Patent Application
20250092360
The forebrain is an essential functional region of the central nervous system (CNS). Dysfunction of forebrain neurons can lead to severe neurological diseases. GABAergic interneurons of the forebrain are the major inhibitory neurons of the CNS. GABAergic interneurons contribute to key aspects of the functional maturation of the cortex. Cortical GABA interneurons originate from the medial ganglionic eminence (MGE), a ventral forebrain territory. Dysfunction of GABAergic interneurons has been reported to result in neurodegenerative or psychiatric diseases (Fishell and Rudy (2011) Annu. Rev. Neurosci. 34:535:567; Le Magueresse and Monyer (2013) Neuron 77:388-405). The ability to obtain neural progenitor cells from human pluripotent stem cells (hPSCs) that are committed to the forebrain lineage, including those than can develop into GABAergic interneurons, thus provides a means for potentially treating such forebrain-related neurological and psychiatric diseases.
Early approaches for obtaining neural progenitor cells from hPSCs used feeder cell layers and culture media containing serum and/or other undefined components, which is unsuitable for clinical use. More recently, chemically defined feeder-free systems have been developed. For example, an approach has been reported in which hPSCs are first differentiated to induce forebrain neuroepithelial cells using an embryoid body protocol, which requires ten days, followed by culture of the resulting cells with an SHH agonist, which leads to NKX2-1-expressing MGE progenitors and maturation of GABAergic interneuron subtypes over the course of several weeks (Liu et al. (2013) Nat Protoc. 8:1670-1679; Yuan et al. (2015) Sci Rep 5:18550). Additional forebrain differentiation protocols have been reported in the art, including approaches referred to as the adherent differentiation protocol (Yan et al. (2013) Stem Cells Transl. Med. 2:862-870) and the nonadherent differentiation protocol (Crompton et al. (2013) Stem Cell Res. 11:1206-1221).
Wnt inhibition has been incorporated into strategies for differentiation forebrain neural progenitors and the timing of Wnt inhibition has been reported to modulate the differentiation of medial ganglionic eminence progenitors of GABAergic interneurons (Ihnatovych et al. (2018) Stem Cells International, vol. 2018, Article ID 3983090).
More recently, feeder-free protocols using additional chemical components have been developed. One approach involves an eight-day neural induction phase leading to neuroectodermal progenitors (rosettes) followed by a further eight-day regionalization phase leading to forebrain progenitors by day 16 (Comella-Bolla et al. (2020) Mol. Neurobiol. 57:2766-2798).
Additional protocols for differentiation of forebrain neural progenitors include use of SMAD 2/3 inhibition, such as by inclusion of a TGFβ antagonist in the culture media (see e.g., Nicholas et al. (2013) Cell Stem Cell 12:573-586; U.S. Patent Publication No. 2016/0272940; U.S. Patent Publication No. 2019/0062700; U.S. Patent Publication No. 2021/0040443).
Additional protocols for differentiation of forebrain neural progenitors are also described in U.S. Patent Publication No. 2015/0361393 and U.S. Patent Publication No. 2017/0292112.
Furthermore, there are various subtypes of cortical GABAergic interneurons that are distinguishable based on the expression of molecular markers, including calbindin (CB) interneurons, calretinin (CR) interneurons, parvalbumin (PV) interneurons and somatostatin (SST) interneurons (see e.g., Wonders and Anderson (2006) Nature Rev. Neurosci. 7:687-696). Thus, the ability to differentiate neural progenitor cells specifically into subtypes of GABAergic interneurons is of great interest.
Accordingly, while some progress has been, there remains a need for efficient and robust methods and compositions for generating forebrain-committed neural progenitor cells and mature GABAergic interneurons, particularly of specific subtypes, from human pluripotent stem cells.
This disclosure provides methods of generating somatostatin+ interneurons from human forebrain neural progenitor cells, and culture media and other compositions for use in such methods. Human forebrain neural progenitor cells can be obtained from pluripotent stem cells in a three-stage nine-day protocol that sequentially generates forebrain neural stem cells (FB-NSCs), ventral forebrain neural stem cells (VFB-NSCs) and medial ganglionic eminence neural progenitor cells (MGE-NPCs). The MGE-NPCs then can be further differentiated into mature somatostatin+ interneurons in a two-stage protocol requiring 17 additional days. The methods use chemically defined culture media that allows for generation of FB-NSCs within three days of culture, VFB-NSCs within six days of culture, MGE-NPCs within nine days of culture, immatured neurons within 12 days of culture and mature somatostatin positive interneurons within 26 days of culture.
