PRODUCTION METHOD FOR SHEET-LIKE RETINAL TISSUE
The present disclosure provides a method for manufacturing a cell aggregate comprising retinal tissue having a layer structure.
1 . A method for manufacturing retinal tissue having an epithelial structure, comprising
suspension-culturing or adhesion-culturing a dispersed retinal cell population in a culture medium comprising a Wnt signaling pathway agonist,
wherein the retinal cell population comprises one or more cells selected from the group consisting of a retinal progenitor cell and a photoreceptor progenitor cell,
wherein the culture medium further comprises one or more substances selected from the group consisting of a ROCK inhibitor, a SHH signaling pathway agonist and an FGF signaling pathway agonist, and
wherein the method further comprises (i) differentiating pluripotent stem cells to obtain a cell aggregate comprising one or more cells selected from the group consisting of the retinal progenitor cell and the photoreceptor progenitor cell, and (ii) dispersing the cell aggregate comprising one or more cells selected from the group consisting of a retinal progenitor cell and a photoreceptor progenitor cell to obtain the dispersed retinal cell population.
2 . The manufacturing method according to claim 1 , wherein the Wnt signaling pathway agonist is one or more substances selected from the group consisting of CHIR99021, BIO, Wnt2b and Wnt3a.
3 . (canceled)
4 . The manufacturing method according to claim 3 , wherein the ROCK inhibitor is one or more substances selected from the group consisting of Y-27632, fasudil (HA1077) and H-1152.
5 . The manufacturing method according to claim 3 , wherein the SHH signaling pathway agonist is one or more substances selected from the group consisting of SAG, PMA and SHH.
6 . The manufacturing method according to claim 3 , wherein the FGF signaling pathway agonist is one or more fibroblast growth factors selected from the group consisting of FGF2, FGF4 and FGF8.
7 . The manufacturing method according to claim 1 , comprising performing the adhesion culture in the culture medium comprising the Wnt signaling pathway agonist, wherein the retinal tissue having an epithelial structure is a sheet-shaped retinal tissue.
8 . The manufacturing method according to claim 7 , wherein the adhesion culture is performed using a culture container coated with an extracellular matrix and/or a temperature-responsive polymer.
9 . The manufacturing method according to claim 8 , wherein a culture surface of the culture container is coated with the temperature-responsive polymer, and an upper surface of the temperature-responsive polymer is coated with the extracellular matrix.
10 . The manufacturing method according to claim 8 , wherein the extracellular matrix is one or more substances selected from the group consisting of collagen, laminin, fibronectin, Matrigel and vitronectin.
11 . The manufacturing method according to claim 8 , further comprising the step of exposing the culture container coated with the temperature-responsive polymer to a temperature that changes the properties of the temperature-responsive polymer to thereby detach the sheet-shaped retinal tissue from the culture container.
12 - 13 . (canceled)
14 . The manufacturing method according to claim 1 , further comprising the step of increasing the percentage of a retinal progenitor cell comprised in the dispersed retinal cell population before suspension-culturing or adhesion-culturing the dispersed retinal cell population.
15 . The method according to claim 14 , wherein contamination with or differentiation into retinal pigment epithelial cells is suppressed.
16 . The manufacturing method according to claim 14 , wherein the step of increasing the percentage of a retinal progenitor cell comprises the step of contacting the dispersed retinal cell population with a substance that binds to one or more antigens selected from the group consisting of CD9, CD39, CD90 and CXCR4 to obtain a cell population expressing the antigens.
17 . The manufacturing method according to claim 16 , wherein the step of increasing the percentage of a retinal progenitor cell comprises the step of further contacting the dispersed retinal cell population with a substance that binds to one or more antigens selected from the group consisting of SSEA1, CD66b, CD69 and CD84 to obtain a cell population having expression levels of the antigens that are equal to or less than a reference.
18 . The manufacturing method according to claim 14 , wherein the step of increasing the percentage of a retinal progenitor cell comprises the following steps:
(1) culturing pluripotent stem cells in the presence of one or more selected from the group consisting of a Shh signaling pathway agonist, ATP and an A2A receptor agonist to manufacture a cell aggregate;
(2) differentiating the cell aggregate into a retinal progenitor cell; and
(3) dispersing the cell aggregate and contacting it with a substance that binds to CD39.
19 . (canceled)
20 . The manufacturing method according to claim 1 , wherein the retinal progenitor cell and/or the photoreceptor progenitor cell occupies 80% or more of the total number of cells comprised in the retinal cell population.
21 . The manufacturing method according to claim 1 , wherein from the start of the suspension culture or the adhesion culture, the dispersed retinal cell population is cultured in the culture medium comprising the Wnt signaling pathway agonist.
22 . The manufacturing method according to claim 1 , wherein in the epithelial structure, the orientation of cells is a direction roughly perpendicular to the layer direction.
23 . The manufacturing method according to claim 1 , further comprising the step of dissecting a size necessary for transplantation from the retinal tissue having an epithelial structure obtained by the suspension culture or the adhesion culture.
24 . The manufacturing method according to claim 1 , wherein the epithelial structure is a multilayered structure.
25 - 36 . (canceled)