Method for producing cell aggregate including glial progenitor cells
The method for producing a cell aggregate including glial progenitor cells according to the present invention comprises: (1) a step of subjecting pluripotent stem cells to suspension culture in an embryoid-body-forming culture medium containing one or more SMAD signaling inhibitors and one or more Wnt signaling activators in the absence of feeder cells for 5 days to 10 days, to form a cell aggregate; (2) a step of subjecting the cell aggregate obtained in (1) to suspension culture in an embryoid-body-forming culture medium containing retinoic acid; (3) a step of subjecting the cell aggregate obtained in (2) to suspension culture in an embryoid-body-forming culture medium or neuron-and-glia-proliferating culture medium containing retinoic acid and one or more SHH signaling activators; and (4) a step of subjecting the cell aggregate obtained in (3) to suspension culture in a neuron-and-glia-proliferating culture medium containing no retinoic acid and one or more SHH signaling activators.
1 . A method for producing a cell aggregate including glial progenitor cells, comprising:
(1) a step of subjecting pluripotent stem cells to suspension culture in an embryoid-body-forming culture medium containing SMAD signaling inhibitors and a Wnt signaling activator in the absence of feeder cells for 7 days, to form a cell aggregate, wherein the SMAD signaling inhibitors are a TGFβ inhibitor and a BMP inhibitor, wherein the TGFβ inhibitor is SB431542, wherein the BMP inhibitor is LDN-193189, and wherein the Wnt signaling activators is CHIR99021;
(2) a step of subjecting the cell aggregate obtained in (1) to suspension culture in an embryoid-body-forming culture medium containing retinoic acid for 7 days;
(3) a step of subjecting the cell aggregate obtained in (2) to suspension culture in an embryoid-body-forming culture medium or neuron-and-glia-proliferating culture medium containing retinoic acid and a SHH signaling activator, wherein the SHH signaling activator is purmorphamine; and
(4) a step of subjecting the cell aggregate obtained in (3) to suspension culture in a neuron-and-glia-proliferating culture medium containing no retinoic acid and purmorphamine.
2 . The production method according to claim 1 , wherein, in step (1), pluripotent stem cells are cultured by using a culture vessel having a plurality of pores uniform in shape.
3 . The production method according to claim 1 , wherein, in step (1), step (1) is sustained until a cell aggregate satisfying at least one of the following conditions:
1) having an RNA expression level of at least one of SOX1, PAX6, HES4, and HES5 increased by 100 times or more;
2) having an RNA expression level of OCT3/4 decreased by 200 times or more; and
3) having an RNA expression level of NANOG decreased by 400 times or more,
as compared with the cell aggregate at initiation of step (1), is obtained, and step (2) is then initiated.
4 . The production method according to claim 1 , wherein, in step (2), step (2) is sustained until a cell aggregate satisfying at least one of the following conditions:
1) having an RNA expression level of at least one of ASCL1, DCX, HEY1, ZBTB20, βμl tubulin, ELAVL3, and SLIT1 increased by 5 times or more; and
2) having an RNA expression level of at least one of HOXB3, HOXA4, HOXB4, HOXB6, and HOXB8 increased by 5 times or more,
as compared with the cell aggregate at initiation of step (2), is obtained, and step (3) is then initiated.
5 . The production method according to claim 1 , wherein an oxygen concentration is 3% to 10% in steps (1) and (2).
6 . The production method according to claim 1 , wherein, in step (3), step (3) is sustained until a cell aggregate satisfying at least one of the following conditions:
1) Having an RNA expression level of at least one of HEY2, NKX6.2, and NKX2.2 increased by 5 times or more; and
2) having an RNA expression level of OLIG1 and/or OLIG2 increased by 10 times or more,
as compared with the cell aggregate at initiation of step (3), is obtained, and step (4) is then initiated.
7 . The production method according to claim 1 , wherein step (3) is performed for 4 days to 11 days.
8 . The production method according to claim 1 , wherein, in step (4), step (4) is sustained until a cell aggregate satisfying at least one of the following conditions:
1) Having an RNA expression level of at least one of NFIA, NFIB, SLC1A3, S100B, and FABP7 increased by 10 times or more; and
2) Having an RNA expression level of PAX6 decreased by 5 times or more,
as compared with the cell aggregate at initiation of step (4), is obtained, and step (5) is then initiated.
9 . The production method according to claim 1 , wherein step (4) is performed for 4 days or more.
10 . The production method according to claim 1 , wherein, in step (4), the cell aggregate obtained in step (3) is dispersed at initiation of step (4), and the dispersed cells are then subjected to suspension culture, to form a cell aggregate again.
11 . The production method according to claim 1 , comprising step (5), wherein, in step (5), the cell aggregate obtained in step (4) is dispersed at initiation of step (5), and the dispersed cells are then subjected to suspension culture for 5 days to 100 days, to form a cell aggregate again.
