Methods and compositions for generating human forebrain neural progenitor cells and maturing them into parvalbumin-positive interneurons - Patents.com

Abstract

A method is provided for generating human forebrain neural progenitor cells and mature forebrain neurons from human pluripotent stem cells using a chemically defined medium. The method allows for the initial generation of OTX2+ FEZF2+ SIX3+ forebrain neural stem cells, which can then be cultured to generate NKX2-1+ ventral forebrain neural stem cells and subsequently ASCL1+ medial ganglia primitive neural progenitor cells (MGE-NPCs). The MGE-NPCs are further differentiated into immature neurons and mature GABAergic interneurons. Culture media, isolated cell populations, and kits are also provided.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 273,742, filed October 29, 2021, and U.S. Provisional Patent Application No. 63 / 391,206, filed July 21, 2022. The entire contents of those prior applications are incorporated herein by reference in their entireties.

[0002] Government Licensed Rights This invention was made with Government support under Grant No. W911NF-17-3-0003 awarded by the U.S. Army ACC-AGP-RTP. The U.S. Government has certain rights in the invention. [Background technology]

[0003] 2. Background of the Invention The forebrain is an essential functional region of the central nervous system (CNS). Dysfunction of forebrain neurons can result in severe neurological diseases. GABAergic interneurons of the forebrain are the main inhibitory neurons of the CNS. GABAergic interneurons contribute to important aspects of cortical functional maturation. Cortical GABA interneurons originate from the medial ganglia primordium (MGE) in the ventral forebrain region. Dysfunction of GABAergic interneurons has been reported to lead to neurodegenerative or psychiatric diseases (Fishell and Rudy (2011) Annu. Rev. Neurosci. 34:535:567 (Non-Patent Document 1); Le Magueresse and Monyer (2013) Neuron 77:388-405 (Non-Patent Document 2)). Thus, the ability to derive forebrain lineage-committed neural progenitor cells, including cells that can develop into GABAergic interneurons, from human pluripotent stem cells (hPSCs) provides a means to potentially treat such forebrain-associated neurological and psychiatric disorders.

[0004] The early approach to obtain neural precursor cells from hPSCs used feeder cell layers and culture media containing serum and / or other undefined components, which are inappropriate for clinical use. Recently, chemically defined feeder-free systems have been developed. For example, an approach was reported in which hPSCs were first differentiated to induce forebrain neuroepithelial cells using an embryoid body protocol, which takes 10 days, and the resulting cells were then cultured with SHH agonists, resulting in the maturation of NKX2-1-expressing MGE precursor cells and GAB interneuron subtypes within a few weeks (Liu et al. (2013) Nat Protoc. 8:1670-1679 (Non-Patent Document 3); Yuan et al. (2015) Sci Rep 5:18550 (Non-Patent Document 4)). Additional forebrain differentiation protocols have been reported in the art, including approaches referred to as adherent differentiation protocols (Yan et al. (2013) Stem Cells Transl. Med. 2:862-870 (Non-Patent Document 5)) and non-adherent differentiation protocols (Crompton et al. (2013) Stem Cell Res. 11:1206-1221 (Non-Patent Document 6)).

[0005] Wnt inhibition has been incorporated into strategies to differentiate forebrain neural progenitor cells, and the timing of Wnt inhibition has been reported to modulate the differentiation of medial ganglia primordium progenitor cells of GABAergic interneurons (Ihnatovych et al. (2018) Stem Cells International, vol. 2018, Article ID 3983090).

[0006] More recently, feeder-free protocols using additional chemical components have been developed. One approach involves an 8-day neural induction phase resulting in neuroectodermal progenitor cells (rosettes), followed by an additional 8-day regionalization phase, which results in forebrain progenitor cells by day 16 (Comella-Bolla et al. (2020) Mol. Neurobiol. 57:2766-2798).

[0007] Further protocols for differentiation of forebrain neural progenitor cells include the use of SMAD2 / 3 inhibition, such as by inclusion of a TGFβ antagonist in the culture medium (see, e.g., Nicholas et al. (2013) Cell Stem Cell 12:573-586 (Non-Patent Document 9); U.S. Patent Application Publication No. 2016 / 0272940 (Patent Document 1); U.S. Patent Application Publication No. 2019 / 0062700 (Patent Document 2); U.S. Patent Application Publication No. 2021 / 0040443 (Patent Document 3)).

[0008] Further protocols for differentiation of forebrain neural progenitor cells are also described in US Patent Application Publication No. 2015 / 0361393 (Patent Document 4) and US Patent Application Publication No. 2017 / 0292112 (Patent Document 5).

[0009] Thus, despite some progress, there remains a need for efficient and robust methods and compositions for generating forebrain committed neural progenitor cells and mature GABAergic interneurons from human pluripotent stem cells. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] US Patent Application Publication No. 2016 / 0272940 [Patent Document 2] US Patent Application Publication No. 2019 / 0062700 [Patent Document 3] U.S. Patent Application Publication No. 2021 / 0040443 [Patent Document 4] US Patent Application Publication No. 2015 / 0361393 [Patent Document 5] US Patent Application Publication No. 2017 / 0292112 [Non-patent literature]

[0011]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Summary of the Invention

[0012] The present disclosure provides a method for generating human forebrain neural progenitor cells, including forebrain neural stem cells (FB-NSCs), ventral forebrain neural stem cells (VFB-NSCs) and medial ganglia primitive neural progenitor cells (MGE-NPCs), in a three-step protocol requiring nine days, and MGE-NPCs can be further differentiated into mature GABAergic interneurons in a two-step protocol requiring an additional 17 days. The method uses a chemically defined medium that allows the generation of FB-NSCs within three days of culture, VFB-NSCs within six days of culture, MGE-NPCs within nine days of culture, immature neurons within 12 days of culture, and mature parvalbumin-positive interneurons within 26 days of culture. The defined medium used to obtain different types of neural progenitor cells and mature neurons contains small molecule agents that agonize or antagonize specific signaling pathway activity in pluripotent stem cells, resulting in promotion of differentiation along the forebrain neural lineage, leading to cell maturation and expression of forebrain neural progenitor cell-associated biomarkers. The disclosed method uses a differentiation medium with different components than earlier protocols, avoiding the need for certain specific reagents (e.g., the disclosed method does not require dual SMAD inhibition). The disclosed method also has the advantage that the use of small molecule agents in the medium allows precise control of culture components, avoiding the need for a neural induction phase, and the time required for further maturation into mature GABAergic interneurons following differentiation into MGE-NPCs is significantly reduced compared to prior art protocols.

[0013] Thus, in one aspect, the present disclosure relates to a method for generating human OTX2+ FEZF2+ SIX3+ forebrain neural stem cells (FB-NSCs), comprising culturing human pluripotent stem cells in a medium comprising a BMP pathway antagonist, a MEK pathway antagonist, a WNT pathway antagonist, an AKT pathway antagonist, a SHH pathway agonist, and a PKC pathway antagonist from day 0 to day 3 to obtain human OTX2+ FEZF2+ SIX3+ FB-NSCs.

[0014] The method may further include further culturing the FB-NSCs in a medium containing a BMP pathway antagonist, a MEK pathway antagonist, a WNT pathway antagonist, and a SHH pathway agonist from day 3 to day 6 to obtain human NKX2-1+ ventral forebrain neural stem cells (VFB-NSCs).

[0015] The method may further include further culturing the VFB-NSCs in a medium containing a TAK1 pathway antagonist, an SHH pathway agonist, a TGF-β pathway antagonist, a TRK pathway antagonist, a Notch pathway antagonist, and an IGF1 pathway agonist from day 6 to day 9 to obtain human ASCL1+ medial ganglia primitive neural progenitor cells (MGE-NPCs).

[0016] The method may further include further culturing the MGE-NPCs in a medium containing a CREB or PKA pathway agonist, valproic acid or an analog, substance P or an analog, a GDNF pathway agonist, and an mTOR pathway agonist from day 9 to day 12 to obtain human immature neurons.

[0017] The method can further include further culturing the human immature neurons in a medium containing a BDNF pathway agonist, an IGF-1 pathway agonist, ascorbic acid or an analog, sodium pyruvate or an analog, LPA or an analog, an N2 supplement, and a NEAA supplement from day 12 to day 26 to obtain mature parvalbumin+ interneurons.

[0018] In one embodiment, the human pluripotent stem cells are induced pluripotent stem cells (iPSCs). In one embodiment, the human pluripotent stem cells are embryonic stem cells. In one embodiment, the human pluripotent stem cells are attached to vitronectin-coated plates during culture.

[0019] In one embodiment, the BMP pathway antagonist is selected from the group consisting of LDN193189, DMH1, DMH2, Dorsopmorphin, K02288, LDN214117, LDN212854, follistatin, ML347, noggin, and combinations thereof. In one embodiment, the BMP pathway antagonist is present in the medium at a concentration in the range of 100-500 nM. In one embodiment, the BMP pathway antagonist is LDN193189 present in the medium at a concentration of 250-275 nM.

[0020] In one embodiment, the MEK pathway antagonist is selected from the group consisting of PD0325901, binimetinib (MEK162), cobimetinib (XL518), selumetinib, trametinib (GSK1120212), CI-1040 (PD-184352), refametinib, ARRY-142886 (AZD-6244), PD98059, U0126, BI-847325, RO 5126766, and combinations thereof. In one embodiment, the MEK pathway antagonist is present in the medium at a concentration in the range of 50-150 nM. In one embodiment, the MEK pathway antagonist is PD0325901 present in the medium at a concentration of 100-110 nM.

[0021] In one embodiment, the WNT pathway antagonist is selected from the group consisting of XAV939, ICG001, capmatinib, endo-IWR-1, IWP-2, IWP-4, MSAB, CCT251545, KY02111, NCB-0846, FH535, LF3, WIKI4, triptonide, KYA1797K, JW55, JW 67, JW74, Cardionogen 1, NLS-StAx-h, TAK715, PNU 74654, iCRT3, WIF-1, DKK1, and combinations thereof. In one embodiment, the WNT pathway antagonist is present in the medium at a concentration in the range of 50-150 nM. In one embodiment, the WNT pathway antagonist is XAV939 present in the medium at a concentration of 100-110 nM.

[0022] In one embodiment, the SHH pathway agonist is selected from the group consisting of purmorphamine, GSA 10, SAG, and combinations thereof. In one embodiment, the SHH pathway agonist is present in the medium at a concentration in the range of 250-750 nM. In one embodiment, the SHH pathway agonist is purmorphamine present in the medium at a concentration of 500-550 nM.

[0023] In one embodiment, the AKT pathway antagonist is selected from the group consisting of MK2206, GSK690693, Perifosine (KRX-0401), Ipatasertib (GDC-0068), Capivasertib (AZD5363), PF-04691502, AT 7867, Triciribine (NSC154020), ARQ751, Miransertib (ab235550), Borussertib, Cerisertib, and combinations thereof. In one embodiment, the AKT pathway antagonist is present in the medium at a concentration in the range of 50-200 nM. In one embodiment, the AKT pathway antagonist is MK2206 present in the medium at a concentration of 138 nM.

[0024] In one embodiment, the PKC pathway antagonist is selected from the group consisting of Go 6983, sotrastaurin, enzastaurin, staurosporine, LY31615, Go 6976, GF 109203X, Ro 31-8220 mesylate, and combinations thereof. In one embodiment, the PKC pathway antagonist is present in the medium at a concentration in the range of 50-200 nM. In one embodiment, the PKC pathway antagonist is Go 6983 present in the medium at a concentration of 110 nM.

[0025] In one embodiment, the TAK1 pathway antagonist is selected from the group consisting of takinib, dehydoabietic acid, NG25, sarsasapogenin, and combinations thereof. In one embodiment, the TAK1 pathway antagonist is present in the medium at a concentration in the range of 1-5uM. In one embodiment, the TAK1 pathway antagonist is takinib, which is present in the medium at a concentration of 2uM.

[0026] In one embodiment, the TGFβ pathway antagonist is selected from the group consisting of A 83-01, SB-431542, GW788388, SB525334, TP0427736, RepSox, SD-208, and combinations thereof. In one embodiment, the TGFβ pathway antagonist is present in the medium at a concentration in the range of 250-750 nM. In one embodiment, the TGFβ pathway antagonist is A 83-01 present in the medium at a concentration of 500 nM.

[0027] In one embodiment, the TRK pathway antagonist is selected from the group consisting of GNF-5837, BMS-754807, UNC2020, taretrectinib, altilatinib, ceritrectinib, PF 06273340, and combinations thereof. In one embodiment, the TRK pathway antagonist is present in the medium at a concentration in the range of 25-75 nM. In one embodiment, the TRK pathway antagonist is GNF-5837 present in the medium at a concentration of 50 nM.

[0028] In one embodiment, the Notch pathway antagonist is selected from the group consisting of GSI-XX, RO4929097, semagacestat, dibenzazepine, LY411575, crenigacestat, IMR-1, IMR-1A, FLI-06, DAPT, valproic acid, YO-01027, CB-103, tangeretin, BMS-906024, avagacestat, brucein D, and combinations thereof. In one embodiment, the Notch pathway antagonist is present in the medium at a concentration in the range of 50-150 nM. In one embodiment, the Notch pathway antagonist is GSI-XX present in the medium at a concentration of 100 nM.

[0029] In one embodiment, the IGF1 pathway agonist is selected from the group consisting of IGF1, IGF1-Ado, X10, mecasermin, and combinations thereof. In one embodiment, the IGF1 pathway agonist is present in the medium at a concentration in the range of 5-15 ng / ml. In one embodiment, the IGF1 pathway agonist is IGF1 present in the medium at a concentration of 10 ng / ml.

[0030] In one embodiment, the CREB or PKA pathway agonist is cAMP, dibutyryl-cAMP, 8-Br-cAMP, cAMPS-Sp, CW 008, forskolin, 8-CPT-cAMP, CW 008, N6-benzoyl-adenosine 3',5'-cyclic monophosphate sodium salt, adenosine 3',5'-cyclic monophosphate sodium salt monohydrate, (S)-adenosine cyclic 3',5'-(hydrogen phosphorothioate) triethylammonium, Sp-adenosine 3',5'-cyclic monophosphorothioate triethylammonium salt, Sp-5,6-DCI-cBiMPS, 8-bromoadenosine 3',5'-cyclic The CREB or PKA pathway agonist is selected from the group consisting of monophosphorothioate Sp-isomer sodium salt, 8-bromo-adenosine 3',5'-cyclic monophosphorothioate Sp-isomer sodium salt, Sp-8-pCPT-cyclic GMPS sodium, 8-bromoadenosine 3',5'-cyclic monophosphate, N6-monobutyryladenosine 3':5'-cyclic monophosphate sodium salt, 8-PIP-cAMP, Sp-cAMPS, and combinations thereof. In one embodiment, the CREB or PKA pathway agonist is present in the medium at a concentration in the range of 0.5-2.5 μM. In one embodiment, the CREB or PKA pathway agonist is cAMP present in the medium at a concentration of 1.0-1.5 μM.

[0031] In one embodiment, the valproic acid or analog is selected from the group consisting of valproic acid, valproate, sodium valproate, and semisodium valproate. In one embodiment, the valproic acid or analog is present in the medium at a concentration in the range of 250-750 nM. In one embodiment, the valproic acid is present in the medium at a concentration in the range of 450-550 nM.

[0032] In one embodiment, substance P is present in the medium at a concentration in the range of 100-150 nM.

[0033] In one embodiment, the GDNF pathway agonist is selected from the group consisting of GDNF, BT13, BT44, and combinations thereof. In one embodiment, the GDNF pathway agonist is present in the medium at a concentration in the range of 5-50 ng / ml. In one embodiment, the GDNF pathway agonist is GDNF present in the medium at a concentration of 10 ng / ml.

[0034] In one embodiment, the mTOR pathway agonist is selected from the group consisting of MHY1458, NV-5138, testosterone, 3-benzyl-5-((2-nitrophenoxy)methyl)-dihydrofuran-2(3H)-one (3BDO), 3BDO, L-leucine, NV-5138 hydrochloride, NV-5138, L-leucine-d1, L-leucine-2-13C,15N, leucine-13C6, L-leucine-d7, L-leucine-d10, L-leucine-d2, 1-leucine-d3, L-leucine-18O2, L-leucine-13C, L-leucine-2-13C, L-leucine-13C6-15N, L-leucine-15N, L-leucine-1-13C,15N, and combinations thereof. In one embodiment, the mTOR pathway agonist is present in the medium at a concentration in the range of 1-3 μM. In one embodiment, the mTOR agonist is MHY1458, which is present in the medium at a concentration of 2 μM.

[0035] In one embodiment, the BDNF pathway agonist is selected from the group consisting of BDNF, rotigotine, 7,8-DHF, ketamine, tricyclic dimeric peptide-6 (TDP6), LM22A-4, and combinations thereof. In one embodiment, the BDNF pathway agonist is present in the medium at a concentration in the range of 5-50 ng / ml. In one embodiment, the BDNF pathway agonist is BDNF present in the medium at a concentration of 10 ng / ml.

[0036] In one embodiment, the ascorbic acid or analogue is selected from the group consisting of vitamin C, 2-phospho-L-ascorbic acid, L-ascorbic acid, sodium ascorbyl phosphate, magnesium ascorbyl phosphate, ascorbyl glucoside, tetrahexyldecyl ascorbate (THD), ethylated L-ascorbic acid, and combinations thereof. In one embodiment, the ascorbic acid or analogue is present in the medium at a concentration in the range of 100-400 μM. In one embodiment, the 2-phospho-L-ascorbic acid is present in the medium at a concentration of 200 μM.

[0037] In one embodiment, sodium pyruvate is present in the medium at a concentration of 100-300 μM.

[0038] In one embodiment, the LPA or analogue is selected from the group consisting of lysophosphatidic acid (LPA), 2-[[3-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)propyl]thio]benzoic acid, 1-oleoyl lysophosphatidic acid (O-LPA), UCM-05194, and combinations thereof. In one embodiment, the LPA or analogue is present in the medium at a concentration in the range of 100-400 nM. In one embodiment, the LPA or analogue is O-LPA present in the medium at a concentration in the range of 200-300 nM.

