Method for manufacturing telencephalon or progenitor tissue thereof

Abstract

The present invention provides a method of producing more mature telencephalon or a progenitor tissue thereof, in vitro, from mammalian pluripotent stem cells, comprising obtaining a telencephalon marker-positive aggregate by culturing an aggregate of pluripotent stem cells in suspension in the presence of a Wnt signal inhibitor and a TGFβ signal inhibitor, and further culturing the telencephalon marker-positive aggregate in suspension under a high oxygen partial pressure condition. In one embodiment, the suspension culture under a high oxygen partial pressure condition is performed in the presence of a Wnt signal enhancer and a bone morphogenetic factor signal transduction pathway activating substance.

1 . An artificial cell aggregate comprising a telencephalon marker-positive neuroepithelium-like structure, wherein not less than 70% of the cells contained in the aggregate are Foxg1 positive, and wherein the neuroepithelium-like structure in the aggregate shows a pseudostratified columnar epithelial structure having a cerebral ventricle-like cavity inside. 
     
     
         2 . The cell aggregate according to  claim 1 , wherein the telencephalon marker-positive neuroepithelium-like structure is a semispherical and Foxg1-positive neuroepithelium-like structure. 
     
     
         3 . The cell aggregate according to  claim 1 , wherein the neuroepithelium-like structure has a Pax6 positive and Sox2 positive cell layer in the luminal side. 
     
     
         4 . The cell aggregate according to  claim 3 , wherein the neuroepithelium-like structure contains phosphorylated Histone H3 positive mitotic cells in its innermost part. 
     
     
         5 . The cell aggregate according to  claim 1 , wherein outside of cell layers of the neuroepithelium-like structure, the aggregate contains cells which express a post-mitotic neuron marker Tuj1 and early cortical plate markers Ctip2 and Tbr1. 
     
     
         6 . The cell aggregate according to  claim 5 , wherein the aggregate contains Reelin-positive Cajal-Retzius cells outside of the cell layers of the neuroepithelium-like structure, and has a Laminin-rich layer near a superficial layer of the aggregate. 
     
     
         7 . A method of producing a cell aggregate comprising telencephalon, comprising steps (a), (b), and (c):
 (a) culturing dispersed pluripotent stem cells in suspension to allow for aggregate formation,   (b) culturing the aggregate obtained in (a) in suspension in the presence of a wingless type mouse mammary tumor virus integration site (Wnt) signal inhibitor and a transforming growth factor β (TGFβ) signal inhibitor to induce expression of Foxg1 gene until not less than 50% of the cell aggregates in the culture are Foxg1 positive, thereby producing a Foxg1-positive aggregate, and   (c) further culturing the Foxg1-positive aggregate in suspension, wherein an oxygen concentration in a medium used in the suspension culturing is adjusted to be exceeding the concentration under an oxygen partial pressure in the air.   
     
     
         8 . The method according to  claim 7 , wherein the oxygen concentration in the culture medium in step (c) is adjusted to be equivalent to the concentration under an oxygen partial pressure of 30-60%. 
     
     
         9 . The method according to  claim 7 , wherein suspension culturing in step (c) is performed under an oxygen partial pressure of 30-60%. 
     
     
         10 . The method according to  claim 7 ,
 wherein the Wnt signal inhibitor is selected from a group consisting of IWR-1-endo(4-[(3aR,4S,7R,7aS)-1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl]-N-8-quinolinyl-benzamide), IWP-2, XAV939, Dkk1, Cerberus protein, Wnt receptor inhibitors, soluble Wnt receptors, Wnt antibodies, casein kinase inhibitor and dominant negative Wnt protein, and   wherein the TGFβ, signal inhibitor is selected from a group consisting of SB431542 (4-(5-benzol[1,3]dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)-benzamide), LY-364947, SB-505 and A-83-01.   
     
     
         11 . The method according to  claim 7 , wherein the suspension culturing in step (c) is performed in a medium not containing a Wnt signal inhibitor and a TGFβ, signal inhibitor, and in the presence of a Wnt signal enhancer. 
     