12 . The production method according to claim 1 , comprising step (5), wherein, in step (5), step (5) is sustained until one or more markers selected from an O4 antigen, NG2, OLIG2, PDGFRα, SOX10, SPON1, FAM181B, TIMP4, SOX6, GRIK3, LHFPL3, KLF9, an A2B5 antigen, CNP, and PLP are expressed.
13 . The production method according to claim 1 , comprising step (5), wherein, in step (5), step (5) is sustained until one or more proteins selected from the group consisting of SPARCL1, MIF, MCP-1, IL-8, SCF, M-CSF, HGF, GRO-α, LIF, IFN-γ, and TRAIL are detected in the medium for culture of the cell aggregate.
14 . The production method according to claim 1 , wherein the pluripotent stem cells are induced pluripotent stem cells.
15 . The production method according to claim 1 , wherein the pluripotent stem cells are human induced pluripotent stem cells.
16 . The production method according to claim 1 , wherein the cell aggregate including glial progenitor cells has the following characteristics:
(a) including oligodendrocyte progenitor cells, astrocyte progenitor cells, and neural progenitor cells;
(b) expressing a spinal region marker; and
(c) including no feeder cell and containing no xenogeneic-cell-derived component derived from feeder cells.
17 . A method for producing a cell population including oligodendrocytes, astrocytes, and neural cells, comprising a step of culturing a cell aggregate including glial progenitor cells with use of a maturation culture medium for 5 days to 60 days, wherein the cell aggregate is produced by the production method according to claim 1 .
18 . The production method according to claim 17 , wherein the cell population including oligodendrocytes, astrocytes, and neural cells includes:
(i) cells expressing one or more markers selected from the group consisting of an O4 antigen, GalC, MBP, APC, GSTI, CNP, PLP, OLIG2, SOX10, PDGFRα, and NG2;
(ii) cells expressing one or more markers selected from the group consisting of βIII tubulin, MAP2, and ELAVL3; and
(iii) cells expressing one or more markers selected from the group consisting of SLC1A3, S100B, AQP4, GFAP, and NG2.
19 . The production method according to claim 17 , wherein the maturation culture medium is a culture medium containing at least one of T3, NT-3, and LIF.
20 . The production method according to claim 19 , wherein the maturation culture medium further contains CNTF.
21 . The production method according to claim 1 , wherein the cell aggregate has the following characteristics:
(a) including oligodendrocyte progenitor cells, astrocyte progenitor cells, and neural progenitor cells;
(b) including cells expressing a spinal region marker;
(c) including no feeder cell and containing no xenogeneic-cell-derived component derived from feeder cells; and
(d) having an ability to differentiate into a cell population including oligodendrocytes, astrocytes, and neural cells.
22 . The production method according to claim 21 , wherein the spinal region marker is one or more markers selected from the group consisting of HOXB3, HOXB4, HOXB6, and HOXD8.
23 . The production method according to claim 21 , further comprising cells expressing one or more markers selected from the group consisting of NKX2.1, NKX2.2, NKX6.1, and NKX6.2.
24 . The production method according to claim 21 , having the following characteristics:
(I) including cells expressing one or more markers selected from the group consisting of NFIA, NFIB, SOX9, HEY1, HEY2, FABP7, ZBTB20, SLC1A3, S100B, MLC1, SLIT1, TIMP3, SPARCL1, GFAP, and AQP4;
(II) including cells expressing one or more markers selected from the group consisting of OLIG2, PDGFRα, SOX10, SPON1, FAM181B, TIMP4, SOX6, GRIK3, LHFPL3, KLF9, an A2B5 antigen, CNP, and PLP;
(III) including cells expressing one or more markers selected from the group consisting of DCX, Bill tubulin, MAP2, ELAVL3, NTRK2, GRIA2, PTPRO, and EPHA3;
(IV) including cells expressing one or more markers selected from the group consisting of SOX1, SOX2, NESTIN, MEIS1, MEIS2, DLL3, and ASCL1; and
(V) having an ability to differentiate into a cell population including (i) cells expressing one or more markers selected from the group consisting of an O4 antigen, GalC, MBP, APC, GSTπ, CNP, PLP, OLIG2, SOX10, PDGFRα, and NG2, (ii) cells expressing one or more markers selected from the group consisting of βIII tubulin, MAP2, and ELAVL3, and (iii) cells expressing one or more markers selected from the group consisting of SLC1A3, S100B, AQP4, GFAP, and NG2.
25 . The production method according to claim 21 , further (VI) comprising cells expressing one marker or a plurality of markers selected from the group consisting of C1ORF61 and SERPINE2.
26 . The production method according to claim 21 , further comprising cells expressing or secreting one or more markers selected from the group consisting of SPARCL1, MIF, MCP-1, IL-8, SCF, M-CSF, HGF, GRO-α, LIF, IFN-γ, and TRAIL.
27 . The production method according to claim 1 , wherein the following step is performed prior to step (1):
a step of subjecting pluripotent stem cells to maintenance culture in the absence of feeder cells.