[0039] In one embodiment, the N2 supplement is present in the medium at a concentration ranging from 0.5% to 1.5%.

[0040] In one embodiment, the NEAA supplement is present in the medium at a concentration ranging from 0.5% to 1.5%.

[0041] In another aspect, the present disclosure provides a method for producing a method for manufacturing a semiconductor device comprising: (a) culturing human pluripotent stem cells in a medium containing a BMP pathway antagonist, a MEK pathway antagonist, a WNT pathway antagonist, an AKT pathway antagonist, a SHH pathway agonist, and a PKC pathway antagonist from day 0 to day 3 to obtain human OTX2+ FEZF2+ SIX3+ forebrain neural stem cells (FB-NSCs); and (b) Further culturing the human OTX2+ FEZF2+ SIX3+ FB-NSCs in a medium containing a BMP pathway antagonist, a MEK pathway antagonist, a WNT pathway antagonist, and a SHH pathway agonist, and lacking an AKT pathway antagonist and a PKC pathway antagonist, from day 3 to day 6 to obtain human NKX2-1+ ventral forebrain neural stem cells (VFB-NSCs). The present invention provides a method for generating human NKX2-1 ventral forebrain neural stem cells (VFB-NSCs), comprising:

[0042] In one embodiment, the BMP pathway antagonist is LDN193189, the MEK pathway antagonist is PD0325901, the WNT pathway antagonist is XAV939, the SHH pathway agonist is purmorphamine, the AKT pathway antagonist is MK2206, and the PKC pathway antagonist is Go 6983.

[0043] In one embodiment, LDN193189 is present in the medium at a concentration in the range of 100-500 nM, PD0325901 is present in the medium at a concentration in the range of 50-150 nM, XAV939 is present in the medium at a concentration in the range of 50-150 nM, purmorphamine is present in the medium at a concentration in the range of 250-750 nM, MK2206 is present in the medium at a concentration in the range of 50-150 nM in step (a), and Go 6983 is present in the medium at a concentration in the range of 50-150 nM in step (a). In one embodiment, LDN193189 is present in the medium at a concentration of 275 nM in step (a) and 250 nM in step (b). PD0325901 is present in the culture medium at a concentration of 110 nM in step (a) and 100 nM in step (b), XAV939 is present in the culture medium at a concentration of 110 nM in step (a) and 100 nM in step (b), purmorphamine is present in the culture medium at a concentration of 550 nM in step (a) and 500 nM in step (b), MK2206 is present in the culture medium at a concentration of 138 nM in step (a), and Go 6983 is present in the culture medium at a concentration of 110 nM in step (a).

[0044] In yet another aspect, the present disclosure provides a method for producing a method for manufacturing a semiconductor device comprising: (a) culturing human pluripotent stem cells in a medium containing a BMP pathway antagonist, a MEK pathway antagonist, a WNT pathway antagonist, an AKT pathway antagonist, a SHH pathway agonist, and a PKC pathway antagonist from day 0 to day 3 to obtain human OTX2+ FEZF2+ SIX3+ forebrain neural stem cells (FB-NSCs); (b) further culturing the human OTX2+ FEZF2+ SIX3+ FB-NSCs in a medium containing a BMP pathway antagonist, a MEK pathway antagonist, a WNT pathway antagonist, and a SHH pathway agonist, and lacking an AKT pathway antagonist and a PKC pathway antagonist, from day 3 to day 6 to obtain human NKX2-1+ ventral forebrain neural stem cells (VFB-NSCs); and (c) Further culture the human NKX2-1+ VFB-NSCs in medium containing a TAK1 pathway antagonist, an SHH pathway agonist, a TGF-β pathway antagonist, a TRK pathway antagonist, a Notch pathway antagonist, and an IGF1 pathway agonist from day 6 to day 9 to obtain human ASCL1+ medial ganglia epiphysis neural progenitor cells (MGE-NPCs). The present invention relates to a method for generating human ASCL1+ medial ganglia primordium neural progenitor cells (MGE-NPCs), comprising:

[0045] In one embodiment, the BMP pathway antagonist is LDN193189, the MEK pathway antagonist is PD0325901, the WNT pathway antagonist is XAV939, the SHH pathway agonist is purmorphamine, the AKT pathway antagonist is MK2206, the PKC pathway antagonist is Go 6983, the TAK1 pathway antagonist is takinib, the TGF-β pathway antagonist is A 83-01, the TRK pathway antagonist is GNF-5837, the Notch pathway antagonist is GSI-XX, and the IGF1 pathway agonist is IGF-1.

[0046] In one embodiment, LDN193189 is present in the medium at a concentration in the range of 100-500 nM, PD0325901 is present in the medium at a concentration in the range of 50-150 nM, XAV939 is present in the medium at a concentration in the range of 50-150 nM, purmorphamine is present in the medium at a concentration in the range of 250-750 nM, MK2206 is present in the medium at a concentration in the range of 50-150 nM in step (a), Go 6983 is present in the medium at a concentration in the range of 50-150 nM in step (a), and takinib is present in the medium at a concentration in the range of 1-5 uM in step (c), 83-01 is present in the medium in step (c) at a concentration in the range of 250 to 750 nM, GNF-5837 is present in the medium in step (c) at a concentration in the range of 25 to 75 nM, GSI-XX is present in the medium in step (c) at a concentration in the range of 50 to 150 nM, and IGF-1 is present in the medium in step (c) at a concentration in the range of 5 to 15 ng / ml. In one embodiment, LDN193189 is present in the medium at a concentration of 275 nM in step (a) and 250 nM in step (b), PD0325901 is present in the medium at a concentration of 110 nM in step (a) and 100 nM in step (b), XAV939 is present in the medium at a concentration of 110 nM in step (a) and 100 nM in step (b), purmorphamine is present in the medium at a concentration of 550 nM in step (a) and 500 nM in step (b), MK2206 is present in the medium at a concentration of 138 nM in step (a), Go 6983 is present in the medium at a concentration of 110 nM in step (a), and takinib is present in the medium at a concentration of 2 uM in step (c), 83-01 is present in the medium in step (c) at a concentration of 500 nM, GNF-5837 is present in the medium in step (c) at a concentration of 50 nM, GSI-XX is present in the medium in step (c) at a concentration of 100 nM, and IGF-1 is present in the medium in step (c) at a concentration within the range of 10 ng / ml.

[0047] In yet another aspect, the present disclosure provides a method for producing a method for manufacturing a semiconductor device comprising: (a) culturing human MGE-NPCs in a medium containing a CREB or PKA pathway agonist, a valproic acid or analog, a substance P or analog, a GDNF pathway agonist, and an mTOR pathway agonist from day 0 to day 3 to obtain human immature neurons; and (b) culturing human immature neurons in a medium containing a BDNF pathway agonist, an IGF-1 pathway agonist, ascorbic acid or an analog, sodium pyruvate or an analog, LPA or an analog, N2 supplement, and NEAA supplement from day 3 to day 17 to obtain human mature parvalbumin+ interneurons; The present invention relates to a method for generating human mature parvalbumin+ interneurons from human medial ganglia primordial neural progenitor cells (MGE-NPCs), comprising:

[0048] In one embodiment, the CREB or PKA pathway agonist is cAMP, the valproic acid or analog is valproic acid, the substance P or analog is substance P, the GDNF pathway agonist is GDNF, the mTOR pathway agonist is MHY1458, the BDNF pathway agonist is BDNF, the IGF-1 pathway agonist is IGF-1, the ascorbic acid or analog is 2-phospho-L-ascorbic acid, the sodium pyruvate or analog is sodium pyruvate, and the LPA or analog is O-LPA.

[0049] In one embodiment, cAMP is present at a concentration ranging from 1.0-1.5 μM, valproic acid is present at a concentration ranging from 450-550 nM, substance P is present at a concentration ranging from 100-150 nM, GDNF is present at a concentration ranging from 5-50 ng / ml, MHY1458 is present at a concentration ranging from 1-3 μM, BDNF is present at a concentration ranging from 5-50 ng / ml, IGF-1 is present at a concentration ranging from 5-50 ng / ml, 2-phospho-L-ascorbic acid is present at a concentration ranging from 100-400 μM, O-LPA is present at a concentration ranging from 100-400 μM, sodium pyruvate is present at a concentration ranging from 100-300 μM, N2 supplement is present at a concentration ranging from 0.5% to 1.5%, and NEAA supplement is present at a concentration ranging from 0.5% to 1.5%.

[0050] In one embodiment, cAMP is present at a concentration of 1.0 μM, valproic acid is present at a concentration of 500 nM, substance P is present at a concentration of 100 nM, GDNF is present at a concentration of 10 ng / ml, MHY1458 is present at a concentration of 2 μM, BDNF is present at a concentration of 10 ng / ml, IGF-1 is present at a concentration of 10 ng / ml, 2-phospho-L-ascorbic acid is present at a concentration of 200 μM, sodium pyruvate is present at a concentration of 100 μM, O-LPA is present at a concentration of 200 μM, N2 supplement is present at a concentration of 1.0%, and NEAA supplement is present at a concentration of 1.0%.

[0051] In another aspect, the present disclosure relates to various media for generating human forebrain neural stem cells or progenitor cells.In one aspect, the present disclosure provides a medium for obtaining human OTX2+ FEZF2+ SIX3+ forebrain neural stem cells (FB-NSCs), comprising BMP pathway antagonist, MEK pathway antagonist, WNT pathway antagonist, AKT pathway antagonist, SHH pathway agonist, and PKC pathway antagonist.In one aspect, the present disclosure provides a medium for obtaining human NKX2-1+ ventral forebrain neural stem cells (VFB-NSCs), comprising BMP pathway antagonist, MEK pathway antagonist, WNT pathway antagonist, and SHH pathway agonist. In one aspect, the present disclosure provides a medium for obtaining human ASCL1+ medial ganglia primitive neural progenitor cells (MGE-NPCs), comprising a TAK1 pathway antagonist, an SHH pathway agonist, a TGF-β pathway antagonist, a TRK pathway antagonist, a Notch pathway antagonist, and an IGF1 pathway agonist. In one aspect, the present disclosure provides a medium for obtaining human immature neurons, comprising a CREB or PKA pathway agonist, a valproic acid or analog, a substance P or analog, a GDNF pathway agonist, and an mTOR pathway agonist. In one aspect, the present disclosure provides a medium for obtaining human mature GABAergic interneurons, comprising a BDNF pathway agonist, an IGF-1 pathway agonist, an ascorbic acid or analog, a sodium pyruvate or analog, an LPA or analog, an N2 supplement, and an NEAA supplement.

[0052] In another aspect, the present disclosure relates to the isolated cell culture of human forebrain neural stem cell or progenitor cell.In one aspect, the present disclosure provides the isolated cell culture of human OTX2+ FEZF2+ SIX3+ forebrain neural stem cell (FB-NSC), the cell culture comprises human OTX2+ FEZF2+ SIX3+ FB-NPC cultured in the medium comprising BMP pathway antagonist, MEK pathway antagonist, WNT pathway antagonist, AKT pathway antagonist, SHH pathway agonist and PKC pathway antagonist. In one aspect, the present disclosure provides an isolated cell culture of human NKX2-1+ ventral forebrain neural stem cells (VFB-NSCs), the cell culture comprises human NKX2-1+ VFB-NPCs cultured in a medium comprising a BMP pathway antagonist, a MEK pathway antagonist, a WNT pathway antagonist, and a SHH pathway agonist.In one aspect, the present disclosure provides an isolated cell culture of human ASCL1+ medial ganglia primitive neural progenitor cells (MGE-NPCs), the cell culture comprises human ASCL1+ MGE-NPCs cultured in a medium comprising a TAK1 pathway antagonist, a SHH pathway agonist, a TGF-β pathway antagonist, a TRK pathway antagonist, a Notch pathway antagonist, and a IGF1 pathway agonist. In one embodiment, the present disclosure provides an isolated cell culture of human immature neurons, the cell culture comprises human immature neurons cultured in a medium comprising a CREB or PKA pathway agonist, a valproic acid or analog, a substance P or analog, a GDNF pathway agonist, and an mTOR pathway agonist.In one embodiment, the present disclosure provides an isolated cell culture of human mature GABAergic interneurons, the cell culture comprises human mature GABAergic interneurons cultured in a medium comprising a BDNF pathway agonist, an IGF-1 pathway agonist, an ascorbic acid or analog, a sodium pyruvate or analog, an LPA or analog, an N2 supplement, and an NEAA supplement.

[0053] In yet another aspect, the present disclosure relates to human forebrain neural stem or progenitor cells generated by the method of the present disclosure. In one aspect, the present disclosure provides human OTX2+ FEZF2+ SIX3+ forebrain neural stem cells (FB-NSCs) generated by the method of the present disclosure. In one aspect, the present disclosure provides human NKX2-1+ ventral forebrain neural stem cells (VFB-NSCs) generated by the method of the present disclosure. In one aspect, the present disclosure provides human ASCL1+ medial ganglia primitive neural progenitor cells (MGE-NPCs) generated by the method of the present disclosure. In one aspect, the present disclosure provides human immature neurons generated by the method of the present disclosure. In one aspect, the present disclosure provides human parvalbumin+ mature interneurons generated by the method of the present disclosure.

[0054] The disclosed methods and compositions are useful for generating human forebrain neural stem or progenitor cells for research or therapeutic purposes, such as in the treatment of neurological disorders (eg, transplantation to treat epilepsy).

[0055] Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. [Brief description of the drawings]