     
         12 . The method according to  claim 11 ,
 wherein the Wnt signal inhibitor is selected from a group consisting of IWR-1-endo(4-[(3aR,4S,7R,7aS)-1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl]-N-8-quinolinyl-benzamide), IWP-2, XAV939, Dkk1, Cerberus protein, Wnt receptor inhibitors, soluble Wnt receptors, Wnt antibodies, casein kinase inhibitor and dominant negative Wnt protein,   wherein the TGFβ, signal inhibitor is selected from a group consisting of SB431542 (4-(5-benzol[1,3]dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)-benzamide), LY-364947, SB-505 and A-83-01, and   wherein the Wnt signal enhancer is selected from a group consisting of GSK-3β inhibitor, recombinant Wnt3a, Wnt agonist, and R-Spondin.   
     
     
         13 . The method according to  claim 7 , wherein the suspension culturing in step (c) is performed in a medium not containing a Wnt signal inhibitor and a TGFβ, signal inhibitor, and in the presence of a Wnt signal enhancer and a bone morphogenetic factor signal transduction pathway activating substance. 
     
     
         14 . The method according to  claim 13 ,
 wherein the Wnt signal inhibitor is selected from a group consisting of IWR-1-endo(4-[(3aR,4S,7R,7aS)-1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl]-N-8-quinolinyl-benzamide), IWP-2, XAV939, Dkk1, Cerberus protein, Wnt receptor inhibitors, soluble Wnt receptors, Wnt antibodies, casein kinase inhibitor and dominant negative Wnt protein,   wherein the TGFβ, signal inhibitor is selected from a group consisting of SB431542 (4-(5-benzol[1,3]dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)-benzamide), LY-364947, SB-505 and A-83-01,   wherein the Wnt signal enhancer is selected from a group consisting of GSK-3β inhibitor, recombinant Wnt3a, Wnt agonist, and R-Spondin, and   wherein the bone morphogenetic factor signal transduction pathway activating substance is selected from a group consisting of BMP2, BMP4, BMP7 and GDF5.   
     
     
         15 . The method according to  claim 7 , comprising treating the cell aggregate with a sonic hedgehog (shh) signal agonist. 
     
     
         16 . The method according to  claim 15 , wherein the shh signal agonist is selected from a group consisting of protein belonging to the Hedgehog family, Shh, Shh receptor agonist, Purmorphamine and SAG (N-Methyl-N′-(3-pyridinylbenzyl)-N′-(3-chlorobenzo[b]thiophene-2-carbonyl)-1,4-diaminocyclohexane). 
     
     
         17 . The method according to  claim 7 , comprising treating the cell aggregate with fibroblast growth factor 8 (FGF8). 
     
     
         18 . A method of producing a cell aggregate comprising telencephalon, comprising steps (a), (b), (c) and (d):
 (a) culturing dispersed pluripotent stem cells in suspension to allow for aggregate formation,   (b) culturing the aggregate obtained in (a) in suspension in the presence of a wingless type mouse mammary tumor virus integration site (Wnt) signal inhibitor and a transforming growth factor β (TGFβ) signal inhibitor to induce expression of Foxg1 gene until not less than 50% of the cell aggregates in the culture are Foxg1 positive, thereby producing a Foxg1-positive aggregate,   (c) further culturing the Foxg1-positive aggregate obtained in (b) in suspension in the presence of a Wnt signal enhancer and a bone morphogenetic factor signal transduction pathway activating substance, and   (d) further culturing the Foxg1-positive aggregate obtained in (c) in suspension in the absence of a Wnt signal enhancer and a bone morphogenetic factor signal transduction pathway activating substance,   wherein an oxygen concentration in a medium used for the suspension culturing in (c) and (d) is adjusted to be exceeding the concentration under an oxygen partial pressure in the air.   
     
     
         19 . The method according to  claim 7 , wherein the obtained cell aggregate comprises a tissue selected from the group consisting of cerebral cortex, basal ganglion, hippocampus and choroid plexus. 
     
     
         20 . A method of producing a mature hippocampal neuron, comprising:
 (i) obtaining a cell aggregate comprising a hippocampus by the method according to  claim 19 , and   (ii) dispersing the cell aggregate obtained in (i) and further subjecting the dispersed cells to adhesion culture to induce a mature hippocampal neuron from the cells.