Cortical interneurons and other neuronal cells produced by the directed differentiation of pluripotent and multipotent cells
Provided are cortical interneurons and other neuronal cells and in vitro methods for producing such cortical interneurons and other neuronal cells by the directed differentiation of stem cells and neuronal progenitor cells. The present disclosure relates to novel methods of in vitro differentiation of stem cells and neural progenitor cells to produce several type neuronal cells and their precursor cells, including cortical interneurons, hypothalamic neurons and pre-optic cholinergic neurons. The present disclose describes the derivation of these cells via inhibiting SMAD and Wnt signaling pathways and activating SHH signaling pathway. The present disclosure relates to the novel discovery that the timing and duration of SHH activation can be harnessed to direct controlled differentiation of neural progenitor cells into either cortical interneurons, hypothalamic neurons or pre-optic cholinergic neurons. The present disclosure also relates to compositions of cortical interneurons, hypothalamic neurons or pre-optic cholinergic neurons, and their precursors, that are highly enriched and can be used in variety of application. These cells can be used therapeutically to treat neurodegenerative and neuropsychiatric disorders, and can be used for disease modeling and drug screening.
1. An in vitro method for differentiating human stem cells comprising:
contacting human stem cells selected from the group consisting of human pluripotent stem cells, human multipotent stem cells, and combinations thereof with at least one inhibitor of Small Mothers Against Decapentaplegic (SMAD) signaling and with at least one Wingless (Wnt) antagonist; and
contacting the cells with at least one activator of Sonic Hedgehog (SHH) signaling to obtain differentiated cells expressing FOXG1, wherein the initial contact of the cells with the at least one activator of SHH signaling is at least 6 days from the initial contact of the cells with the at least one inhibitor of SMAD signaling.
2. The method of claim 1 , wherein said stem cells are selected from the group consisting of human embryonic stem cells, human adult stem cells, human neural stem cells, human induced pluripotent cells, human primary progenitor cells, and human induced progenitor cells.
3. The method of claim 1 , wherein said contacting with the at least one inhibitor of SMAD signaling and said contacting with the at least one Wnt antagonist are carried out simultaneously or sequentially, and each has a duration between about 5 days and about 30 days.
4. The method of claim 3 , wherein the contact of the cells with the at least one Wnt antagonist is initiated within 5 days from the initial contact of the cells with the at least one inhibitor of SMAD signaling.
5. The method of claim 1 , wherein said at least one inhibitor of SMAD signaling is selected from the group consisting of SB431542, LDN-193189, Noggin PD169316, SB203580, LY364947, A77-01, A-83-01, BMP4, GW788388, GW6604, SB-505124, lerdelimumab, metelimumab, GC-I008, AP-12009, AP-110I4, LY550410, LY580276, LY364947, LY2109761, SB-505124, E-616452 (RepSox ALK inhibitor), SD-208, SMI6, NPC-30345, KÏ26894, SB-203580, SD-093, activin-M108A, P144, soluble TBR2-Fc, DMH-1, Dorsomorphin dihydrochloride, derivatives thereof, and combinations thereof.
6. The method of claim 5 , wherein the at least one inhibitor of SMAD signaling comprises SB431542 and LDN-193189.
7. The method of claim 6 , wherein
(i) SB431542 is provided at a concentration within the range between about 0.1 μM and about 1 mM in a culture medium comprising said cells;
(ii) SB431542 is provided at a concentration within the range between about 0.1 μM and about 100 μM in a culture medium comprising said cells;
(iii) LDN-193189 is provided at a concentration within the range between about 1 nM and about 10 μM in a culture medium comprising said cells;
(iv) LDN-193189 is provided at a concentration within the range between about 1 nM and about 1 μM in a culture medium comprising said cells;
or a combination thereof.
8. The method of claim 1 , wherein the at least one Wnt antagonist is selected from the group consisting of XAV939, DKK1, DKK-2, DKK-3, Dkk-4, SFRP-1, SFRP-2, SFRP-5, SFRP-3, SFRP-4, WIF-1, Soggy, IWP-2, IWR1, ICG-001, KY0211, Wnt-059, LGK974, IWP-L6, derivatives thereof, and combinations thereof.
9. The method of claim 8 , wherein said at least one Wnt antagonist comprises XAV939.
10. The method of claim 8 , wherein
(i) XAV939 is provided at a concentration within the range between about 10 nM and about 500 μM in a culture medium comprising said cells; or
(ii) XAV939 is provided at a concentration within the range between about 0.2 μM and about 200 μM in a culture medium comprising said cells.
11. The method of claim 1 , wherein said at least one activator of SHH signaling is selected from the group consisting of Smoothened agonist (SAG), SAG analog, SHH, C25-SHH, C24-SHH, purmorphamine, Hg—Ag, derivatives thereof, and combinations thereof.
12. The method of claim 11 , wherein said at least one activator of SHH signaling comprises recombinant SHH and purmorphamine.
13. The method of claim 12 , wherein said recombinant SHH is provided at a concentration within the range between about 5 ng/mL and about 5 μg/mL in a culture medium comprising said cells, purmorphamine is provided at a concentration within the range between about 0.1 μM and about 20 μM in a culture medium comprising said cells.
14. The method of claim 1 , wherein
(i) the contact of the cells with the at least one activator of SHH signaling is concluded from about 5 days to about 30 days from its initiation;
(ii) the initial contact of the cells with the at least one activator of SHH signaling is between about 8 days and about 18 days from the initial contact of the cells with the at least one inhibitor of SMAD signaling, and the contact of the cells with the at least one activator of SHH signaling is concluded between about 8 days and about 16 days from its initiation;
(iii) the initial contact of the cells with the at least one activator of SHH signaling is no later than about 20 days from the initial contact of the cells with the at least one inhibitor of SMAD signaling;
(iv) the initial contact of the cells with the at least one activator of SHH signaling is no later than about 10 days from the initial contact of the cells with the at least one inhibitor of SMAD signaling;
(v) the initial contact of the cells with the at least one activator of SHH signaling is at least about 8 days and up to about 18 days from the initial contact of the cells with the at least one inhibitor of SMAD signaling; and/or
(vi) the initial contact of the cells with the at least one activator of SHH signaling is between 6 days and 8 days from the initial contact of the cells with the at least one inhibitor of SMAD signaling, and the contact of the cells with the at least one activator of SHH signaling is concluded between about 8 days and about 24 days from its initiation.
15. The method of claim 1 , wherein said differentiated cells further express at least one marker selected from the group consisting of SST, PV, GABA, calbindin, LHX6, OLIG2, NKX6.2, VGLUT1, MAP2, CTIP2, SATB2, TBR1, DLX2, ASCL1, and combinations thereof.
16. The method of claim 1 , wherein said differentiated cells further express at least one marker selected from the group consisting of ChAT, NGF, Ach, VAChT, LHX8, 1, p75 and combinations thereof.
17. The method of claim 1 , wherein said contact of the cells with said at least one inhibitor of SMAD signaling, with said at least one Wnt antagonist, and with said at least one activator of SHE signaling are carried out in a culture medium.
18. The method of claim 17 , wherein the culture medium is selected from the group consisting of essential 6 media, KSR-based media, N2 media, and combinations thereof.
19. The method of claim 1 , wherein the differentiated cells expressing FOXG1 are cortical interneuron precursors.
20. The method of claim 1 , comprising
contacting said cells with said at least one inhibitor of SMAD signaling and with at least one Wnt antagonist in a serum-supplemented culturing medium.
21. The method of claim 1 , further comprising maturing the differentiated cells.