[0056] [Figure 1] Figure 1 shows results from an HD-DoE model of a 12-factor experiment optimized for maximum expression of FEZF2. The top section of the model shows the predicted expression levels of 53 preselected genes when optimized for FEZF2. The bottom section of the model shows the effectors tested in the model and their contribution to maximum expression of FEZF2. The value column refers to the concentration of each effector required to mimic the model. [Diagram 2] Results from an HD-DoE model of a 12-factor experiment optimized for maximum expression of SIX3 are shown. The top and bottom sections are similar to those described for Figure 1. The conditions highlight the effectors LDN193189 and TTNPB with factor contributions 24.9 and 23.7 as important inputs for high expression of SIX3. [Diagram 3] Results from the HD-DoE model of a 12-factor experiment optimized for maximum expression of OTX2 are shown. The top and bottom sections are similar to those described for Figure 1. This condition highlights the effector TTNPB with factor contribution 28.8 as the critical input minimum expression of OTX2. [Figure 4] Dynamic profiles of expression levels of OTX2, FEZF2 and SIX3 genes versus concentration of 12 effectors tested. The positive impact of LDN193189 and XAV939 on OTX2 expression and their factor contributions are shown by the slope of the plot for each effector. [Diagram 5] Figure 1 shows results from an HD-DoE model of a 12-factor experiment optimized for maximal expression of FEZF2. The top and bottom sections are similar to those described for Figure 1. The conditions highlight the effector MK2206 with factor contribution 12.2 as a critical input to maximal expression of FEZF2. [Figure 6] Figure 1 shows the dynamic profile of FEZF2 expression levels versus the concentrations of 12 effectors tested. The positive effects of LDN193189, XAV939, PD0325901, MK2206, purmorphamine and GO6983 on FEZF2 expression and their factorial contributions are shown by the slope of the plot for each effector. [Figure 7] Figure 1 shows results from an HD-DoE model of a 12-factor experiment applied to stage 1 neural stem cells to create a recipe for stage 2 of differentiation. The model is optimized for maximum expression of NKX2-1. The top and bottom sections are similar to the description for Figure 1. This setup highlights the negative role of SANT-1 in the expression of NKX2-1 with factor contribution 26.3. [Figure 8]Figure 2 shows the dynamic profile of expression levels of NKX2-1, PAX6 and NKX2-2 genes against the concentration of 12 effectors. The positive effects of PD0325901 and XAV939 and the negative effects of SANT-1 and TTNPB on NKX2-1 and their factor contributions are shown by the slope of the plot for each effector. [Figure 9] Shown are results from an HD-DoE model of a 12-factor experiment applied to stage 2 neural stem cells to create a recipe for stage 3 of differentiation. The model is optimized for maximum expression of ASCL1. The top and bottom sections are similar to the description for Figure 1. This setup highlights the positive role of A8301 and GSI-XX with factor contributions 18 and 14. [Figure 10] Figure 1 shows the dynamic profile of expression levels of ASCL1, DLX1 and LHX6 genes versus concentration of 12 effectors. The positive effects of A8301, GSI-XX and purmorphamine on the expression levels of ASCL1 and LHX6 and their factorial contributions are shown by the slope of the plot for each effector. [Figure 11A] Figures 11A-11B show the dynamic profile of expression levels of OTX2, FEZF2 and SIX3 genes against the concentration of six validated effectors in the recipe of stage 1 of differentiation. Figure 11A shows the expression levels of the gene of interest in the presence of all finalized effectors. Figure 11B shows the expression levels of the gene of interest in the absence of one finalized effector at a time and the presence of the others. [Figure 11B] See legend to Figure 11A. [Figure 12] Figure 1 shows the dynamic profile of FEZF2 expression levels versus the concentration of six validated effectors in the differentiation stage 1 recipe. At each time, the expression levels of FEZF2 are shown in the absence of one finalized effector and the presence of the others. [Figure 13A]Figure 13A-13B show the dynamic profile of expression levels of NKX2-1, PAX6 and NKX2-2 genes against the concentration of four validated effectors in the recipe of stage 2 of differentiation. Figure 13A shows the expression levels of the gene of interest in the presence of all finalized effectors. Figure 13B shows the expression levels of the gene of interest in the absence of one finalized effector at a time and the presence of the others. [Figure 13B] See legend to Figure 13A. [Figure 14] Figure 1 shows the interaction plots of the two effectors in the 12-factor HD-DoE model used for the optimization of the expression level of NKX2-1. The blue plot shows the expression level of NKX2-1 when the concentration of XAV939 is increased while the concentration of purmorphamine is kept at its highest value (500 nM). The green plot shows the expression level of NKX2-1 when the concentration of XAV93 is increased while the concentration of purmorphamine is kept at 0. [Figure 15A] Figure 15A-15B show the dynamic profile of the expression levels of ASCL1, DLX1 and LHX6 genes against the concentration of six validated effectors in the recipe of stage 3 of differentiation. Figure 15A shows the expression levels of the gene of interest in the presence of all six factors. Figure 15B shows the expression levels of the gene of interest in the absence of one finalized effector at a time and the presence of the others. [Figure 15B] See legend to Figure 15A. [Figure 16]Figures 16A-16C show photographs of fluorescent images of ventral forebrain-derived neural progenitor cells at the end of stage 1, 2 and 3 treatment. Cells are stained with forebrain biomarkers including SIX3, OTX2, dorsal forebrain marker PAX6, ventral forebrain biomarkers NKX2-1, DLX5 and MASH1, MGE-specific biomarkers LHX6 and SOX6, pan-neuronal biomarker βIII-tubulin, GABAergic interneuron biomarker GABA, proliferation biomarker KI67 and glial biomarkers OLIG2 and GFAP. Figure 16A shows photographs of fluorescent images of cells at day 3 (end of stage 1). Figure 16B shows photographs of fluorescent images of cells at day 6 (end of stage 2). Figure 16C shows photographs of fluorescent images of cells at day 9 (end of stage 3). [Figure 17] FIG. 1 is a schematic diagram of an exemplary culture method of the present disclosure for a three-stage protocol for generating MGE-NPCs from pluripotent stem cells. [Figure 18] FIG. 1 is a schematic diagram of an exemplary culture method of the present disclosure for a two-stage protocol for generating mature GABAergic interneurons from MGE-NPCs. [Figure 19] Figure 1 shows results from a model of a 12-factor experiment optimized for maximum expression of MEF2C. The top section of the model shows the predicted expression levels of 53 preselected genes when optimized for MEF2C. The bottom section of the model shows the effectors tested in the model and their contribution to maximum expression of MEF2C. The value column refers to the concentration of each effector required to mimic the model. [Figure 20] Dynamic profile of MEF2C gene expression levels versus concentration of 12 effectors. The positive effects of cAMP, valproic acid, substance P, and GDNF on MEF2C and their factor contributions are shown by the slope of the plot for each effector. [Figure 21]Results from a model of an eight-factor experiment optimized for maximal expression of PVALB are shown. The top and bottom sections are similar to those described for Figure 19. The conditions highlight the effectors, BDNF, IGF-1, and 2-phospho-L-ascorbic acid, with factor contributions of 14.6, 12.8, and 15.04, as important inputs to maximal expression of PVALB. [Figure 22] Dynamic profile of PVALB expression levels versus the concentration of eight effectors. The positive effects of indolactam-V, oleic acid, 2-phospho-L-ascorbic acid, BDNF, and IGF-1 and their factor contributions are shown by the slope of the plot for each effector. [Figure 23] Figure 1 shows results from a model of an 8-factor experiment applied to stage 4 neural stem cells to create a recipe for stage 5 of differentiation. The model is optimized for maximum expression of PVALB. The top and bottom sections are similar to those described for Figure 19. This setup highlights the positive role of N2 in the expression of PVALB with a factor contribution of 15.9. [Figure 24] Figure 1 shows the dynamic profile of the expression level of the PVALB gene versus the concentration of eight effectors. The positive effect of N2 and the negative effects of GSI-XX, THI0019 and heparin on PVALB and their factorial contributions are shown by the slope of the plot for each effector. [Diagram 25] Shown are results from a model of an 8-factor experiment applied to stage 4 neural stem cells to create a recipe for stage 5 of differentiation. The model has been optimized for maximum expression of PVALB. The top and bottom sections are similar to the description for Figure 19. This setup highlights the positive role of forskolin and sodium pyruvate with factor contributions of 3.14 and 17.1. [Figure 26] Figure 1 shows the dynamic profile of the expression level of the PVALB gene versus the concentration of eight effectors. The positive effects of sodium pyruvate and forskolin on the expression level of PVALB and their factorial contributions are shown by the slope of the plot for each effector. [Figure 27A] 27A-27B show the dynamic profile of expression levels of MEF2C, PVALB and somatostatin genes against the concentration of five effectors in the recipe at stage 4 of differentiation. The results show the expression levels of the genes of interest in the presence of all seven factors. [Figure 27B] See legend to Figure 27A. [Figure 28A] 28A-28B show the dynamic profile of expression levels of PVALB and somatostatin genes against the concentrations of three effectors in the recipe at stage 5 of differentiation. The results show the expression levels of the genes of interest in the presence of the indicated factors. [Figure 28B] See legend to Figure 28A. [Figure 29] Figure 1 shows the dynamic profile of expression levels of PVALB and somatostatin genes versus the concentration of N2 effectors in the recipe at stage 5 of differentiation. The results show the expression levels of genes of interest in the presence of N2 factors. [Diagram 30] Figure 1 shows the dynamic profile of expression levels of PVALB and somatostatin genes versus the concentration of sodium pyruvate in the recipe at stage 5 of differentiation. The results show the expression levels of genes of interest in the presence of sodium pyruvate factor. [Figure 31A] 31A-31B show the dynamic profile of expression levels of LHX6 and GAD1 genes versus effector concentration in the recipe at stage 5 of differentiation. The results show the expression levels of the genes of interest in the presence of all factors. [Figure 31B] See legend to Figure 31A. [Diagram 32] Photographs of fluorescent images of differentiated interneuron cells at the end of treatment at stage 4. Cells were stained with forebrain biomarkers including NeuN, dorsal forebrain marker PAX6, GAD65, nestin, MASH1, pan-neuronal biomarkers βIII-tubulin, SOX2, and SOX6. Results show fluorescent images of cells at day 3 (end of stage 4). [Diagram 33]Photographs of fluorescent images of differentiated interneuron cells at the end of five treatments are shown. Cells were stained with forebrain biomarkers including GAD65, MASH1, MAP2, synapsin, GABAergic interneuron biomarker GABA, LHX6, neurofilament, parvalbumin and GFAP. Results show fluorescent images of cells at day 17 (end of stage 5). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0057] Detailed Description of the Invention Described herein are methodologies and compositions that allow the generation of not only forebrain neural progenitor cells but also mature GABAergic interneurons from human pluripotent stem cells in chemically defined culture conditions using a small molecule-based approach. The disclosed method generates medial ganglia primitive neural progenitor cells (progenitors to GABAergic interneurons) in a three-stage protocol in which OTX2+ FEZF2+ SIX3+ forebrain neural stem cells (FB-NSCs) are generated in 3 days, followed by NKX2-1+ ventral forebrain neural stem cells (VFB-NSCs) by day 6 of culture, followed by ASCL1+ medial ganglia primitive neural progenitor cells (MGE-NPCs) by day 9 of culture. Thus, the present disclosure allows the access of MGE-NPCs in a significantly shorter time than prior art protocols using chemically defined culture conditions. MGE-NPCs can be further differentiated following a two-stage protocol in which MGE-NPCs are first differentiated into immature neurons in 3 days (on day 12 of culture following a 9-day protocol to generate MGE-NPCs) and subsequently into mature parvalbumin+ interneurons in 14 days (on day 26 of culture).

[0058] As described in Example 1, a High-Dimensional Design of Experiments (HD-DoE) approach was used to simultaneously test multiple process inputs (e.g., small molecule agonists or antagonists) on output responses such as gene expression. These experiments allowed the identification of chemically defined media containing agonists and / or antagonists of specific signaling pathways sufficient to generate forebrain neural stem and progenitor cells, including FB-NSCs, VFB-NSCs, and MGE-NPCs, under defined conditions and in a short time. The optimized media was further validated by factor lethality analysis, which examined the effect of removing individual agonists or antagonists, as described in Example 2. The phenotype of cells generated by the differentiation protocol described in Example 3 was further confirmed by immunohistochemistry.

[0059] FIG. 17 illustrates a schematic diagram of an embodiment of a method of the disclosure for generating FB-NSCs, VFB-NSCs, and MGE-NPCs using a three-stage protocol.

[0060] FIG. 18 illustrates a schematic diagram of an embodiment of a method of the disclosure for generating immature neurons and mature parvalbumin+ interneurons from MGE-NPCs using a two-stage protocol.

[0061] Various aspects of the invention are described in further detail in the following subsections.

[0062] I. cell The starting cells used in the culture of the present disclosure are human pluripotent stem cells. As used herein, the term "human pluripotent stem cells" (abbreviated as hPSC) refers to human stem cells that have the ability to differentiate into a variety of different cell types. As used herein, the term "pluripotent" refers to cells that have the ability to differentiate into cell types characteristic of all three germ cell layers (endoderm, mesoderm and ectoderm) under different conditions. Pluripotent cells are primarily characterized by their ability to differentiate into all three germ layers, for example, using nude mice and teratoma formation assays. Although the preferred test for pluripotency is the demonstration of the ability to differentiate into cells of each of the three germ layers, pluripotency can also be demonstrated by the expression of embryonic stem (ES) cell markers.

[0063] Human pluripotent stem cells include, for example, human embryonic stem cells, such as induced pluripotent stem cells (iPSCs) and ES cell lines. Non-limiting examples of induced pluripotent stem cells (iPSCs) include 19-11-1, 19-9-7 or 6-9-9 cells (e.g., as described in Yu, J. et al. (2009) Science 324:797-801). Non-limiting examples of human embryonic stem cell lines include ES03 cells (WiCell Research Institute) and H9 cells (Thomson, JA et al. (1998) Science 282:1145-1147). Human pluripotent stem cells (PSCs) express cell markers that can be used to identify cells as PSCs. Non-limiting examples of pluripotent stem cell markers include TRA-1-60, TRA-1-81, TRA-2-54, SSEA1, SSEA3, SSEA4, CD9, CD24, OCT3, OCT4, NANOG and / or SOX2. Because the methods of generating forebrain neural progenitor cells of the present disclosure are used to differentiate (mature) a starting pluripotent stem cell population, in various embodiments the forebrain neural progenitor cell population generated by the methods of the present disclosure lack expression of one or more stem cell markers, such as one or more stem cell markers selected from the group consisting of TRA-1-60, TRA-1-81, TRA-2-54, SSEA1, SSEA3, SSEA4, CD9, CD24, OCT3, OCT4, NANOG and / or SOX2.

[0064] The pluripotent stem cells are subjected to culture conditions described herein that induce cell differentiation. As used herein, the term "differentiation" refers to the development of cells from a more primitive stage to more mature (i.e., less primitive) cells that typically exhibit the phenotypic characteristics of lineage commitment to a particular cell lineage.

[0065] As used herein, "neural stem cell" refers to a cell that is committed to a neural lineage but is more differentiated than a pluripotent stem cell in that it still has the capacity to differentiate into different cell types along the neural lineage.

[0066] As used herein, "neural progenitor cells" refer to cells that are more differentiated than neural stem cells but are capable of further differentiation into specific neural cell types.

[0067] In some embodiments, cells can be identified and characterized based on the expression of one or more biomarkers, such as specific biomarkers for neural progenitor cells or forebrain-committed neural cells. A "positive" biomarker is one that is expressed on the cell of interest, while a "negative" biomarker is one that is not expressed on the cell of interest.

[0068] Non-limiting examples of biomarkers whose expression can be assessed in characterizing cells of interest include genes involved in the development and patterning of the anterior neuroectoderm and forebrain, including OTX2 (Hoch et al. (2015) Cell Reports 12:482-494), SIX3 (Lagutin et al. (2003) Genes & Development 17:368-379) and FEZF2 (Zhang et al. (2014) Development 141:4794-47805), genes expressed in the ventral forebrain, including NKX2-1, and the absence of the dorsal forebrain marker PAX6 (i.e., PAX6 as a negative biomarker) (Stoykova et al. (2000) J. Neurosci. 20:8042-8050), and genes expressed in neuronal progenitor cells of the medial ganglia primordium (MGE) region, including ASCL1, LHX6 and DLX1 (Silberberg et al. (2015) Cell Reports 12:482-494). al. (2016) Neuron 92:59-74). In some embodiments, the FB-NSCs are OTX2+ FEZF2+ SIX3+. In some embodiments, the VFB-NSCs are NKX2-1+. In some embodiments, the VFB-NSCs are NKX2-1+ and PAX6 negative (PAX6-). In some embodiments, the MBE-NPCs are ASCL1+. In some embodiments, the MBE-NPCs are positive for at least two markers selected from ASCL1, LHX6, and DLX1. In some embodiments, the MBE-NPCs are ASCL1+ LHX6+ DLX1+.

[0069] Other biomarkers included βIII-tubulin (a pan-neuronal marker), KI67 (a proliferation marker), DLX5 (a neuronal progenitor marker expressed in the LGE and MGE regions of the forebrain (Wang et al. (2010) J. Neurosci. 30:5334-5345)), OLIG2 (an oligodendrocyte marker), GFAP (a glial marker), DCX (a marker identifying immature neurons), SOX6 (a marker expressed in postmitotic progenitor cells in the MGE region (Batista-Brito et al. (2009) Neuron 63:466-481)) and GABA (a marker specifically expressed by GABAergic interneurons).

[0070] As used herein, a slight "low" level of expression of a biomarker of interest by a cell is intended to refer to a level that is at most 20% above background levels, more preferably less than 20%, less than 15%, less than 10% or less than 5% above background levels (wherein background levels correspond, for example, to the expression level of a negative control marker that is not believed to be expressed by the cell).

[0071] In some embodiments, the cell generated by the method of the present disclosure is forebrain neural stem cell (FB-NSC).As used herein, "forebrain neural stem cell" or "FB-NSC" refers to stem cell-derived neural stem cell that expresses at least one biomarker selected from OTX2, FEZF2 and SIX3, preferably two or all three biomarkers.FB-NSC may also express additional biomarkers, including but not limited to βIII-tubulin and / or KI67.

[0072] In some embodiments, the cells generated by the method of the present disclosure are ventral forebrain neural stem cells (VFB-NSCs), which are more differentiated (more mature) than FB-NSCs and are committed along the ventral lineage.As used herein, "ventral forebrain neural stem cells" or "VFB-NSCs" refer to stem cell-derived neural cells that express biomarker NKX2-1.In one embodiment, VFB-NSCs do not express biomarker PAX6 or express it at slightly low levels.In addition, VFB NSCs may also express additional biomarkers, including but not limited to βIII-tubulin and / or KI67.

[0073] In some embodiments, the cell generated by the method of the present disclosure is medial ganglia primordial neural progenitor cell (MGE-NPC).As used herein, "medial ganglia primordial neural progenitor cell" or "MGE-NPC" refers to stem cell-derived neural cell that expresses at least one biomarker selected from ASCL1, LHX6 and DLX1, preferably two or all three biomarkers.MGE-NPC may also express additional biomarkers, including but not limited to βIII-tubulin and / or KI67.

[0074] The lineage-committed MGE-NPCs generated by the methods of the present disclosure can be further cultured in vitro to generate mature GABAergic interneurons, for example, according to the culture protocols described herein. As used herein, mature GABAergic neurons refer to neuronal-derived cells that express the biomarker parvalbumin and may also express LHX6, MAP2, GABA, and / or GAD1.

[0075] II. Media components The disclosed methods for generating FB-NSCs, VFB-NSCs, and MGE-NPCs, as well as immature neurons and parvalbumin+ interneurons from MGE-NPCs, include culturing human pluripotent stem cells in a medium that includes a specific agonist and / or antagonist of a cell signaling pathway. In various embodiments, the medium lacks serum, lacks exogenously added growth factors, lacks animal products, is serum-free, xeno-free, and / or feeder layer-free. In various embodiments, the medium lacks a SMAD2 / 3 inhibitor or antagonist, lacks a dual SMAD inhibitor or antagonist, or lacks a TGFβ pathway antagonist.

[0076] As described in Example 1, the medium containing BMP pathway antagonist, MEK pathway antagonist, WNT pathway antagonist, AKT pathway antagonist, SHH pathway agonist and PKC pathway antagonist is sufficient to generate OTX2+ FEZF2+ SIX3+ forebrain neural stem cell (FB-NSC) in only 3 days (referred to as "stage 1" of differentiation protocol herein).Further differentiation of FB-NSC in the medium containing BMP pathway antagonist, MEK pathway antagonist, WNT pathway antagonist and SHH pathway agonist is sufficient to generate NKX2-1+ VFB-NSC in another 3 days (referred to as "stage 2" herein). Further differentiation of VFB-NSCs in medium containing TAK1 pathway antagonist, SHH pathway agonist, TGF-β pathway antagonist, TRK pathway antagonist, Notch pathway antagonist, and IGF1 pathway agonist was sufficient to generate ASCL1+ medial ganglia primitive neural progenitor cells (MGE-NPCs) in another 3 days (referred to herein as "Stage 3"), for a total of 3-stage, 9-day protocol to generate MGE-NPCs. MGE-NPCs can be further differentiated into immature neurons by additional culture for 3 days in medium containing CREB or PKA pathway agonist, valproic acid or its analog, substance P or its analog, GDNF pathway agonist, and mTOR pathway agonist (referred to herein as "Stage 4"). Finally, the immature neurons can be further differentiated into mature GABAergic interneurons by further culturing for 2 weeks (14 days) in medium containing a BDNF pathway agonist, an IGF-1 pathway agonist, ascorbic acid or an analog, sodium pyruvate, O-LPA or an analog, N2 supplement and nonessential amino acids (referred to herein as "Stage 5").

[0077] As used herein, an "agonist" of a cell signaling pathway is intended to refer to an agent that stimulates (upregulates) the cell signaling pathway. Stimulation of a cell signaling pathway can be initiated extracellularly, for example, by the use of an agonist (e.g., an agonist can be a receptor ligand) that activates a cell surface receptor involved in the signaling pathway. Additionally or alternatively, stimulation of cell signaling can be initiated intracellularly, for example, by the use of a small molecule agonist that interacts intracellularly with a component of the signaling pathway.

[0078] As used herein, an "antagonist" of a cell signaling pathway is intended to refer to an agent that inhibits (downregulates) a cell signaling pathway. Inhibition of a cell signaling pathway can be initiated extracellularly, for example, by using an antagonist that blocks cell surface receptors involved in the signaling pathway. Alternatively or additionally, inhibition of cell signaling can be initiated intracellularly, for example, by using a small molecule antagonist that interacts intracellularly with a component of the signaling pathway.

