing the human retinal tissue in a medium comprising (a) methionine at a concentration not less than 25 mg/L and not more than 75 mg/L, (b) corticosterone at a concentration of not less than 1 nM and not more than 1 μM, and (c) L-glutamic acid at a concentration of not more than 25 μM, wherein the human retinal tissue is derived from pluripotent stem cells, wherein the human retinal tissue is in a differentiation stage from an early developmental stage until a developmental stage when the proportion of NRL positive cells to the number of photoreceptor progenitor cells present in the human retinal tissue reaches 1-3%, and wherein the human retinal tissue does not have a rosette structure.
2 . The method according to claim 1 , wherein the medium comprises L-aspartic acid, and wherein the concentration of the L-aspartic acid in the medium is less than 50 μM.
3 . The method according to claim 1 , wherein the medium comprises at least one kind of antioxidant selected from the group consisting of glutathione, catalase, Superoxide dismutase, alpha-tocopherol, and L-cysteine at a concentration within the following concentration ranges:
glutathione: not more than 100 ng/ml,
catalase: not more than 100 U/mL,
Superoxide dismutase: not more than 100 U/mL,
alpha-tocopherol: not more than 50 nM,
cysteine: not more than 0.26 mM.
4 . The method according to claim 1 , wherein the medium comprises progesterone at a concentration of not more than 100 nM.
5 . The method according to claim 1 , wherein the medium comprises at least one kind of nucleic acid synthesis promoter selected from the group consisting of hypoxanthine, thymidine and vitamin B12 at the following concentration range:
hypoxanthine: less than 15 μM,
thymidine: less than 1.5 μM,
vitamin B12: less than 0.68 mg/L (0.5 μM).
6 . The method according to claim 1 , wherein the medium comprises (a) a medium comprising 30 mg/L glycine, 2 mg/L L-alanine, 84 mg/L L-arginine hydrochloride, 0.83 mg/L L-asparagine·H 2 O, 31.5 mg/L L-cysteine, 42 mg/L L-histidine hydrochloride·H 2 O, 105 mg/L L-isoleucine, 105 mg/L L-leucine, 146 mg/L L-lysine hydrochloride, 30 mg/L L-methionine, 66 mg/L L-phenylalanine, 7.76 mg/L L-proline, 42 mg/L L-serine, 95 mg/L L-threonine, 16 mg/L L-tryptophan, 72 mg/L L-tyrosine, 94 mg/L L-valine, 4 mg/L choline chloride, 4 mg/L D-calcium pantothenate, 4 mg/L folic acid, 4 mg/L niacinamide, 4 mg/L pyridoxal hydrochloride, 0.4 mg/L riboflavin, 4 mg/L thiamine hydrochloride, 0.0068 mg/L vitamin B12, 7.2 mg/L i-inositol, 200 mg/L calcium chloride (anhydrous), 0.1 mg/L ferric nitrate (Fe(NO 3 ) 3 ·9H 2 O), 77.3 mg/L magnesium chloride (anhydrous), 400 mg/L potassium chloride, 2200 mg/L sodium bicarbonate, 3000 mg/L sodium chloride, 125 mg/L sodium phosphate monobasic (NaH 2 PO 4 ·H 2 O), 0.194 mg/L zinc sulfate (ZnSO 4 ·7H 2 O), 4500 mg/L D-glucose (dextrose), 2600 mg/L HEPES, 8.1 mg/L phenol red, and 25 mg/L sodium pyruvate blended with (b) supplement comprising biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, BSA (fatty acid free fraction V), catalase, insulin, transferrin, superoxide dismutase, corticosterone, D-galactose, ethanolamine, glutathione (reduced), L-carnitine, linoleic acid, progesterone, putrescine, sodium selenite, and T3 (triodo-L-thryonine) at a volume ratio of not less than 50%.
7 . The method according to claim 1 , wherein the human retinal tissue is in an initial state of development at the time of start of culture.
8 . The method according to claim 7 , wherein the human retinal tissue is cultured for a period up to emergence of a rod photoreceptor progenitor cell.
9 . The method according to claim 1 , wherein the early developmental stage is a stage where retinal progenitor cells have emerged but ganglion cells have not emerged.
10 . The method according to claim 1 , wherein the early developmental stage is a stage where RX positive and PAX6 positive cells are present, and TUJ1 positive cells and BRN3 positive cells are not present.
11 . The method according to claim 1 , wherein the human retinal tissue in an early developmental stage is produced according to the following steps:
(1) the first step of forming a cell aggregate by culturing pluripotent stem cells in suspension in a serum-free medium, and
(2) the second step of obtaining an aggregate containing a retinal progenitor cell or a neural retinal progenitor by culturing in suspension the aggregate formed in the first step in a serum-free medium or serum-containing medium not containing an SHH signal transduction pathway agonist and containing a BMP signal transduction pathway agonist.
12 . The method according to claim 1 , wherein L-glutamic acid is present in the medium at a concentration of not more than 12.5 μM.