Automated method for preparing retinal pigment epithelium cells

An automated method for large-scale production of retinal pigment epithelium (RPE) cells and for promoting directed differentiation of human pluripotent stem cells into retinal pigment epithelium (RPE) cells, wherein the method comprises the use of only three differentiating agents to direct the differentiation of human pluripotent stem cells into RPE cells in the sequential steps of:

• (a) culturing human pluripotent stem cells in a medium supplemented with a first differentiating agent consisting of one Nicotinamide (NA) mimetic compound to generate differentiating cells, for at least 3 days;
• (b) culturing said differentiating cells obtained in step a) in a medium supplemented with a second differentiating agent, consisting of one member of transforming growth factor β (TGF β) superfamily to further differentiating said differentiating cells, for at least 3 days;
• (c) culturing said further differentiating cells obtained in step b) in a medium supplemented with a third differentiating agent, consisting of one activator of the Wnt canonical pathway to induce said further differentiating cells to differentiate into RPE cells, for 20 to 50 days,
• wherein the Nicotinamide (NA) mimetic compound is Nicotimamide,
• wherein the one member of transforming growth factor β (TGF β) superfamily is selected from the group consisting of the TGFβ subfamily, Activin, Nodal, growth and differentiation factors (GDF), bone morphogenetic protein (BMP), and antiMullerian hormone (AMH),
• wherein the one activator of the Wnt canonical pathway is a GSK-3 inhibitor selected from the group consisting of 3F8, 1-Azakenpaullone, 10Z-Humenialdisine, Alsterpaullone, A-1070722, AR-A014418, AZD1080, AZD2858, Bikinin, BIO, Cazpaullone, CT98014, CT98023, CT99021 (Chir99021), Chir98014, Dibromocantharelline, GSKJ2, HMK-32, Hymenialdesine, Indirubin, Indirubin-3′-oxime, IM-12, Kenpaullone, L803, L803-mts, Lithium carbonate, LY2090314, Manzamine A, Meridianin, NSC693868, NP031115, Palinurine, SB216763, SB415286, TCS21311, TC-G-24, TCS2002, TDZD-8, Tideglusib, Tricantine and TWS119, and
• wherein the automated method uses an apparatus for large-scale production of cells comprising:
• a) robotic means for handling culture vessels;
• b) means for inoculating cells into a culture;
• c) means for changing or adding medium to a culture;
• and d) programmable control means.