The defined culture media used to obtain the different types of neural progenitor cells and matured neurons comprise small molecule agents that either agonize or antagonize particular signaling pathway activity in the pluripotent stem cells such that differentiation along the forebrain neural lineage is promoted, leading to cellular maturation and expression of forebrain neural progenitor-associated biomarkers. The methods of the disclosure use culture media for differentiation that comprise different components than those of earlier protocols, avoiding the need for certain reagents (e.g., the methods of the disclosure do not require dual SMAD inhibition). The methods of the disclosure also have the advantage that the use of small molecule agents in the culture media allows for precise control of the culture components, the need for a neural induction phase is avoided and the time needed for differentiation to MGE-NPCs, followed by further differentiation to mature somatostatin+ interneurons, is significantly shortened compared to prior art protocols.
Accordingly, in one aspect, the disclosure pertains to a method of generating human OTX2+ FEZF2+ SIX3+ forebrain neural stem cells (FB-NSCs) comprising: culturing human pluripotent stem cells in a culture media comprising 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 on days 0-3 to obtain human OTX2+ FEZF2+ SIX3+ FB-NSCs.
The method can further comprise further culturing the FB-NSCs on days 3-6 in a culture comprising a BMP pathway antagonist, a MEK pathway antagonist, a WNT pathway antagonist and an SHH pathway agonist to obtain human NKX2-1+ ventral forebrain neural stem cells (VFB-NSCs).
The method can further comprise further culturing the VFB-NSCs on days 6-9 in a culture media 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 to obtain human ASCL1+ medial ganglionic eminence neural progenitor cells (MGE-NPCs).
The method can further comprise further culturing the MGE-NPCs on days 9-12 in a culture media comprising an IGF-1 pathway agonist, a Notch pathway antagonist, at least one FGFR pathway antagonist (e.g., an FGFR1 pathway antagonist and an FGFR4 pathway antagonist), a GDNF pathway agonist, and an AKT pathway antagonist.
The method can further comprise further culturing the human immatured neurons on days 12-26 in a culture media comprising at least one PKC pathway agonist (e.g., a benzolactam-derived PKC pathway agonist and a phorbol ester PKC pathway agonist), a BDNF pathway agonist, an IGF-1 pathway agonist, an ascorbic acid pathway agonist, a PPAR-γ pathway agonist, and an N2 supplement to obtain mature somatostatin+ interneurons.
In an embodiment, the human pluripotent stem cells are induced pluripotent stem cells (iPSCs). In an embodiment, the human pluripotent stem cells are embryonic stem cells. In an embodiment, the human pluripotent stem cells are attached to vitronectin-coated plates during culturing.
In an embodiment, the BMP pathway antagonist is selected from the group consisting of LDN193189, DMH1, DMH2, Dorsopmorphin, K02288, LDN214117, LDN212854, Folistatin, ML347, Noggin, and combinations thereof. In an embodiment, the BMP pathway antagonist is present in the culture media at a concentration within a range of 100-500 nM. In an embodiment, the BMP pathway antagonist is LDN193189, which is present in the culture media at a concentration of 250-275 nM.
In an 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 an embodiment, the MEK pathway antagonist is present in the culture media at a concentration within a range of 50-150 nM. In an embodiment, the MEK pathway antagonist is PD0325901, which is present in the culture media at a concentration of 100-110 nM.
In an 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 an embodiment, the WNT pathway antagonist is present in the culture media at a concentration within a range 50-150 nM. In an embodiment, the WNT pathway antagonist is XAV939, which is present in the culture media at a concentration of 100-110 nM.
