Generation of midbrain-specific organoids from human pluripotent stem cells

A method of deriving and maintaining a midbrain-like organoid comprising:

• (a) culturing pluripotent stem cells in a first cell culture medium to obtain neuronal lineage embryoid bodies, wherein culturing under (a) is for 3 to 5 days;
• (b) culturing the neuronal lineage embryoid bodies from (a) in a second cell culture medium to obtain midbrain regionalized tissues, wherein culturing under (b) is for 3 to 5 days, wherein the midbrain regionalized tissues are OTX2 positive and negative for FOXG1 and GBX2;
• (c) embedding and culturing the midbrain regionalized tissues from (b) in an extracellular matrix in a third cell culture medium to obtain developing neuroepithelial tissues, wherein culturing under (c) is for 1 to 2 days;
• (d) culturing the neuroepithelial tissues from (c) to obtain a midbrain-like organoid, wherein the neuroepithelial tissues of operation (d) are cultured in a fourth cell culture medium, and,
• (e) maintaining the midbrain-like organoid from (d) wherein the midbrain-like organoid is cultured in a neuronal culture media comprising neurotrophic factor,
• wherein the first cell culture medium comprises:
• (i) TGF-β inhibitor and/or SMAD2/3 inhibitor;
• and
• (ii) WNT-signaling activator;
• wherein the second cell culture medium comprises:
• (iii) TGF-β inhibitor and/or SMAD2/3 inhibitor;
• (iv) WNT-signaling activator;
• (v) hedgehog signaling protein;
• (vi) fibroblast growth factor;
• wherein the third cell culture medium comprises:
• (vii) hedgehog signaling protein;
• and
• (viii) fibroblast growth factor,
• wherein the fourth cell culture medium comprises:
• (ix) neurotrophic factor;
• (x) ascorbic acid;
• and
• (xi) activator of cAMP-dependent pathways,
• and wherein the resulting midbrain-like organoid is OTX2 positive and negative for FOXG1 and GBX2.