[0079] The agonist and antagonist used in the method of the present disclosure are known in the art and commercially available.They are used in medium at effective concentrations to achieve desired outcome, for example, the generation of forebrain neural stem or progenitor cells that express forebrain markers of interest.The non-limiting examples of suitable agonist and antagonist agents and effective concentration ranges are further described below.

[0080] Antagonists of the BMP (bone morphogenetic protein) pathway include agents, molecules, compounds, or substances that can inhibit (downregulate) the BMP signaling pathway, which is biologically activated by the binding of BMP to a BMP receptor that is an activin receptor-like kinase (ALK) (e.g., type I BMP receptor, including but not limited to ALK2 and ALK3). In one embodiment, the BMP pathway antagonist is selected from the group consisting of LDN193189, DMH1, DMH2, dorsomorphin, K02288, LDN214117, LDN212854, follistatin, ML347, noggin, and combinations thereof. In one embodiment, the BMP pathway antagonist is present in the medium at a concentration within the range of 100-500 nM, 100-400 nM, 150-350 nM, or 200-300 nM. In one embodiment, the BMP pathway antagonist is LDN193189. In one embodiment, the BMP pathway antagonist is LDN193189 present in the medium at a concentration in the range of 100-500 nM, 100-400 nM, 150-350 nM, or 200-300 nM. In one embodiment, the BMP pathway antagonist is LDN193189 present in the medium at a concentration of 275 nM in step (a) (stage 1) and 250 nM in step (b) (stage 2).

[0081] Antagonists of the MEK pathway include agents, molecules, compounds, or substances that can inhibit (downregulate) one or more signaling pathways of components of the MAPK / ERK pathway (also known as the Ras-Raf-MEK-ERK pathway). In one embodiment, the MEK pathway antagonist is selected from the group consisting of PD0325901, binimetinib (MEK162), cobimetinib (XL518), selumetinib, trametinib (GSK1120212), CI-1040 (PD-184352), refametinib, ARRY-142886 (AZD-6244), PD98059, U0126, BI-847325, RO 5126766, and combinations thereof. In one embodiment, the MEK pathway antagonist is present in the medium at a concentration in the range of 25-300 nM, 50-150 nM, 50-250 nM, 75-200 nM, or 100-120 nM. In one embodiment, the MEK pathway antagonist is PD0325901. In one embodiment, the MEK pathway antagonist is PD0325901 present in the medium at a concentration in the range of 25-300 nM, 50-150 nM, 50-250 nM, 75-200 nM, or 100-120 nM. In one embodiment, the MEK pathway antagonist is PD0325901 present in the medium at a concentration of 110 nM in step (a) (stage 1) and 100 nM in step (b) (stage 2).

[0082] Antagonists of WNT pathway include agents, molecules, compounds, or substances that can inhibit (downregulate) canonical Wnt / β-catenin signaling pathway, which is biologically activated by binding of Wnt-protein ligand with Frizzled family receptor.In one embodiment, WNT pathway antagonists are selected from the group consisting of XAV939, ICG001, capmatinib, endo-IWR-1, IWP-2, IWP-4, MSAB, CCT251545, KY02111, NCB-0846, FH535, LF3, WIKI4, triptonide, KYA1797K, JW55, JW 67, JW74, cardiogen 1, NLS-StAx-h, TAK715, PNU 74654, iCRT3, WIF-1, DKK1, and combinations thereof. In one embodiment, the WNT pathway antagonist is present in the medium at a concentration in the range of 10-500 nM, 50-250 nM, or 50-150 nM. In one embodiment, the WNT pathway antagonist is XAV939. In one embodiment, the WNT pathway antagonist is XAV939 present in the medium at a concentration of 10-500 nM, 50-250 nM, or 50-150 nM. In one embodiment, the WNT pathway antagonist is XAV939 present in the medium at a concentration of 110 nM in step (a) (stage 1) and 100 nM in step (b) (stage 2).

[0083] Agonists of the SHH (Sonic Hedgehog) pathway include agents, molecules, compounds, or substances that can stimulate (activate) signal transduction through the SHH pathway, which biologically involves the binding of SHH to the Patched-1 (PTCH1) receptor and transduction through the Smoothened (SMO) transmembrane protein. In one embodiment, the SHH pathway agonist is selected from the group consisting of purmorphamine, GSA 10, SAG, and combinations thereof. In one embodiment, the SHH pathway agonist is present in the medium at a concentration within the range of 100-1000 nM, 200-800 nM, 250-750 nM, or 500-600 nM. In one embodiment, the SHH pathway agonist is purmorphamine. In one embodiment, the SHH pathway agonist is purmorphamine present in the medium at a concentration of 100-1000 nM, 200-800 nM, 250-750 nM, or 500-600 nM. In one embodiment, the SHH pathway agonist is purmorphamine present in the medium at a concentration of 550 nM in step (a) (stage 1) and 500 nM in steps (b) and (c) (stages 2 and 3).

[0084] Antagonists of the AKT pathway include agents, molecules, compounds, or substances that can inhibit (downregulate) the signaling pathway of one or more of the serine / threonine kinase AKT family members, including AKT1 (also called PKB or RacPK), AKT2 (also called PKBβ or RacPK-β), and AKT3 (also called PKBγ or thymoma viral protooncogene 3). In one embodiment, the AKT pathway antagonist is selected from the group consisting of MK2206, GSK690693, perifosine (KRX-0401), ipatasertib (GDC-0068), capivasertib (AZD5363), PF-04691502, AT 7867, triciribine (NSC154020), ARQ751, milansertib (ab235550), borsertib, cerisertib, and combinations thereof. In one embodiment, the AKT pathway antagonist is present in the medium at a concentration in the range of 25-300 nM, 50-200 nM, 75-200 nM, or 125-150 nM. In one embodiment, the AKT pathway antagonist is MK2206. In one embodiment, the AKT pathway antagonist is MK2206 present in the medium at a concentration in the range of 25-300 nM, 50-200 nM, 75-200 nM, or 125-150 nM. In one embodiment, the AKT pathway antagonist is MK2206 present in the medium at a concentration of 138 nM in step (a) (stage 1).

[0085] Antagonists of PKC (protein kinase C) pathway include agents, molecules, compounds, or substances that can inhibit (downregulate) PKC signaling pathway, which is biologically mediated by PKC family members. The PKC family of serine / threonine kinases includes 15 isozymes, encompassing the "classical" PKC subcategory, including isoforms α, β1, β2, and γ. In one embodiment, the PKC pathway antagonist inhibits the activity of at least one (in other embodiments, at least two or three) PKC enzymes selected from PKCα, PKCβ1, PKCβ2, and PKCγ. In one embodiment, the PKC pathway antagonist is selected from the group consisting of Go 6983, sotrastaurin, enzastaurin, staurosporine, LY31615, Go 6976, GF 109203X, Ro 31-8220 mesylate, and combinations thereof. In one embodiment, the PKC pathway antagonist is present in the medium at a concentration in the range of 10-500 nM, 50-300 nM, 50-150 nM, or 75-150 nM. In one embodiment, the PKC pathway antagonist is Go 6983. In one embodiment, the PKC pathway antagonist is Go 6983 present in the medium at a concentration of 10-500 nM, 50-300 nM, 50-150 nM, or 75-150 nM. In one embodiment, the PKC pathway antagonist is Go 6983 present in the medium at a concentration of 110 nM in step (a) (stage 1).

[0086] Antagonists of the TAK1 (also known as MAP3K7) pathway include agents, molecules, compounds, or substances that can inhibit (downregulate) signal transduction through TAK1 (MAP3K7). In one embodiment, the TAK1 pathway antagonist is selected from the group consisting of takinib, dehydroabietic acid, NG25, sarsasapogenin, and combinations thereof. In one embodiment, the TAK1 pathway antagonist is present in the medium at a concentration in the range of 1-5uM, 1-3uM, or 1.5-2.5uM. In one embodiment, the TAK1 pathway antagonist is takinib. In one embodiment, the TAK1 pathway antagonist is takinib present in the medium at a concentration of 1-5uM, 1-3uM, or 1.5-2.5uM. In one embodiment, the TAK1 pathway antagonist is takinib, which is present in the medium at a concentration of 2 uM in step (c) (ie, stage 3) of the method.

[0087] Antagonists of the TGFβ (transforming growth factor beta) pathway include agents, molecules, compounds, or substances capable of inhibiting (downregulating) signaling through TGFβ receptor family members, the serine / threonine kinase receptor family. In one embodiment, the TGFβ pathway antagonist is selected from the group consisting of A 83-01, SB-431542, GW788388, SB525334, TP0427736, RepSox, SD-208, and combinations thereof. In one embodiment, the TGFβ pathway antagonist is present in the medium at a concentration in the range of 300-800 nM, 250-750 nM, 300-650 nM, or 400-600 nM. In one embodiment, the TGFβ pathway antagonist is A 83-01. In one embodiment, the TGFβ pathway antagonist is A 83-01 present in the medium at a concentration of 300-800 nM, 250-750 nM, 300-650 nM or 400-600 nM. In one embodiment, the TGFβ pathway antagonist is A 83-01 present in the medium at a concentration of 500 nM in step (c) (i.e., stage 3) of the method.

[0088] Antagonists of the TRK pathway include agents, molecules, compounds, or substances capable of inhibiting (downregulating) signaling through TrkA, TrkB, and / or TrkC tyrosine kinases. In one embodiment, the TRK pathway antagonist is selected from the group consisting of GNF-5837, BMS-754807, UNC2020, taretrectinib, altilatinib, ceritrectinib, PF 06273340, and combinations thereof. In one embodiment, the TRK pathway antagonist is present in the medium at a concentration in the range of 30-80 nM, 25-75 nM, 30-65 nM, or 40-60 nM. In one embodiment, the TRK pathway antagonist is GNF-5837. In one embodiment, the TRK pathway antagonist is GNF-5837 present in the medium at a concentration of 30-80 nM, 25-75 nM, 30-65 nM or 40-60 nM. In one embodiment, the TRK pathway antagonist is GNF-5837 present in the medium at a concentration of 50 nM in step (c) (i.e., stage 3) of the method.

[0089] Antagonists of the Notch pathway include agents, molecules, compounds, or substances capable of inhibiting (downregulating) signaling through or activity of Notch transcription factors. In one embodiment, the Notch pathway antagonist is selected from the group consisting of GSI-XX, RO4929097, semagacestat, dibenzazepine, LY411575, crenigacestat, IMR-1, IMR-1A, FLI-06, DAPT, valproic acid, YO-01027, CB-103, tangeretin, BMS-906024, avagacestat, brucein D, and combinations thereof. In one embodiment, the TRK pathway antagonist is present in the medium at a concentration in the range of 25-200 nM, 50-150 nM, or 75-125 nM. In one embodiment, the Notch pathway antagonist is GSI-XX. In one embodiment, the Notch pathway antagonist is GSI-XX present in the medium at a concentration of 25-200 nM, 50-150 nM, or 75-125 nM. In one embodiment, the Notch pathway antagonist is GSI-XX present in the medium at a concentration of 100 nM in step (c) (i.e., stage 3) of the method.

[0090] IGF1 (insulin-like growth factor 1) pathway agonists include agents, molecules, compounds, or substances capable of stimulating (activating) signaling through the IGF1 pathway. In one embodiment, the IGF1 pathway agonist is selected from the group consisting of IGF1, IGF1-Ado, X10, mecasermin, and combinations thereof. In one embodiment, the IGF1 pathway agonist is present in the medium at a concentration in the range of 2-20 ng / ml, 5-15 ng / ml, or 7.5-12.5 ng / ml. In one embodiment, the IGF1 pathway agonist is IGF1. In one embodiment, the IGF1 pathway agonist is IGF1 present in the medium at a concentration of 2-20 ng / ml, 5-15 ng / ml, or 7.5-12.5 ng / ml. In one embodiment, the IGF1 pathway agonist is IGF1 present in the medium at a concentration of 10 ng / ml in step (c) (stage 3) of the method. In one embodiment, the IGF1 pathway agonist is IGF1, which is present in the culture medium at step (e) (stage 5) of the method at a concentration of 10 ng / ml.

[0091] CREB or PKA pathway agonists include agents, molecules, compounds, or substances that can stimulate (activate) signaling through the CREB or PKA pathway. In one embodiment, the CREB or PKA pathway agonist is selected from the group consisting of cAMP, dibutyryl-cAMP, 8-Br-cAMP, cAMPS-Sp, CW 008, forskolin, 8-CPT-cAMP, CW 008, N6-benzoyl-adenosine 3',5'-cyclic monophosphate sodium salt, adenosine 3',5'-cyclic monophosphate sodium salt monohydrate, (S)-adenosine cyclic 3',5'-(hydrogen phosphorothioate) triethylammonium, Sp-adenosine 3',5'-cyclic monophosphorothioate triethylammonium salt, Sp-5,6-DCI-cBiMPS, 8-bromoadenosine 3',5'-cyclic The CREB or PKA pathway agonist is selected from the group consisting of monophosphorothioate Sp-isomer sodium salt, 8-bromo-adenosine 3',5'-cyclic monophosphorothioate Sp-isomer sodium salt, Sp-8-pCPT-cyclic GMPS sodium, 8-bromoadenosine 3',5'-cyclic monophosphate, N6-monobutyryladenosine 3':5'-cyclic monophosphate sodium salt, 8-PIP-cAMP, Sp-cAMPS, and combinations thereof. In one embodiment, the CREB or PKA pathway agonist is present in the medium at a concentration in the range of 0.5-2.5 μM, 0.75-2.0 μM, or 1.0-1.5 μM. In one embodiment, the CREB or PKA pathway agonist is cAMP. In one embodiment, the CREB or PKA pathway agonist is cAMP present in the medium at a concentration of 0.5-2.5 μM, 0.75-2.0 μM, or 1.0-1.5 μM. In one embodiment, the CREB or PKA pathway agonist is cAMP present in the medium at a concentration of 1.0 μM in stage 4 of the method.

[0092] The medium of stage 4 comprises valproic acid or an analogue thereof. In one embodiment, the valproic acid or analogue thereof is selected from the group consisting of valproic acid, valproate, sodium valproate, and semisodium valproate. In one embodiment, the valproic acid or analogue is present in the medium at a concentration in the range of 250-750 nM, 300-600 nM, or 450-550 nM. In one embodiment, valproic acid is used. In one embodiment, valproic acid is used at a concentration of 250-750 nM, 300-600 nM, or 450-550 nM. In one embodiment, valproic acid is used that is present in the medium at a concentration of 500 nM in stage 4 of the method.

[0093] The medium of stage 4 comprises substance P or an analogue thereof. Substance P is an 11 amino acid neuropeptide of which numerous analogues have been described in the art (e.g., reviewed in Datar et al. (2004) Curr. Top. Med. Chem. 4:75-103). In one embodiment, substance P or an analogue thereof is present in the medium at a concentration in the range of 50-250 nM, 75-200 nM or 100-150 nM. In one embodiment, substance P is used. In one embodiment, substance P is used at a concentration of 50-250 nM, 75-200 nM or 100-150 nM. In one embodiment, substance P is used that is present in the medium at a concentration of 100 nM in stage 4 of the method.

[0094] Agonists of the glial cell line-derived neurotrophic factor (GDNF) pathway include agents, molecules, compounds, or substances that can stimulate (upregulate) the GDNF signaling pathway. In one embodiment, the GDNF pathway agonist is selected from the group consisting of GDNF, BT13, BT44, and combinations thereof. In one embodiment, the GDNF pathway agonist is present in the medium at a concentration in the range of 1-100 ng / ml, 5-50 ng / ml, 10-25 ng / ml, or 12.5-17.5 ng / ml. In one embodiment, the GDNF pathway agonist is GDNF. In one embodiment, the GDNF pathway agonist is GDNF present in the medium at a concentration of 1-100 ng / ml, 5-50 ng / ml, 10-25 ng / ml, or 12.5-17.5 ng / ml. In one embodiment, the GDNF pathway agonist is GDNF, which is present in the medium at stage 4 of the method at a concentration of 10 ng / ml.

[0095] Agonists of the mTOR (mammalian target of rapamycin) pathway include agents, molecules, compounds, or substances that can stimulate (upregulate) signaling through mTOR, a member of the PI3K-related kinase family, which is a central component of the mTORC1 and mTORC2 complexes. In one embodiment, the mTOR pathway agonist is selected from the group consisting of MHY1458, NV-5138, testosterone, 3-benzyl-5-((2-nitrophenoxy)methyl)-dihydrofuran-2(3H)-one (3BDO), 3BDO, L-leucine, NV-5138 hydrochloride, NV-5138, L-leucine-d1, L-leucine-2-13C,15N, leucine-13C6, L-leucine-d7, L-leucine-d10, L-leucine-d2, 1-leucine-d3, L-leucine-18O2, L-leucine-13C, L-leucine-2-13C, L-leucine-13C6-15N, L-leucine-15N, L-leucine-1-13C,15N, and combinations thereof. In one embodiment, the mTOR pathway agonist is present in the medium at a concentration in the range of 1-5 μM, 1-3 μM, or 1.5-2.5 μM. In one embodiment, the mTOR pathway agonist is MHY1458. In one embodiment, the mTOR pathway agonist is MHY1458 present in the medium at a concentration of 1-5 μM, 1-3 μM, or 1.5-2.5 μM. In one embodiment, the mTOR pathway agonist is MHY1458 present in the medium at a concentration of 2 μM in stage 4 of the method.

[0096] Agonists of the brain-derived neurotrophic factor (BDNF) pathway include agents, molecules, compounds, or substances that can stimulate (upregulate) the BDNF signaling pathway. In one embodiment, the BDNF pathway agonist is selected from the group consisting of BDNF, rotigotine, 7,8-DHF, ketamine, tricyclic dimer peptide-6 (TDP6), LM22A-4, and combinations thereof. In one embodiment, the BDNF pathway agonist is present in the medium at a concentration in the range of 1-100 ng / ml, 5-50 ng / ml, 10-25 ng / ml, or 12.5-17.5 ng / ml. In one embodiment, the BDNF pathway agonist is BDNF. In one embodiment, the BDNF pathway agonist is BDNF present in the medium at a concentration of 1-100 ng / ml, 5-50 ng / ml, 10-25 ng / ml, or 12.5-17.5 ng / ml. In one embodiment, the BDNF pathway agonist is BDNF, which is present in the medium at stage 5 of the method at a concentration of 10 ng / ml.