In an embodiment, the SHH pathway agonist is selected from the group consisting of Purmorphamine, GSA 10, SAG, and combinations thereof. In an embodiment, the SHH pathway agonist is present in the culture media at a concentration within a range of 250-750 nM. In an embodiment, the SHH pathway agonist is Purmorphamine, which is present in the culture media at a concentration of 500-550 nM.
In an 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 an embodiment, the AKT pathway antagonist is present in the culture media at a concentration within a range of 50-200 nM. In an embodiment, the AKT pathway antagonist is MK2206, which is present in the culture media at a concentration of 138 nM.
In an 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 an embodiment, the PKC pathway antagonist is present in the culture media at a concentration within a range of 50-200 nM. In an embodiment, the PKC pathway antagonist is Go 6983, which is present in the culture media at a concentration of 110 nM.
In an embodiment, the TAK1 pathway antagonist is selected from the group consisting of Takinib, Dehydoabietic acid, NG25, Sarsasapogenin, and combinations thereof. In an embodiment, the TAK1 pathway antagonist is present in the culture media at a concentration within a range of 1-5 μM. In an embodiment, the TAK1 pathway antagonist is Takinib, which is present in the culture media at a concentration of 2 μM.
In an 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 an embodiment, the TGFβ pathway antagonist is present in the culture media at a concentration within a range of 250-750 nM. In an embodiment, the TGFβ pathway antagonist is A 83-01, which is present in the culture media at a concentration of 500 nM.
In an embodiment, the TRK pathway antagonist is selected from the group consisting of GNF-5837, BMS-754807, UNC2020, Taletrectinib, Altiratinib, Selitrectinib, PF 06273340, and combinations thereof. In an embodiment, the TRK pathway antagonist is present in the culture media at a concentration within a range of 25-75 nM. In an embodiment, the TRK pathway antagonist is GNF-5837, which is present in the culture media at a concentration of 50 nM.
In an 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, Bruceine D, and combinations thereof. In an embodiment, the Notch pathway antagonist is present in the culture media at a concentration within a range of 50-150 nM. In an embodiment, the Notch pathway antagonist is GSI-XX, which is present in the culture media at a concentration of 100 nM.
In an embodiment, the IGF1 pathway agonist is selected from the group consisting of IGF1, IGF1-Ado, X10, mecasermin, and combinations thereof. In an embodiment, the IGF1 pathway agonist is present in the culture media at a concentration within a range of 5-15 ng/ml. In an embodiment, the IGF1 pathway agonist is IGF1, which is present in the culture media at a concentration of 10 ng/ml.
In another aspect, the disclosure provides a method of generating human NKX2-1 ventral forebrain neural stem cells (VFB-NSCs) comprising:
In an 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.
In an embodiment, LDN193189 is present in the culture media at a concentration within a range of 100-500 nM, PD0325901 is present in the culture media at a concentration within a range of 50-150 nM, XAV939 is present in the culture media at a concentration within a range of 50-150 nM, Purmorphamine is present in the culture media at a concentration within a range of 250-750 nM, MK2206 is present in the culture media in step (a) at a concentration within a range of 50-150 nM, and Go 6983 is present in the culture media in step (a) at a concentration within a range of 50-150 nM. In an embodiment, LDN193189 is present in the culture media at a concentration of 275 nM in step (a) and 250 nM in step (b), PD0325901 is present in the culture media at a concentration of 110 nM in step (a) and 100 nM in step (b), XAV939 is present in the culture media at a concentration of 110 nM in step (a) and 100 nM in step (b), Purmorphamine is present in the culture media at a concentration of 550 nM in step (a) and 500 nM in step (b), MK2206 is present in the culture media in step (a) at a concentration of 138 nM, and Go 6983 is present in the culture media in step (a) at a concentration of 110 nM.
In yet another aspect, the disclosure pertains to a method of generating human ASCL1+ medial ganglionic eminence neural progenitor cells (MGE-NPCs) comprising:
In an 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.