[0097] The medium for stage 5 includes ascorbic acid or an analogue thereof. In one embodiment, the ascorbic acid or an analogue thereof is selected from the group consisting of vitamin C, 2-phospho-L-ascorbic acid, L-ascorbic acid, sodium ascorbyl phosphate, magnesium ascorbyl phosphate, ascorbyl glucoside, tetrahexyldecyl ascorbate (THD), ethylated L-ascorbic acid, and combinations thereof. In one embodiment, the ascorbic acid or analogue is present in the medium at a concentration in the range of 50-500 μM, 100-400 μM, or 200-300 μM. In one embodiment, 2-phospho-L-ascorbic acid is used. In one embodiment, 2-phospho-L-ascorbic acid is used at a concentration of 50-500 μM, 100-400 μM, or 200-300 μM. In one embodiment, 2-phospho-L-ascorbic acid is used which is present in the medium at a concentration of 200 μM in stage 5 of the method.

[0098] The medium at stage 5 comprises sodium pyruvate or an analogue thereof. In one embodiment, sodium pyruvate or an analogue thereof is used at a concentration of 50-500 μM, 100-300 μM, or 150-250 μM. In one embodiment, sodium pyruvate is used that is present in the medium at a concentration of 200 μM at stage 5 of the method.

[0099] The medium of stage 5 comprises lysophosphatidic acid (LPA) or an analog thereof. In one embodiment, the LPA or analog thereof is selected from the group consisting of lysophosphatidic acid, 2-[[3-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)propyl]thio]benzoic acid, 1-oleoyl lysophosphatidic acid sodium salt, UCM-05194, and combinations thereof. In one embodiment, the LPA or analog is present in the medium at a concentration in the range of 50-500 nM, 100-400 nM, or 200-300 nM. In one embodiment, O-LPA is used. In one embodiment, O-LPA is used at a concentration of 50-500 nM, 100-400 nM, or 200-300 nM. In one embodiment, O-LPA is used, which is present in the medium at a concentration of 200 nM in stage 5 of the method.

[0100] Stage 5 medium further comprises N2 supplement. As used herein, "N2 supplement" refers to a mixture of cell culture supplements for promoting the proliferation of neural derived cells. Various N2 supplements are known in the art, including but not limited to N-2 supplement (ThermoFisher Scientific), N2 supplement-A (Stem Cell Technologies), N-2 MAX medium supplement (R&D Systems), and N-2 supplement (CSH Protocols). N2 supplements include not only transferrin family molecules, but also additional proliferation-promoting molecules. In one embodiment, N2 supplements include holo-transferrin, insulin (recombinant full chain), progesterone, putrescine, selenium, retroprogesterone, medrogestone, norethindrone, chlormadinone acetate, cyproterone acetate, medroxyprogesterone acetate, and megestrol acetate. In one embodiment, the N2 supplement is present in the medium at a concentration in the range of 0.1%-5%, 0.5%-3%, or 1-2% nM. In one embodiment, the N2 supplement is present in the medium at a concentration of 1% in stage 5 of the method.

[0101] The medium at stage 5 further comprises a non-essential amino acid (NEAA) supplement. As used herein, "NEAA supplement" refers to an amino acid mixture for promoting cell growth. Various NEAA supplements are known in the art, including, but not limited to, MEM non-essential amino acid solution (ThermoFisher Scientific), OriCell™ NEAA cell culture supplement (Cyagen), and SuperCult® non-essential amino acid (NEAA) cell culture supplement (Creative Bioarray). In one embodiment, the NEAA supplement comprises alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine, and tyrosine. In one embodiment, the NEAA supplement is present in the medium at a concentration in the range of 0.1%-5%, 0.5%-3%, or 1-2%. In one embodiment, the NEAA supplement is present in the medium at a concentration of 1% in stage 5 of the method.

[0102] When agonist or antagonist is used in more than one step of the method, in one embodiment, it is the same specific agonist or antagonist that is used for each step that the agent is present in the medium.In another embodiment, different agonists or antagonists that affect the same signal transduction pathway are used in different steps of the method.For example, for the BMP antagonist used in steps (a) and (b) (stage 1 and 2), in one embodiment, the same BMP antagonist is used in steps (a) and (b).In another embodiment, different BMP antagonists are used in steps (a) and (b).Similarly, for the IGF-1 pathway agonist used in steps (c) and (e) (stage 3 and 5), in one embodiment, the same IGF-1 pathway agonist is used in steps (c) and (e).In another embodiment, different IGF-1 pathway agonists are used in steps (c) and (e).

[0103] When agonist or antagonist is used in more than one step of the method, in one embodiment, the same agonist or antagonist is used in each step where the active substance is present in the medium.In another embodiment, different concentrations of the same agonist or antagonist are used in different steps of the method.For example, for the BMP antagonist used in steps (a) and (b) (stage 1 and 2), in one embodiment, the same BMP antagonist is used in steps (a) and (b).In another embodiment, different concentrations of the same BMP antagonist are used in steps (a) and (b).Similarly, for the IGF-1 agonist used in steps (c) and (e) (stage 3 and 5), in one embodiment, the same IGF-1 agonist is used in steps (c) and (e).In another embodiment, different concentrations of the same IGF-1 agonist are used in steps (c) and (e).

[0104] III. Culture conditions In combination with the chemically defined optimal medium described in subsection II above, the disclosed method of generating FB-NSCs, VFB-NSCs, MGE-NPCs, immature neurons and mature GABAergic interneurons utilizes standard culture conditions established in the art for cell culture. For example, cells can be cultured at 37° C. under 5% O2 and 5% CO2 conditions. Cells can be cultured in standard culture vessels or plates, such as 96-well plates. In certain embodiments, the starting pluripotent stem cells are attached to a plate, preferably coated with an extracellular matrix material, such as vitronectin. In one embodiment, stem cells are cultured on a vitronectin-coated culture surface (e.g., a vitronectin-coated 96-well plate).

[0105] Pluripotent stem cells can be cultured in commercially available medium before differentiation.For example, stem cells can be cultured in Essential 8 Flex medium (Thermo Fisher # A2858501) for at least 1 day before starting differentiation protocol.In a non-limiting exemplary embodiment, stem cells are passaged at a density of 150,000 cells / cm2 on a 96-well plate coated with vitronectin (Thermo Fisher # A14700), and cultured in Essential 8 Flex medium for 1 day before differentiation.

[0106] To initiate the differentiation protocol, the medium in which the stem cells are cultured is exchanged for basal differentiation medium supplemented with signaling pathway agonists and / or antagonists, as described above in subsection II. The basal differentiation medium can include, for example, a commercially available base supplemented with additional standard media components necessary to maintain cell viability and proliferation, but lacking serum (basal differentiation medium is a serum-free medium) or any other exogenously added growth factors, such as FGF2, PDGF, or HGF. In a non-limiting exemplary embodiment, the basal differentiation medium contains 1x IMDM (Thermo Fisher #12440046), 1x F12 (Thermo Fisher #11765047), poly(vinyl alcohol) (Sigma #p8136) at 1 mg / ml, chemically defined lipid concentrate (Thermo Fisher #11905031) at 1%, 1-thioglycerol (Sigma #M6145) at 450 uM, insulin (Sigma #11376497001) at 0.7 ug / ml, and transferrin (Sigma #10652202001) at 15 ug / ml (also referred to herein as "CDM2" medium, as used in the exemplary differentiation protocols shown in Figures 17 and 18).

[0107] The medium is typically replaced with fresh medium periodically, for example, in one embodiment, the medium is replaced every 24 hours.

[0108] To generate FB-NSCs, VFB-NSCs, MGE-NPCs, immature neurons and mature GABAergic interneurons, the starting stem cells are cultured in optimized medium for a sufficient time for cell differentiation and expression of markers associated with lineage-committed FB-NSCs, VFBNSCs, -MGE-NPCs, immature neurons or mature GABAergic interneurons. As described in the Examples, it has been discovered that culturing pluripotent stem cells in a five-stage method, one optimized for generating FB-NSCs, a second optimized for generating VFB-NSCs, a third optimized for generating MGE-NPCs, a fourth optimized for generating immature neurons and a fifth optimized for generating mature GABAergic interneurons, can result in the production of MGE-NPCs in as little as 9 days of culture and mature interneurons in as little as 26 days of culture. The culture period for stage 1 (resulting in FB-NSCs) is from day 0 to day 3, for stage 2 (resulting in VFB-NSCs) is from day 3 to day 6, for stage 3 (resulting in MGE-NPCs) is from day 6 to day 9, for stage 4 (resulting in immature neurons) is from day 9 to day 12, and for stage 5 (resulting in mature interneurons) is from day 12 to day 26.

[0109] Thus, in the first stage of the method of generating FB-NSCs, also referred to herein as "step (a)" or "stage 1", pluripotent stem cells are cultured in stage 1 optimized medium from day 0 to day 3, or starting on day 0 and continuing through day 3, or for 72 hours (3 days), or for at least 60 hours, or at least 64 hours, or at least 68 hours, or at least 70 hours, or for at least 72 hours, or for 60 hours, or for 64 hours, or for 68 hours, or for 70 hours, or for 72 hours.

[0110] Thus, in the second stage of the method of generating VFB-NSCs, also referred to herein as "step (b)" or "stage 2", the FB-NSCs generated in step (a) are further cultured in stage 2 optimized medium from day 4 to day 6, or starting on day 4 and continuing through day 6, or starting on day 4 and continuing for 72 hours (3 days), or starting on day 4 and continuing for at least 60 hours, or at least 64 hours, or at least 68 hours, or at least 70 hours, or at least 72 hours, or starting on day 4 and continuing for 60 hours, or 64 hours, or 68 hours, or 70 hours, or 72 hours.

[0111] Thus, in the third stage of the method of generating MGE-NPCs, also referred to herein as "step (c)" or "stage 3", the VFB-NSCs generated in step (b) are further cultured in stage 3 optimized medium from day 6 to day 9, or starting on day 6 and continuing through day 9, or starting on day 6 and continuing for 72 hours (3 days), or starting on day 6 and continuing for at least 60 hours, or at least 64 hours, or at least 68 hours, or at least 70 hours, or at least 72 hours, or starting on day 6 and continuing for 60 hours, or 64 hours, or 68 hours, or 70 hours, or 72 hours.

[0112] Thus, in the fourth stage of the method of generating immature neurons, also referred to herein as "step (d)" or "stage 4", the MGE-NPCs generated in step (c) are further cultured in stage 4 optimized medium from day 9 to day 12, or starting on day 9 and continuing through day 12, or starting on day 9 and continuing for 72 hours (3 days), or starting on day 9 and continuing for at least 60 hours, or at least 64 hours, or at least 68 hours, or at least 70 hours, or at least 72 hours, or starting on day 9 and continuing for 60 hours, or 64 hours, or 68 hours, or 70 hours, or 72 hours.

[0113] Thus, in the fifth stage of the method of generating mature GABAergic interneurons, also referred to herein as "step (e)" or "stage 5," the immature neurons generated in step (d) are further cultured in stage 5 optimized medium from day 12 to day 26, or starting on day 12 and continuing through day 26, or starting on day 12 and continuing in culture for a time sufficient to generate parvalbumin+ mature interneurons (e.g., 14 days or 2 weeks of culture in stage 5 medium).

[0114] IV. use The disclosed methods and compositions for generating FB-NSCs, VFB-NSCs, MGE-NPCs, immature neurons, and mature GABAergic interneurons allow for efficient and robust availability of these cell populations for a variety of uses. For example, the methods and compositions can be used to study the development and biology of forebrain neural progenitor cells, including differentiation into GABAergic interneurons, to aid in the understanding and potential treatment of neurodegenerative and psychiatric diseases and disorders involving dysfunction of GABAergic interneurons. For example, the FB-NSCs, VFB-NSCs, MBE-NPCs, immature neurons, and mature GABAergic interneurons generated using the disclosed methods can be further purified by methods established in the art using agents that bind to surface markers expressed on the cells. Thus, in one aspect, the disclosure provides a method for isolating FB-NSCs, VFB-NSCs, MGE-NPCs, immature neurons, or mature GABAergic interneurons, comprising: contacting the FB-NSCs, VFB-NSCs, MGE-NPCs, immature neurons, or mature GABAergic interneurons produced by the methods of the present disclosure with at least one binding agent that binds to a cell surface marker expressed by the FB-NSCs, VFB-NSCs, MGE-NPCs, immature neurons, or mature GABAergic interneurons; and Isolating cells that bind to the binding agent, thereby isolating FB-NSCs, VFB-NSCs, MBE-NPCs, immature neurons, or mature GABAergic interneurons. The present invention provides a method comprising:

[0115] In one embodiment, the binding agent is an antibody, such as a monoclonal antibody (mAb) that binds to a cell surface marker. Cells that bind to the antibody can be isolated by methods known in the art, including, but not limited to, fluorescence activated cell sorting (FACS) and magnetic cell sorting (MACS).

[0116] Forebrain neural lineage progenitor cells are also envisioned for use in the treatment of neurodegenerative or psychiatric diseases and disorders by delivering cells to subjects with disease or disorder, including any of these diseases or disorders involving the dysfunction of GABAergic interneurons.It has been shown that the transplantation of embryonic medial ganglia primoria (MGE) cells into adult brain results in the dispersion and migration of transplanted cells and the differentiation into neurons that express GABA (Alvarez-Dolado et al. (2006) J. Neurosci. 26:7380-7389).Therefore, in one embodiment, forebrain neural lineage progenitor cells are used to treat epilepsy.It has been shown that the transplantation of GABAergic interneuron progenitor cells into postnatal neocortex of mice reduces epileptic seizures (Baraban et al. (2009) Proc. Natl. Acad. Sci. USA 106:15472-15477). The use of neural progenitor cells in the treatment of epilepsy is also reviewed in Shetty and Upadhya (2016) Neurosci. Biobehav. Rev. 62:35-47 and Lybrand et al. (2020) Neuropharm. 168:107781.

[0117] Loss or reduced activity of GABAergic interneuron is also associated with cognitive impairment in Alzheimer's disease or autism patients.Therefore, the forebrain lineage cells of the present disclosure can also be used for the treatment of disorders associated with cognitive impairment that may benefit from the functional restoration of GABAergic interneuron, including but not limited to Alzheimer's disease and autism.

[0118] The cells of the present disclosure are also useful for screening potential drugs or developing novel cell therapies for treating diseases or disorders involving dysfunction of GABAergic interneurons.

[0119] V. composition In other aspects, the disclosure provides compositions relating to methods for generating FB-NSCs, VFB-NSCs, MGE-NPCs, immature neurons and mature GABAergic interneurons, including media and cell cultures, as well as isolated progenitor cells and cell populations.

[0120] In one aspect, the present disclosure provides a medium for obtaining human OTX2+ FEZF2+ SIX3+ forebrain neural stem cells (FB-NSCs), comprising a BMP pathway antagonist, a MEK pathway antagonist, a WNT pathway antagonist, an AKT pathway antagonist, a SHH pathway agonist, and a PKC pathway antagonist. Suitable agents and their concentrations include those described in subsection II.

[0121] In one aspect, the present disclosure provides a medium for obtaining human NKX2-1+ ventral forebrain neural stem cells (VFB-NSCs), comprising a BMP pathway antagonist, a MEK pathway antagonist, a WNT pathway antagonist, and a SHH pathway agonist. Suitable agents and their concentrations include those described in subsection II.

[0122] In one aspect, the present disclosure provides a medium for obtaining human ASCL1+ medial ganglia primitive neural progenitor cells (MGE-NPCs) comprising a TAK1 pathway antagonist, a SHH pathway agonist, a TGF-β pathway antagonist, a TRK pathway antagonist, a Notch pathway antagonist, and an IGF1 pathway agonist. Suitable agents and concentrations thereof include those described in subsection II.

[0123] In one aspect, the disclosure provides a medium for obtaining human immature neurons from MGE-NPCs, comprising a CREB or PKA pathway agonist, a valproic acid or analog, a substance P or analog, a GDNF pathway agonist, and an mTOR pathway agonist. Suitable agents and concentrations thereof include those described in subsection II.

[0124] In one aspect, the present disclosure provides a medium for obtaining human mature GABAergic interneurons, comprising a BDNF pathway agonist, an IGF-1 pathway agonist, ascorbic acid or an analog, sodium pyruvate or an analog, LPA or an analog, an N2 supplement, and a NEAA supplement. Suitable agents and concentrations thereof include those described in subsection II.

[0125] In one aspect, the present disclosure provides an isolated cell culture of human OTX2+ FEZF2+ SIX3+ forebrain neural stem cells (FB-NSCs), comprising human OTX2+ FEZF2+ SIX3+ FB-NPCs cultured in a medium containing a BMP pathway antagonist, a MEK pathway antagonist, a WNT pathway antagonist, an AKT pathway antagonist, a SHH pathway agonist, and a PKC pathway antagonist.Suitable agents and their concentrations include those described in subsection II.

[0126] In one aspect, the present disclosure provides an isolated cell culture of human NKX2-1+ ventral forebrain neural stem cells (VFB-NSCs), comprising human NKX2-1+ VFB-NPCs cultured in a medium that includes a BMP pathway antagonist, a MEK pathway antagonist, a WNT pathway antagonist, and a SHH pathway agonist.Suitable agents and their concentrations include those described in subsection II.

[0127] In one aspect, the disclosure provides an isolated cell culture of human ASCL1+ medial ganglia primitive neural progenitor cells (MGE-NPCs), comprising human ASCL1+ MGE-NPCs cultured in a medium comprising a TAK1 pathway antagonist, a SHH pathway agonist, a TGF-β pathway antagonist, a TRK pathway antagonist, a Notch pathway antagonist, and an IGF1 pathway agonist. Suitable agents and concentrations thereof include those described in subsection II.

[0128] In one aspect, the disclosure provides an isolated cell culture of human immature neurons, comprising human immature neurons cultured in a medium comprising a CREB or PKA pathway agonist, a valproic acid or analog, a substance P or analog, a GDNF pathway agonist, and an mTOR pathway agonist. Suitable agents and concentrations thereof include those described in subsection II.

[0129] In one aspect, the present disclosure provides an isolated cell culture of human mature GABAergic interneurons, comprising human mature GABAergic interneurons cultured in a medium comprising a BDNF pathway agonist, an IGF-1 pathway agonist, an ascorbic acid or analog, a sodium pyruvate or analog, an LPA or analog, an N2 supplement, and a NEAA supplement. Suitable agents and concentrations thereof include those described in subsection II.

[0130] In one aspect, the present disclosure provides human OTX2+ FEZF2+ SIX3+ forebrain neural stem cells (FB-NSCs) generated by the methods of the present disclosure (i.e., step (a) or stage 1 of the culture protocol).

[0131] In one aspect, the present disclosure provides human NKX2-1+ ventral forebrain neural stem cells (VFB-NSCs) generated by the methods of the present disclosure (i.e., steps (a) and (b), or stages 1 and 2, of the culture protocol).

[0132] In one aspect, the present disclosure provides human ASCL1+ medial ganglia primordium neural progenitor cells (MGE-NPCs) generated by the methods of the disclosure (i.e., steps (a), (b) and (c), or stages 1, 2 and 3, of the culture protocol).

[0133] In one aspect, the present disclosure provides human immature neurons generated by the methods of the disclosure (i.e., steps (a), (b), (c), and (d) or stages 1, 2, 3, and 4 of the culture protocol).

[0134] In one aspect, the present disclosure provides human mature parvalbumin+ GABAergic interneurons produced by the methods of the disclosure (i.e., steps (a), (b), (c), (d) and (e) or stages 1, 2, 3, 4 and 5 of the culture protocol).

[0135] The present invention is further illustrated by the following examples, which should not be construed as further limiting. The contents of the figures and all references, patents, and published patent applications cited throughout this application are hereby expressly incorporated by reference. EXAMPLES

[0136] Example 1 Development of a protocol for the generation of stem cell-derived medial ganglia primordium neural progenitor cells expressing NKX2-1 and ASCL1 We developed a three-stage recipe for the generation of medial ganglia primordium-derived neural progenitors that can direct human pluripotent stem cells after 9 days in culture to progenitor cells that express NKX2-1 and ASCL1, which can be further differentiated into mature GABAergic interneurons.

[0137] This example utilizes the high-dimensional experimental design (HD-DoE) method previously described in Bukys et al. (2020) Iscience 23:101346. This method employs computerized design geometry to simultaneously test multiple process inputs, providing mathematical modeling of deep effector / response space. This method allows for the discovery of combinatorial signaling inputs that control complex processes, such as during cell differentiation. This allows for the testing of multiple plausible key process parameters, as such parameters affect output responses, such as gene expression. Since gene expression provides a hallmark of the phenotype, for example of human cells, this method can be applied to identify and understand which signaling pathways control cell fate. In this example, the HD-DOE method was applied to discover conditions for inducing genes expressed by midbrain neural progenitor cells directly from a pluripotent stem cell state.

[0138] To develop recipes for each stage, we tested and modeled the effects of agonists and antagonists (herein referred to as effectors) of multiple signaling pathways on the expression of two sets of 53 preselected genes after 3 days of treatment. These effectors are small molecules or proteins that are commonly used during the stepwise differentiation of stem cells to specific fates. The selection of effectors was based on the latest literature on neural induction and differentiation of stem cells into neural progenitor cells in the forebrain region of the developing brain.

[0139] To test effectors, we designed experiments with at least eight factors that could assess the response of cells to 48 or more different combinations of effectors at a range of concentrations.To analyze the model, we focused on the expression of genes involved in the development and patterning of the anterior neuroectoderm and forebrain, including OTX2 (Hoch et al. (2015) Cell Reports 12:482-494), SIX3 (Lagutin et al. (2003) Genes & Development 17:368-379) and FEZF2 (Zhang et al. (2014) Development 141:4794-47805). At later stages, we focused on dorsoventral patterning of the telencephalon and the absence of ventral forebrain expressed genes including NKX2-1 and the dorsal forebrain marker PAX6 (Stoykova et al. (2000) J. Neurosci. 20:8042-8050). The influence of each effector on gene expression levels is defined by a parameter called the factor contribution, which is calculated for each effector during modeling.

[0140] Stage 1 differentiation into forebrain-committed neural stem cells To identify recipes for stage 1 of differentiation, 96 different combinations of effectors were robotically prepared using compression of experimental designs by D-optimality. Effector combinations were prepared in basal medium, which were subsequently added to the cells, which were then differentiated. RNA extraction was performed after 3 days, and gene expression was obtained using quantitative PCR analysis. The data was normalized and modeled into effector designs using partial least squares regression analysis. As a result, gene-specific models were generated, which described the ability of effectors to control the expression of individual genes, both combinatorially and individually, after the models were adjusted to maximize Q2 predictive power. Solutions in the test space could then be explored to address desirability.

[0141] Optimizing for maximum expression of 3300 for SIX3 and expression values ​​of 1355 for FEZF2 resulted in a robust solution. The model was derived from testing 12 effectors including AGN193109, LDN193189, CHIR99021, XAV939, IGF2, purmorphamine, 4-oxo-RA, TTNPB, SR11237, FGF8, PD0325901 and A8301. As shown in Figure 1, three factors, namely LDN193189, an inhibitor of the BMP signaling pathway, XAV939, an inhibitor of the WNT signaling pathway, and purmorphamine, an agonist of the SHH pathway, had a positive effect on the expression of SIX3 with a factor contribution of 16, 5 and 5, respectively. The model also showed that TTNPB, an RA agonist, had a significant negative effect on the expression of SIX3 with a factor contribution of 21. Within the specifications to achieve 80% of maximum expression of SIX3, this complex medium formulation had a Cpk value (process capability index) of 0.6, corresponding to a risk of non-compliance of 3.4%.

[0142] To find any additional factors that can boost the expression of forebrain genes, the model was also optimized separately for the maximum expression of FEZF2 at an expression value of 3576. Within the specification to achieve 80% of the maximum expression of FEZF2, this complex medium composition had a Cpk value (process capability index) of 0.61, corresponding to a risk of non-compliance of 3.3%. As shown in Figure 2, LDN193189 was again identified as having a significant positive impact on the expression of FEZF2 with a factor contribution of 25; MEK inhibitors PD0325901, TTNPB and purmorphamine also had a positive impact on its expression with factor contributions of 10, 24 and 7. Maximum expression of OTX2 at 6348 was also modeled, and within the specification to achieve 80% of the maximum expression of OTX2, this complex medium composition had a Cpk value (process capability index) of 0.49, corresponding to a risk of non-compliance of 6.6%. As shown in Figure 3, the model showed LDN193189 as the highest positive effector with a factor contribution of 8. XAV939 and PD0325901 also showed positive effects, but their factor contributions were lower. The effector with the highest factor contribution was TTNPB, which had a significant negative effect with a factor contribution of 29.

[0143] To find a set of factors with the potential to optimize all three genes in this experiment, a new evaluation was performed by dynamic profile analysis, focusing on the maximum expression of OTX2, FEZF2 and SIX3. The results are shown in Figure 4. Based on this analysis, TTNPB, which was shown to have a negative effect on both OTX2 and SIX3, was removed from the recipe; purmorphamine, which had a negative effect on OTX2 but a positive effect on SIX3 and FEZF2, was included.

[0144] Further HD-DoE experiments were performed to further strengthen the conditions for forebrain differentiation from pluripotency. Further gene regulation models were obtained and used for the preparation of differentiation protocols. Factors in this experiment included LDN193189, PD173074, BLU9931, purmorphamine, SC79, MK2206, ZM336372, PD0325901, CHIR99021, XAV939, UCLA-GP130, tofacitinib and GO6983. As shown in Figure 5, when optimized for FEZF2, LDN193189, purmorphamine, AKT inhibitors MK2206, PD0325901 and XAV939 had a positive impact on its expression with factor contributions of 18, 11, 11, 15 and 12, respectively. Within the specification to achieve 80% of the maximum expression of FEZF2, this complex medium composition had a Cpk value (process capability index) of 0.63, corresponding to a risk of non-compliance of 2.7%. Dynamic profile analysis was also used to evaluate the interactions between effectors. As shown in Figure 6, factors that caused the expression level of FEZF2 to fall outside the target range were removed, and the remaining effectors included LDN193189, PD0325901, XAV939, purmorphamine, MK2206 and GO6983.

[0145] Considering both HD-DoE experiments, conditions maximizing differentiation of cells into forebrain regions with neural stem cell identity, thus associated with robust and high expression of FEZF2, SIX3 and OTX2, included the following effector inputs: LDN193189, PD0325901, XAV939, purmorphamine, MK2206 and GO6983. A representative recipe for stage 1 differentiation is summarized below in Table 1.

[0146] Table 1: Validated effectors for Stage 1 recipes TIFF2024541967000001.tif53128

[0147] Stage 2 Differentiation into Ventral Forebrain Neural Stem Cells To further guide the differentiation of forebrain-committed neural stem cells into ventral forebrain neural stem cells at stage 2, HD-DoE experiments were performed 3 days after the end of stage 1 treatment. At this time, we focused on the maximum expression of NKX2-1 expressed in the ventral forebrain region and the minimum expression of PAX6 expressed in the dorsal forebrain region. The 12-factor experiment included LDN193189, BMP7, PD0325901, MK2206, A8301, XAV939, CHIR99021, purmorphamine, SANT-1, AGN193109, TTNPB and GSI-XX. As shown in Figure 7, when the model was maximized for NKX2-1 expression, six effectors were identified that were able to increase its expression level, including LDN193189, PD0325901, MK2206, XAV939, purmorphamine, and GSI-XX, an inhibitor of the Notch signaling pathway. Using dynamic profile analysis, the model was modified to simultaneously achieve minimal expression levels of PAX6 and NKX2-2. As shown in Figure 8, this resulted in the removal of MK2206 and GSI-XX.

[0148] Considering a model with different optimization settings for maximum differentiation of cells towards the ventral forebrain, the recipe for stage 2 of differentiation included LDN193189, PD0325901, XAV939 and purmorphamine. Representative recipes for stage 2 differentiation are summarized in Table 2 below.

[0149] Table 2: Validated effectors for Stage 2 recipes TIFF2024541967000002.tif38128

[0150] Stage 3 differentiation into medial ganglionic primordium progenitor cells To further guide the cells towards the fate of medial ganglia primordium progenitors, an additional HD-DoE experiment was performed for 3 days on cells that had been treated with stage 1 and stage 2 media for a total of 6 days before the start of the experiment. This experiment included 13 effectors, LDN193189, A8301, GNF5837, AZD3147, GSI-XX, takinib, PD0325901, PD173074, BLU9931, IGF-1, MHY1485, purmorphamine and prostratin. In this model, we focused on maximal expression of genes expressed in neuronal progenitors within the medial ganglia primordium (MGE) region, including ASCL1, LHX6 and DLX1 (Silberberg et al. (2016) Neuron 92:59-74). When the model was optimized for maximum expression of ASCL1 at 1700, A8301, GSI-XX, purmorphamine and takinib, an inhibitor of the TAK1 pathway, had the highest factor contributions of 18.1, 14.1, 12.7 and 10.1, respectively. GNF5837 and IGF-1 also had a positive impact on its expression with factor contributions of 9.4 and 7. MHY1485 also had a positive contribution, but the coefficient was less than 2 (Figure 9). Within the specification to achieve 80% of maximum expression of ASCL1, this complex medium composition had a Cpk value (process capability index) of 0.59, corresponding to a risk of non-compliance of 3.6%.

[0151] Dynamic profile analysis was used to find common factors that have a positive effect on the expression of ASCL1, LHX6 and DLX1. As shown in Figure 10, it was observed that GSI-XX, takinib and GNF5837 had similar effects on the expression of all three genes, while A8301 and purmorphamine showed a positive trend only for ASCL1 and LHX6, whereas they had no significant negative effect on DLX1. IGF-1 had a positive effect on ASCL1 and LHX6, even though it had a negative effect on the expression level of DLX1, so it was added to the recipe. Thus, six effectors were finalized, including A8301, GSI-XX, takinib, GNF5837, IGF-1 and purmorphamine, as ingredients of the stage 3 differentiation medium. A representative recipe for stage 3 differentiation is summarized in Table 3 below.

[0152] Table 3: Validated effectors for stage 3 recipes TIFF2024541967000003.tif53128

[0153] Example 2 : Factor criticality analysis of culture conditions inducing stem cell-derived ventral forebrain neural progenitors To assess the impact of removing each tested factor, dynamic profile analysis was used to compare the expression levels of the gene of interest in the absence of each finalized factor and the presence of the others. This factor fatality analysis revealed the degree of importance of each input effector, since the expression level of the gene of interest revealed whether the desired outcome was achievable.

[0154] In the stage 1 recipe, each of the six finalized factors was removed individually, and the expression levels of forebrain genes in the presence of the other five factors were evaluated compared to the presence of all six factors together. The results are summarized in Figure 11A-B. When LDN193189 was removed, the expression levels of FEZF2, OTX2 and SIX3 were severely reduced from 2000 to 1000, from 5500 to 4000, and from 3000 to 0, respectively. The absence of XAV939 had a similar effect, with the levels of FEZF2, OTX2 and SIX3 reduced to 1700, 5000 and 1000, respectively. When purmorphamine was removed, the levels of FEZF2 and SIX3 were reduced to less than 1000 and 3000, whereas the level of OTX2 increased to 6500. Removal of PD0325901 resulted in a reduction in FEZF2 levels, which had no significant effect on the other two genes. The effect of the absence of MK2206 and GO6983 was also examined, and both factors resulted in a decrease in the expression levels of FEZF2 from 3500 to 2700 and 3200, as shown in FIG.

[0155] In the stage 2 recipe, the expression levels of NKX2-1, PAX6, and NKX2-2 were evaluated when each of the four finalized factors was removed and the other three factors remained present, compared to when all four factors were present. The results are summarized in Figure 13A-B. The absence of PD0325901 reduced the levels of NKX2-1 and NKX2-2 from 5000 to 3200 and 100 to 0, whereas PAX6 increased from 0 to 5000. The level of NKX2-1 did not change significantly in the absence of LDN193189, whereas the levels of PAX6 and NKX2-2 were both reduced below 0. Removing XAV939 reduced the level of NKX2-1 from 5000 to 3000, whereas both PAX6 and NKX2-2 increased to 10000 and 200, respectively. Another observation was the interaction of XAV939 with purmorphamine. In the absence of XAV939, the effect of purmorphamine on NKX2-1 is negative and the levels of NKX2-1 are higher when both factors are present, as shown in the interaction plot in FIG.

[0156] In the stage 3 recipe, the expression levels of ASCL1, DLX1 and LHX6 were evaluated when each of the six finalized factors was removed and the other five factors remained present, compared to when all six factors were present. The results are summarized in Figure 15A-B. By removing A8301, the level of ASCL1 was reduced from 1650 to 1000, while the values ​​of DLX1 and LHX6 did not change significantly. Removing GSI-XX had a significant effect on the values ​​of all three genes, reducing the expression levels of ASCL1, DLX1 and LHX6 from 1650, 220 and 240 to 1200, 50 and 140, respectively. Removing takinib had a similar effect on ASCL1, but the expression levels of DLX1 and LHX6 were reduced to 180 and 170. In the absence of purmorphamine, the values ​​of ASCL1 and LHX6 were reduced to 1200 and 190, while DLX1 remained the same. Similar to purmorphamine, removal of GNF5837 resulted in lower expression of ASCL1 at 1300 and LHX6 at 160, but had no significant effect on DLX1. As expected, in the absence of IGF-1, DLX1 values ​​increased to 410, while ASCL1 and LHX6 decreased to 1390 and 220, respectively.

[0157] Example 3 Immunocytochemical verification of NKX2-1 and ASCL1 expressing stem cell-derived ventral forebrain neural progenitors To validate the developed recipe described in Example 1, cells were treated with stage 1, stage 2 and stage 3 differentiation medium, and immunocytochemistry was used to evaluate biomarkers of ventral forebrain region and neural progenitor cells at the end of each stage. Biomarkers included SIX3, OTX2, βIII-tubulin pan-neuronal marker, NKX2-1, PAX6, KI67 proliferation marker, DLX5 neuronal progenitor marker expressed in LGE and MGE regions of forebrain (Wang et al. (2010) J. Neurosci. 30:5334-5345), LHX6, OLIG2 oligodendrocyte marker, MASH1 (ASCL1), GFAP glial marker, DCX marker specifying immature neurons, SOX6 marker expressed in postmitotic progenitor cells of MGE region (Batista-Brito et al. (2009) Neuron 63:466-481) and GABA marker specifically expressed by GABAergic interneurons. The results are shown in Figures 16, 17 and 18.

[0158] Immunocytochemistry images confirmed expression of SIX3 and OTX2 in more than 90% of the cells by the end of stage 1, while PAX6 and OLIG2 were undetectable. KI67 was also detected in the majority of the cells, confirming that the cells were proliferative at this time point (Figure 16). After treatment with stage 2 medium, expression of NKX2-1 and GABA was observed in the majority of the cultured cells, a minority of which also expressed MASH1, confirming the ventral regionalization of the differentiating cells, whereas PAX6 remained undetectable (Figure 17). By day 9, at the end of stage 3, NKX2-1, DLX5, and MASH1 were detected in the majority of the cultured cells, and βIII-tubulin, DCX, LHX6, and SOX6 were detected in more than half of the cultures, confirming the commitment of the cells to the forebrain MGE region (Figure 18). KI67 was also detected in some of the cultured cells, indicating that these cells still had the ability to proliferate and expand; therefore, this recipe results in a mixed culture of proliferative and post-mitotic MGE lineage-committed cells.

[0159] Detection of ventral forebrain neural progenitor markers and absence of dorsal forebrain markers in differentiated cells after 9 days confirmed the validity and robustness of the generated recipe for a three-stage differentiation protocol for MGE lineage-committed neural progenitor cells derived from human induced pluripotent stem cells.

[0160] Example 4 : Development of a protocol for MGE-neuronal progenitor cell-derived neurons expressing parvalbumin and GABA Here, we developed a two-stage recipe for neuronal maturation from medial ganglionic eminence (MGE)-derived neural progenitor cells that can direct MGE progenitor cells into mature PVALB interneurons expressing parvalbumin and GABA after 17 days in culture.

[0161] To develop recipes for each stage, we evaluated and modeled the effects of agonists and antagonists of multiple signaling pathways, referred to herein as effectors, on the expression of two sets of 53 preselected genes after 3 days of treatment. These effectors are small molecules or proteins. They are commonly used during the stepwise differentiation of stem cells to specific fates. The selection of effectors was based on the current literature on neural induction in the forebrain region of the developing brain and differentiation of stem cells into neural progenitors.

[0162] The effectors in the final Stage 4 and Stage 5 recipes developed herein are shown in Tables 4 and 5, respectively, below.

[0163] Table 4. Validated effectors in Stage 4 recipe TIFF2024541967000004.tif49128

[0164] Table 5. Validated effectors in stage 5 recipe TIFF2024541967000005.tif62128

[0165] To evaluate effectors, we designed experiments with at least eight factors that could evaluate the cell's response to 48 or more different combinations of effectors at a range of concentrations. To analyze the model, we focused on the expression of genes involved in the development and patterning of forebrain interneuron maturation, including LHX6 (Yuan et al. (2018) Elife, 7:e37382), DLX5 (Wang et al. (2010) J. Neuroscience 30:5334-5345), and ASCL1 (Shi et al. (2016) J. Biol. Chem. 291:13560-13570). At the later stage, we focused on the absence of interneuron maturation genes, including parvalbumin, and somatostatin (Horn and Nicoll (2018) Proc. Natl. Acad. Sci. 115:589-594). The influence of each effector on gene expression levels is defined by a parameter called the factor contribution, which is calculated for each effector during modeling.

[0166] To identify recipes for stage 4 of differentiation, 48 different combinations of effectors were robotically prepared, generated using experimental design compression by D-optimality. Effector combinations were prepared in basal medium, which were subsequently added to the cells, which were then differentiated. RNA extraction was performed after 3 days, and gene expression was obtained using quantitative PCR analysis. The data was normalized and modeled using partial least squares regression analysis on the effector design, resulting in the generation of gene-specific models that combinatorially and individually provided a description of the ability of effectors to control the expression of individual genes after model adjustment for maximum predictive power. Solutions within the test space could then be explored to address desirability. Optimizing to a value of 453 for maximum expression of MEF2C (Pai et al. (2020) Elife 9:54903) resulted in a robust solution. This model was derived from testing 12 effectors including cAMP, IGF-1, valproic acid, GSI-XX, LDN193189, substance P, SB431542, PD173074+BLU-554, MK2206, GDNF, PD0325901 and MHY1458. Valproic acid can inhibit histone deacetylase and increase gamma-aminobutyric acid (GABA). MHY1458 is an activator of the mTOR signaling pathway, substance P is a member of the tachykinin neuropeptide family, and cAMP is an activator of the CREB and PKA pathways. GDNF is a ligand for glial cell line-derived neurotrophic factor and can activate the GDNF pathway. They had a positive impact on the expression of MEF2C with a factor contribution of 12, 0.04, 9, 15, and 5, respectively (Figure 19). The model also showed that the BMP inhibitor LDN193189, the TGFβR inhibitor SB431542, the FGFR inhibitor PD17 / BLU, and the Notch inhibitor GSI-XX had significant negative effects on MEF2C expression with factor contributions of 11, 7.9, 7.5, and 11.6. Within the specifications to achieve 80% of maximum expression of MEF2C, this complex medium formulation had a Cpk value (process capability index) of 0.4, corresponding to a risk of non-compliance of 6.7%.

[0167] To find a set of factors with potential to optimize MEF2C in this experiment, a new evaluation was performed by dynamic profile analysis focusing on maximum expression of MEF2C (Figure 20). Based on this analysis, IGF-1, which was shown to have a negative effect on MEF2C, was removed from the recipe; GSI-XX, LDN19, SB431542, and PD17, which also had a negative effect on MEF2C, were also removed.

[0168] To further guide the differentiation of forebrain lineage-committed neural stem cells into PVALB interneuron cells at stage 5, we performed HD-DoE experiments for 3 days after the end of stage 4 treatment. At this time, we focused on the maximum expression of PVALB expressed in parvalbumin (+) interneurons and the minimum expression of somatostatin, another type of interneuron. This eight-factor experiment included JQ1, indolactam-V, oleic acid, BDNF, IGF-1, 2-phospho-L-ascorbic acid, Albumax, and MK2206. When maximizing the model for the expression of PVALB, we identified five effectors that could increase its expression level, including indolactam-V, oleic acid, BDNF, IGF-1, 2-phospho-L-ascorbic acid, and BDNF, a ligand of the BDNFR signaling pathway (Figure 21). We modified the model to simultaneously achieve maximum expression levels of PVALB using dynamic profile analysis, resulting in the inclusion of 2-phospho-L-ascorbic acid, BDNF, IGF1, while removing MK2206, Albumax, and JQ1 (Figure 22). Considering the model with different optimization settings for maximum differentiation of cells toward the ventral forebrain, the recipe for stage 5 of differentiation included BDNF, IGF-1, 2-phospho-L-ascorbic acid from this HD-Doe experiment.

[0169] To examine the effects of other factors on the differentiation of forebrain-committed neural stem cells into PVALB interneuron cells, an additional HD-DoE experiment was performed for 3 days on cells treated with stage 1 to stage 4 medium for a total of 12 days before starting the experiment. This experiment included 8 effectors, prostratin, rosiglitazone, GSI-XX, heparin, GW0742, N2, THI0019, and GDNF. In this model, we focused on the maximum expression of the PVALB gene expressed in PVALB(+) interneurons (Nahar et al. (2021) Front. Psych. 12:679960). When the model was optimized for maximum expression of PVALB, prostratin, GW0742, N2, and GDNF had the highest factor contributions of 13.56, 19.499, 15.9, and 8.2, respectively (Figure 23). Since the contribution of N2 has a significant effect on the gene expression of PVALB, it is included in our stage 5 recipe.

[0170] Using dynamic profile analysis, we found common factors that had a positive effect on the expression of PVALB (Figure 24). We observed that GW0742, N2, prostratin, and GDNF had similar effects on the expression of the PVALB gene, while GSI-XX, THI0019, and heparin had a negative effect on the gene expression of PVALB. Therefore, they were removed from the recipe.

[0171] To further guide the differentiation of forebrain lineage-committed neural stem cells into PVALB interneuron cells, an additional HD-DoE experiment was performed for 3 days on cells that had been treated with stage 1 to stage 4 medium for a total of 12 days before the start of the experiment. This experiment included 8 effectors, forskolin, arachidonic acid, VPA, BDNF, BT13, O-LPA, GW7646, and sodium pyruvate. In this model, we focused on the maximum expression of the PVALB gene expressed in PVALB(+) interneurons (Nahar et al. (2021) Front. Psych. 12:679960). When optimizing the model for maximum expression of PVALB, forskolin, GW7646, and sodium pyruvate had the highest factor contributions of 3.14, 27.2, and 17.1, respectively (Figure 25). Sodium pyruvate had a significant effect on the expression of PVALB, so it is included in the stage 5 recipe.

[0172] Dynamic profile analysis was used to find common factors that have a positive effect on the expression of PVALB (Figure 26). It was observed that GW7646, sodium pyruvate, and forskolin had similar effects on the expression of the PVALB gene, whereas BT13 had a significant negative effect on the gene expression of PVALB. Therefore, it was removed from the recipe.

[0173] Example 5 : Factor criticality analysis of culture conditions inducing forebrain neural progenitor cell-derived interneurons To assess the impact of removing each tested factor, dynamic profile analysis was used to compare the expression levels of the gene of interest in the absence of each finalized factor but the presence of the others. This factor fatality analysis revealed the degree of importance of each input effector, as the expression levels of the gene of interest revealed whether the desired outcome was achievable.

[0174] To assess the impact of removing each tested factor, we again used dynamic profile analysis to compare the expression levels of the genes of interest in the absence of each finalized factor but in the presence of the others. This factor fatality analysis revealed the degree of importance of each input effector, since the expression levels of the genes of interest reveal whether the desired outcome is achievable.

[0175] In the stage 4 recipe, each of the five finalized factors was removed, and the expression levels of forebrain genes in the presence of the other four factors were evaluated compared to the presence of all five factors. The results are shown in Figure 27. MEF2C was reported to determine the cell fate of PVALB- progenitor cells (Mayer et al. (2018) Nature 555:457-462). We optimize MEF2C expression at this stage. cAMP and substance P were found to increase the expression of MEF2C and decrease the expression of somatostatin, so they are included in the stage 4 recipe. GDNF and valproic acid were found to contribute to the expression of PVALB, but the increase in the expression of somatostatin was minimal, so they are also included in the stage 4 recipe. MHY1458 was found to increase the expression of PVALB, so it is included in the recipe (Figure 27).

[0176] In the stage 5 recipe, each of the seven finalized factors was removed and the expression levels of PVALB and SST were evaluated when the other factors remained present compared to when all seven factors were present. In this HD-DoE experiment, 2-phospho-L-ascorbic acid, BDNF, and IGF-1 contributed positively to the expression of PVALB, whereas they did not change the expression of another interneuron marker, somatostatin. 2-phospho-L-ascorbic acid, BDNF, and IGF-1 were included in the stage 5 recipe (Figure 28). In another HD-DoE experiment, N2 and other effectors were found to contribute to the expression of parvalbumin. Others were also found to increase the expression of the SST interneuron marker, somatostatin. Thus, they were removed from the recipe and N2 was included in the stage 5 recipe (Figure 29). In the final HD-DoE experiment, sodium pyruvate was found to contribute to the expression of PVALB. Sodium pyruvate was included in the stage 5 recipe (Figure 30). Finally, O-LPA was found to contribute to the expression of LHX6 and GAD1 and was therefore included in the recipe (Figure 31).

[0177] Example 6 Immunocytochemical demonstration of parvalbumin-expressing interneurons derived from forebrain neural progenitor cells To validate the recipe developed in Example 4, cells were first treated with stage 1, stage 2 and stage 3 differentiation medium from Example 1, then with stage 4 and stage 5 differentiation medium from Example 4, and biomarkers of forebrain neurons and neural progenitor cells were assessed using immunocytochemistry at the end of each stage. Biomarkers included MAP2, NeuN, neurofilament, βIII-tubulin pan-neuronal marker, PAX6, LGE marker (Kioussi et al. (1999) Proc. Natl. Acad. Sci. 96:14378-14382;Kioussi et al. (1999) Proc. Natl. Acad. Sci. 96:14378-14382), SOX2 neuronal progenitor marker expressed in the ventral forebrain (Hansen et al. (2013) Nature Neurosci. 16:1576-1587), LHX6, cortical interneuron marker, MASH1 (ASCL1), GFAP glial marker, and SOX6 marker expressed in postmitotic progenitor cells in the MGE region (Batista-Brito et al. (2009) Neuron 63:466-481) and GABA markers that are specifically expressed by GABAergic interneurons (Figure 32).

[0178] Immunocytochemistry images confirmed that more than 90% of cells expressed NeuN by the end of stage 4 (Gusel'Nikova et al. (2015) Acta Naturae 7:42-47) and PAX6 was not detected. Expression of Mash1 and SOX6 observed in the majority of cultured cells confirmed the ventral regionalization of the differentiating cells (Figure 32). Detection of GAD65 in the majority of cells confirmed that the cells were interneurons.

[0179] By day 27, when stage 5 was complete, GABA, MASH1, and LHX6 were detected in the majority of cultured cells, and MAP2, neurofilament, and parvalbumin were detected in more than half of the cultures in which the forebrain MGE region of cells were identified as differentiated PVALB(+) interneurons (Figure 33). We also detected neurofilament, synapsin, and GAD65 in the majority of cultured cells, indicating that these cells had mature neuronal functions (Nguyen et al. (2014) J. Neurosci. 34:14948-14960) (Figure 33).

[0180] At day 27, mature neural differentiation markers were detected in differentiated cells and dorsal forebrain markers were absent, confirming the validity and robustness of the developed recipe for a two-stage differentiation protocol for MGE lineage-committed neural progenitor cells into differentiated parvalbumin(+) interneurons.

[0181] Equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims.

Claims

1. A method for generating human OTX2+FEZF2+SIX3+ FB-NSCs, comprising culturing human pluripotent stem cells for at least 60 hours in a medium containing a bone morphogenetic protein (BMP) pathway antagonist, a mitogen-activated protein kinase kinase (MEK) pathway antagonist, a Wingless-related integration site (WNT) pathway antagonist, an Akt pathway antagonist, a Sonic Hedgehog (SHH) pathway agonist, and a protein kinase C (PKC) pathway antagonist to obtain human OTX2+FEZF2+SIX3+ forebrain neural stem cells (FB-NSCs).

2. The method according to claim 1, further culturing the human OTX2+ FEZF2+ SIX3+ FB-NSCs for at least 60 hours in a medium containing a BMP pathway antagonist, a MEK pathway antagonist, a WNT pathway antagonist, and an SHH pathway agonist, but lacking an Akt pathway antagonist and a PKC pathway antagonist, to obtain human NKX2-1+ ventral forebrain neural stem cells (VFB-NSCs).

3. The method according to claim 2, further culturing the human NKX2-1+ VFB-NSCs for at least 60 hours in a medium containing a transforming growth factor β-activated kinase 1 (TAK1) pathway antagonist, an SHH pathway agonist, a transforming growth factor β (TGFβ) pathway antagonist, a tropomyosin receptor kinase (TRK) pathway antagonist, a Notch pathway antagonist, and an insulin-like growth factor 1 (IGF1) pathway agonist to obtain human ASCL1+ medial basal ganglia primordial neural progenitor cells (MGE-NPCs).

4. The method according to claim 3, further culturing the human ASCL1+ MGE-NPC for at least 60 hours in a medium containing cAMP response element-binding protein (CREB) or a protein kinase A (PKA) pathway agonist, valproic acid or an analogue, substance P or an analogue, a glial cell line-derived neurotrophic factor (GDNF) pathway agonist, and a mammalian target of rapamycin (mTOR) pathway agonist to obtain human immature neurons.

5. The method according to claim 4, further culturing the human immature neurons for a sufficient amount of time in a medium comprising a brain-derived neurotrophic factor (BDNF) pathway agonist, an IGF1 pathway agonist, ascorbic acid or an analogue, sodium pyruvate or an analogue, lysophosphatidic acid (LPA) or an analogue, an N2 supplement, and a non-essential amino acid (NEAA) supplement to obtain mature parvalbumin+ interneurons.

6. The aforementioned human pluripotent stem cells, (i) induced pluripotent stem cells (iPSCs) or embryonic stem cells; and / or (ii) Adhering to the vitronectin-coated plate during culture, The method according to claim 1.

7. (i) The BMP pathway antagonist is selected from the group consisting of LDN193189, DMH1, DMH2, Dorsopmorphin, K02288, LDN214117, LDN212854, follistatin, ML347, noggin, and combinations thereof; (ii) The MEK pathway antagonist is selected from the group consisting of PD0325901, binimetinib (MEK162), cobimetinib (XL518), selumetinib, trametinib (GSK1120212), CI-1040 (PD-184352), refametinib, ARRY-142886 (AZD-6244), PD98059, U0126, BI-847325, RO 5126766, and combinations thereof; (iii) The WNT pathway antagonist is selected from the group consisting of XAV939, ICG001, capmatinib, endo-IWR-1, IWP-2, IWP-4, MSAB, CCT251545, KY02111, NCB-0846, FH535, LF3, WIKI4, tryptonide, KYA1797K, JW55, JW67, JW74, cardionogen 1, NLS-StAx-h, TAK715, PNU 74654, iCRT3, WIF-1, DKK1, and combinations thereof; (iv) The SHH pathway agonist is selected from the group consisting of purmorphamine, GSA 10, SAG, and combinations thereof; (v) The Akt pathway antagonist is selected from the group consisting of MK2206, GSK690693, Perifosin (KRX-0401), Ipatasertib (GDC-0068), Capivasertib (AZD5363), PF-04691502, AT 7867, Trisirivine (NSC154020), ARQ751, Miransertib (ab235550), Borussertib, Cerisertib, and combinations thereof; (vi) The PKC pathway antagonist is selected from the group consisting of Go 6983, sotrastaurin, enzastaurin, staurosporine, LY31615, Go 6976, GF 109203X, Ro 31-8220 mesylate, and combinations thereof; (vii) The TAK1 pathway antagonist is selected from the group consisting of taquinib, dehydroabietic acid, NG25, sarsasapogenin, and combinations thereof; (viii) The TGFβ pathway antagonist is selected from the group consisting of A 83-01, SB-431542, GW788388, SB525334, TP0427736, RepSox, SD-208, and combinations thereof; (ix) The TRK pathway antagonist is selected from the group consisting of GNF-5837, BMS-754807, UNC2020, taretrectinib, altilatinib, ceritrectinib, PF 06273340, and combinations thereof; (x) The Notch pathway antagonist is selected from the group consisting of GSI-XX, RO4929097, semagacestat, dibenzoazepine, LY411575, crenigacestat, IMR-1, IMR-1A, FLI-06, DAPT, valproic acid, YO-01027, CB-103, tangeretin, BMS-906024, abagacestat, brucein D, and combinations thereof; (xi) The IGF1 pathway agonist is selected from the group consisting of IGF1, IGF1-Ado, X10, mecasermin, and combinations thereof; (xii) The CREB or PKA pathway agonist is cAMP, dibutyryl-cAMP, 8-Br-cAMP, cAMPS-Sp, CW 008, forskolin, 8-CPT-cAMP, CW 008, N6-benzoyl-adenosine 3',5'-cyclic monophosphate sodium salt, adenosine 3',5'-cyclic monophosphate sodium salt monohydrate, (S)-adenosine cyclic 3',5'-(hydrogen phosphorothioate)triethylammonium, Sp-adenosine 3',5'-cyclic monophosphorothioate triethylammonium salt, Sp-5,6-DCI-cBiMPS, 8-bromoadenosine 3',5'-cyclic Selected from the group consisting of monophosphorothioate Sp-isomer sodium salt, 8-bromo-adenosine 3',5'-cyclic monophosphorothioate Sp-isomer sodium salt, Sp-8-pCPT-cyclic GMPS sodium, 8-bromoadenosine 3',5'-cyclic monophosphate, N6-monobutyryladenosine 3':5'-cyclic monophosphate sodium salt, 8-PIP-cAMP, Sp-cAMPS, and combinations thereof; (xiii) Valproic acid or an analogue is selected from the group consisting of valproic acid, valproate, sodium valproate, and semisodium valproate; (xiv) A substance P or analogue is a substance P; (xv) The GDNF pathway agonist is selected from the group consisting of GDNF, BT13, BT44, and combinations thereof; (xvi) The mTOR pathway agonist is selected from the group consisting of MHY1458, NV-5138, testosterone, 3-benzyl-5-((2-nitrophenoxy)methyl)-dihydrofuran-2(3H)-one (3BDO), 3BDO, L-leucine, NV-5138 hydrochloride, NV-5138, L-leucine-d1, L-leucine-2-13C,15N, leucine-13C6, L-leucine-d7, L-leucine-d10, L-leucine-d2, L-leucine-d3, L-leucine-18O2, L-leucine-13C, L-leucine-2-13C, L-leucine-13C6-15N, L-leucine-15N, L-leucine-1-13C,15N, and combinations thereof; (xvii) The BDNF pathway agonist is selected from the group consisting of BDNF, rotigotine, 7,8-DHF, ketamine, tricyclic dimeric peptide-6 (TDP6), LM22A-4, and combinations thereof; (xviii) Ascorbic acid or its analogues are selected from the group consisting of vitamin C, 2-phospho-L-ascorbic acid, L-ascorbic acid, sodium ascorbyl phosphate, magnesium ascorbyl phosphate, ascorbyl glucoside, tetrahexyldecyl ascorbate (THD), ethylated L-ascorbic acid, and combinations thereof; (xix) Sodium pyruvate or an analogue is sodium pyruvate; and (xx) LPA or analogues, lysophosphatidic acid, 2-[[3-(1,3-dioxo-1H-benzo[de]isoquinoline-2(3H)-yl)propyl Selected from the group consisting of thiobenzoic acid, 1-oleoyl lysophosphatidic acid sodium salt, UCM-05194, and combinations thereof, The method according to any one of claims 1 to 6.

8. (i) The BMP pathway antagonist is present in the culture medium at a concentration in the range of 100 to 500 nM; (ii) The MEK pathway antagonist is present in the culture medium at a concentration in the range of 25 to 300 nM; (iii) The WNT pathway antagonist is present in the culture medium at a concentration in the range of 10 to 500 nM; (iv) The SHH pathway agonist is present in the culture medium at a concentration in the range of 100 to 1000 nM; (v) The Akt pathway antagonist is present in the culture medium at a concentration in the range of 25 to 300 nM; (vi) The PKC pathway antagonist is present in the culture medium at a concentration in the range of 10 to 500 nM; (vii) The TAK1 pathway antagonist is present in the culture medium at a concentration of 1 to 5 μM. (viii) The TGFβ pathway antagonist is present in the culture medium at a concentration in the range of 300 to 800 nM; (ix) The TRK pathway antagonist is present in the culture medium at a concentration in the range of 30 to 80 nM; (x) The Notch pathway antagonist is present in the culture medium at a concentration in the range of 25 to 200 nM; (xi) The IGF1 pathway agonist is present in the culture medium at a concentration in the range of 2 to 20 ng / ml; (xii) The CREB or PKA pathway agonist is present in the culture medium at a concentration in the range of 0.5 to 2.5 μM; (xiii) Valproic acid or an analogue is present in the culture medium at a concentration in the range of 250 to 750 nM; (xiv) Substance P or an analogue is present in the culture medium at a concentration in the range of 50 to 250 nM; (xv) The GDNF pathway agonist is present in the culture medium at a concentration in the range of 1 to 100 ng / ml; (xvi) The mTOR pathway agonist is present in the culture medium at a concentration of 1 to 5 μM; (xvii) The BDNF pathway agonist is present in the culture medium at a concentration in the range of 1 to 100 ng / ml; (xviii) Ascorbic acid or an analogue is present in the culture medium at a concentration in the range of 50 to 500 μM; (xix) Sodium pyruvate or an analogue is present in the culture medium at a concentration in the range of 50 to 500 μM; (xx) LPA or an analogue is present in the culture medium at a concentration in the range of 50 to 500 nM; (xxi)N2 supplement is present in the culture medium at a concentration ranging from 0.1% to 5%; and (xxii) The NEAA supplement is present in the culture medium at a concentration of 0.1% to 5%. The method according to claim 7.

9. (i) The BMP pathway antagonist is LDN193189 present in the culture medium at a concentration of 275 nM or 250 nM; (ii) The MEK pathway antagonist is PD0325901 present in the culture medium at a concentration of 110 nM or 100 nM; (iii) The WNT pathway antagonist is XAV939 present in the culture medium at a concentration of 110 nM or 100 nM; (iv) The SHH pathway agonist is purmorphamine present in the culture medium at a concentration of 550 nM or 500 nM; (v) The Akt pathway antagonist is MK2206, which is present in the culture medium at a concentration of 138 nM; (vi) The PKC pathway antagonist is Go 6983 present in the culture medium at a concentration of 110 nM; (vii) The TAK1 pathway antagonist is a takinib present in the culture medium at a concentration of 2 μM; (viii) The TGFβ pathway antagonist is A83-01 present in the culture medium at a concentration of 500 nM; (ix) The TRK pathway antagonist is GNF-5837 present in the culture medium at a concentration of 50 nM; (x) The Notch pathway antagonist is GSI-XX present in the culture medium at a concentration of 100 nM; (xi) The IGF1 pathway agonist is IGF1 present in the culture medium at a concentration of 10 ng / ml; (xii) The CREB or PKA pathway agonist is cAMP present in the culture medium at a concentration of 1.0 μM; (xiii) Valproic acid or an analogue is valproic acid present in the culture medium at a concentration of 500 nM; (xiv) Substance P or an analogue is substance P present in the culture medium at a concentration of 100 nM; (xv) The GDNF pathway agonist is GDNF present in the culture medium at a concentration of 10 ng / ml; (xvi) The mTOR pathway agonist is MHY1458, which is present in the culture medium at a concentration of 2 μM; (xvii) The BDNF pathway agonist is BDNF present in the culture medium at a concentration of 10 ng / ml; (xviii) Ascorbic acid or an analogue is 2-phospho-L-ascorbic acid present in the culture medium at a concentration of 200 μM; (xix) Sodium pyruvate or an analogue is sodium pyruvate present in the culture medium at a concentration of 200 μM; (xx)LPA or analogue is O-LPA present in the culture medium at a concentration of 200 nM; (xxi)N2 supplement is present in the culture medium at a concentration of 1%; and (xxii) The NEAA supplement is present in the culture medium at a concentration of 1%. The method according to claim 8.

10. (a) to obtain human OTX2+ FEZF2+ SIX3+ FB-NSCs by culturing human pluripotent stem cells for at least 60 hours in a medium containing a BMP pathway antagonist, a MEK pathway antagonist, a WNT pathway antagonist, an Akt pathway antagonist, an SHH pathway agonist, and a PKC pathway antagonist; and (b) Further culture the human OTX2+ FEZF2+ SIX3+ FB-NSC for at least 60 hours in a medium containing a BMP pathway antagonist, a MEK pathway antagonist, a WNT pathway antagonist, and an SHH pathway agonist, but lacking an Akt pathway antagonist and a PKC pathway antagonist, to obtain human NKX2-1+ VFB-NSC. A method for generating human NKX2-1+ VFB-NSCs, including [specific component].

11. (a) Culturing human pluripotent stem cells for at least 60 hours in a medium containing a BMP pathway antagonist, a MEK pathway antagonist, a WNT pathway antagonist, an Akt pathway antagonist, an SHH pathway agonist, and a PKC pathway antagonist to obtain human OTX2+ FEZF2+ SIX3+ FB-NSCs; (b) Further culturing the human OTX2+ FEZF2+ SIX3+ FB-NSC for at least 60 hours in a medium containing a BMP pathway antagonist, a MEK pathway antagonist, a WNT pathway antagonist, and an SHH pathway agonist, but lacking an Akt pathway antagonist and a PKC pathway antagonist, to obtain human NKX2-1+ VFB-NSC; and (c) Further culturing the human NKX2-1+ VFB-NSC for at least 60 hours in a medium containing a TAK1 pathway antagonist, an SHH pathway agonist, a TGFβ pathway antagonist, a TRK pathway antagonist, a Notch pathway antagonist, and an IGF1 pathway agonist to obtain human ASCL1+ MGE-NPC. A method for generating human ASCL1+ MGE-NPCs, including [specific component].

12. (a) to obtain human immature neurons by culturing human ASCL1+MGE-NPCs for at least 60 hours in a medium containing a CREB or PKA pathway agonist, valproic acid or analogue, substance P or analogue, a GDNF pathway agonist, and an mTOR pathway agonist; and (b) Culturing the human immature neurons for a sufficient amount of time in a medium containing a BDNF pathway agonist, an IGF1 pathway agonist, ascorbic acid or analogue, sodium pyruvate or analogue, LPA or analogue, N2 supplement, and NEAA supplement to obtain human mature parvalbumin+ interneurons. A method for generating human mature parvalbumin+ interneurons from human ASCL1+MGE-NPCs, including [specific component].

13. (i) The BMP path antagonist is LDN193189; (ii) The MEK pathway antagonist is PD0325901; (iii) The WNT pathway antagonist is XAV939; (iv) The SHH pathway agonist is purmorphamine; (v) The Akt path antagonist is MK2206; (vi) The PKC pathway antagonist is Go 6983; (vii) The TAK1 pathway antagonist is a takinib; (viii) The TGFβ pathway antagonist is A 83-01; (ix) The TRK pathway antagonist is GNF-5837; (x) The notch pathway antagonist is GSI-XX; (xi) The IGF1 pathway agonist is IGF1; (xii) The CREB or PKA pathway agonist is cAMP; (xiii) The valproic acid or analogue is valproic acid; (xiv) The substance P or analogue is substance P; (xv) The GDNF pathway agonist is GDNF; (xvi) The mTOR pathway agonist is MHY1458; (xvii) The BDNF pathway agonist is BDNF; (xviii) The ascorbic acid or analogue is 2-phospho-L-ascorbic acid; (xix) The sodium pyruvate or analogue is sodium pyruvate; and (xx) The LPA or analogue is O-LPA, The method according to any one of claims 10 to 12.

14. (i) LDN193189 is present in the culture medium at a concentration in the range of 100 to 500 nM; (ii) PD0325901 is present in the culture medium at a concentration in the range of 25 to 300 nM; (iii) XAV939 is present in the culture medium at a concentration in the range of 10 to 500 nM; (iv) Pulmorphamine is present in the culture medium at a concentration in the range of 100 to 1000 nM; (v) MK2206 is present in the culture medium at a concentration in the range of 25 to 300 nM; (vi) Go 6983 is present in the culture medium at a concentration in the range of 10 to 500 nM; (vii) Taquinib is present in the culture medium at a concentration in the range of 1 to 5 μM; (viii) A 83-01 is present in the culture medium at a concentration in the range of 250 to 750 nM; (ix) GNF-5837 is present in the culture medium at a concentration in the range of 25 to 75 nM; (x) GSI-XX is present in the culture medium at a concentration in the range of 25 to 200 nM; (xi) IGF1 is present in the culture medium at a concentration in the range of 2 to 20 ng / ml; (xii) cAMP is present in the culture medium at a concentration in the range of 0.5 to 2.5 μM; (xiii) Valproic acid is present in the culture medium at a concentration in the range of 250 to 750 nM; (xiv) Substance P is present in the culture medium at a concentration in the range of 50 to 250 nM; (xv) GDNF is present in the culture medium at a concentration in the range of 1 to 100 ng / ml; (xvi) MHY1458 is present in the culture medium at a concentration in the range of 1 to 5 μM; (xvii) BDNF is present in the culture medium at a concentration in the range of 1 to 100 ng / ml; (xviii) 2-phospho-L-ascorbic acid is present in the culture medium at a concentration in the range of 50 to 500 μM; (xix) Sodium pyruvate is present in the culture medium at a concentration in the range of 50 to 500 μM; (xx)O-LPA is present in the culture medium at a concentration in the range of 50 to 500 nM; (xxi)N2 supplement is present in the culture medium at a concentration ranging from 0.1% to 5%; and (xxii) The NEAA supplement is present in the culture medium at a concentration ranging from 0.1% to 5%. The method according to claim 13.

15. (i) LDN193189 is present in the culture medium at concentrations of 275 nM in step (a) and 250 nM in step (b); (ii) PD0325901 is present in the culture medium at concentrations of 110 nM in step (a) and 100 nM in step (b); (iii) XAV939 is present in the culture medium at concentrations of 110 nM in step (a) and 100 nM in step (b); (iv) Prumorphamine is present in the culture medium at a concentration of 550 nM in step (a) and 500 nM in steps (b) and (c); (v) MK2206 is present in the culture medium at a concentration of 138 nM; (vi) Go 6983 is present in the culture medium at a concentration of 110 nM; (vii) Taquinib is present in the culture medium at a concentration of 2 μM; (viii) A 83-01 is present in the culture medium at a concentration of 500 nM; (ix) GNF-5837 is present in the culture medium at a concentration of 50 nM; (x) GSI-XX is present in the culture medium at a concentration of 100 nM; (xi) IGF-1 is present in the culture medium at a concentration within the range of 10 ng / ml; (xii) cAMP is present in the culture medium at a concentration of 1.0 μM; (xiii) Valproic acid is present in the culture medium at a concentration of 500 nM; (xiv) Substance P is present in the culture medium at a concentration of 100 nM; (xv) GDNF is present in the culture medium at a concentration of 10 ng / ml; (xvi) MHY1458 is present in the culture medium at a concentration of 2 μM; (xvii) BDNF is present in the culture medium at a concentration of 10 ng / ml; (xviii) 2-phospho-L-ascorbic acid is present in the culture medium at a concentration of 200 μM; (xix) Sodium pyruvate is present in the culture medium at a concentration of 200 μM; (xx)O-LPA is present in the culture medium at a concentration of 200 nM; (xxi)N2 supplement is present in the culture medium at a concentration of 1.0%; and (xxii) The NEAA supplement is present in the culture medium at a concentration of 1.0%. The method according to claim 14.

16. A culture medium for obtaining human OTX2+ FEZF2+ SIX3+ FB-NSCs, containing BMP pathway antagonists, MEK pathway antagonists, WNT pathway antagonists, Akt pathway antagonists, SHH pathway agonists, and PKC pathway antagonists.

17. A culture medium for obtaining human NKX2-1+ VFB-NSCs, containing a BMP pathway antagonist, a MEK pathway antagonist, a WNT pathway antagonist, and an SHH pathway agonist.

18. A culture medium for obtaining human ASCL1+ MGE-NPC, containing a TAK1 pathway antagonist, an SHH pathway agonist, a TGF-β pathway antagonist, a TRK pathway antagonist, a Notch pathway antagonist, and an IGF1 pathway agonist.

19. A culture medium for obtaining human immature neurons, comprising a CREB or PKA pathway agonist, valproic acid or its analogue, substance P or its analogue, a GDNF pathway agonist, and an mTOR pathway agonist.

20. A culture medium for obtaining mature human GABAergic interneurons, comprising a CREB or PKA pathway agonist, valproic acid or its analogue, substance P or its analogue, a GDNF pathway agonist, and an mTOR pathway agonist.

21. An isolated cell culture of human OTX2+ FEZF2+ SIX3+ FB-NSC, comprising human OTX2+ FEZF2+ SIX3+ FB-NPC cultured in a medium containing a BMP pathway antagonist, a MEK pathway antagonist, a WNT pathway antagonist, an Akt pathway antagonist, an SHH pathway agonist, and a PKC pathway antagonist.

22. A cell culture of isolated human NKX2-1+ VFB-NSCs, comprising human NKX2-1+ VFB-NPCs cultured in a medium containing a BMP pathway antagonist, a MEK pathway antagonist, a WNT pathway antagonist, and an SHH pathway agonist.

23. A cell culture of isolated human ASCL1+ MGE-NPC, comprising human ASCL1+ MGE-NPC cultured in a medium containing a TAK1 pathway antagonist, an SHH pathway agonist, a TGFβ pathway antagonist, a TRK pathway antagonist, a Notch pathway antagonist, and an IGF1 pathway agonist.

24. A cell culture of isolated human immature neurons, comprising human immature neurons cultured in a medium containing a CREB or PKA pathway agonist, valproic acid or an analogue, substance P or an analogue, a GDNF pathway agonist, and an mTOR pathway agonist.

25. A cell culture of isolated human mature GABAergic interneurons, comprising human mature GABAergic interneurons cultured in a medium containing a BDNF pathway agonist, an IGF-1 pathway agonist, ascorbic acid or an analogue, sodium pyruvate or an analogue, LPA or an analogue, an N2 supplement, and a NEAA supplement.

26. Human OTX2+ FEZF2+ SIX3+ FB-NSC produced by the method described in claim 1.

27. Human NKX2-1+ VFB-NSC produced by the method described in claim 2.

28. Human ASCL1+ MGE-NPC produced by the method described in claim 3.

29. Human immature neurons generated by the method described in claim 4.

30. Human parvalbumin + mature GABAergic interneurons produced by the method according to claim 5.

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