In an embodiment, LDN193189 is present in the culture media at a concentration within a range of 100-500 nM, PD0325901 is present in the culture media at a concentration within a range of 50-150 nM, XAV939 is present in the culture media at a concentration within a range of 50-150 nM, Purmorphamine is present in the culture media at a concentration within a range of 250-750 nM, MK2206 is present in the culture media in step (a) at a concentration within a range of 50-150 nM, Go 6983 is present in the culture media in step (a) at a concentration within a range of 50-150 nM, Takinib is present in the culture media in step (c) at a concentration within a range of 1-5 μM, A 83-01 is present in the culture media in step (c) at a concentration within a range of 250-750 nM, GNF-5837 is present in the culture media in step (c) at a concentration within a range of 25-75 nM, GSI-XX is present in the culture media in step (c) at a concentration within a range of 50-150 nM and IGF-1 is present in the culture media in step (c) at a concentration within a range of 5-15 ng/ml. In an embodiment, LDN193189 is present in the culture media at a concentration of 275 nM in step (a) and 250 nM in step (b), PD0325901 is present in the culture media at a concentration of 110 nM in step (a) and 100 nM in step (b), XAV939 is present in the culture media at a concentration of 110 nM in step (a) and 100 nM in step (b), Purmorphamine is present in the culture media at a concentration of 550 nM in step (a) and 500 nM in step (b), MK2206 is present in the culture media in step (a) at a concentration of 138 nM, Go 6983 is present in the culture media in step (a) at a concentration of 110 nM, Takinib is present in the culture media in step (c) at a concentration of 2 μM, A 83-01 is present in the culture media in step (c) at a concentration of 500 nM, GNF-5837 is present in the culture media in step (c) at a concentration of 50 nM, GSI-XX is present in the culture media in step (c) at a concentration of 100 nM and IGF-1 is present in the culture media in step (c) at a concentration within a range of 10 ng/ml.
In yet another aspect, the disclosure pertains to a method of generating human mature somatostatin+ interneurons from human medial ganglionic eminence neural progenitor cells (MGE-NPCs) comprising:
In an embodiment, the culture media in step (a) comprises an FGFR1 pathway antagonist and an FGFR4 pathway antagonist. In an embodiment, the culture media in step (b) comprises a benzolactam-derived PKC pathway agonist and a phorbol ester PKC pathway agonist.
In an embodiment, the three-step method of generating MGE-NPCs is combined with the two step method of generating mature somatostatin+ interneurons from the MGE-NPCs, resulting in a five-step method for preparing the mature somatostatin+ interneurons, wherein the first three culturing steps (stages 1, 2 and 3) are performed on days 0-3, 3-6 and 6-9, respectively, and the last two culturing steps (stages 4 and 5) are performed on days 9-12 and 12-24, respectively, as described herein.
In an embodiment, the IGF-1 pathway agonist is selected from the group consisting of IGF1, IGF1-Ado, X10, mecasermin, IGF2, Insulin, Rg5, IGF-1 30-41, Demethylasterriquinone B1, IGF-1 24-41, and combinations thereof. In an embodiment, the IGF-1 pathway agonist is present in the culture media at a concentration within a range of 5-15 ng/ml. In an embodiment, the IGF-1 pathway agonist is IGF-1 which is present in the culture media at a concentration of 10 ng/ml.
In an 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, Bruceine D, BMS 299897, Compound E, DBZ, L-685458, LY 450139, MRK 560, PF 3084014 Hydrobromide, Begacestat, JLK6, L-685458, LY 3039478, and combinations thereof. In an embodiment, the Notch pathway antagonist is present in the culture media at a concentration within a range of 50-150 nM. In an embodiment, the Notch pathway antagonist is GSI-XX which is present in the culture media at a concentration of 100 nM.
In an embodiment, the FGFR1 pathway antagonist is selected from the group consisting of PD173074, PD161570, SU5402, SU6668, AP24534, PD166866, and combinations thereof. In an embodiment, the FGFR1 pathway antagonist is present in the culture media at a concentration within a range of 25-75 nM. In an embodiment, the FGFR1 pathway antagonist is PD173074, which is present in the culture media at a concentration of 50 nM.
In an embodiment, the FGFR4 pathway antagonist is selected from the group consisting of BLU9931, BLU-554, H3B-6527, FGF401, and combinations thereof. In an embodiment, the FGFR4 pathway antagonist is present in the culture media at a concentration within a range of 25-75 nM. In an embodiment, the FGFR4 pathway antagonist is BLU9931, which is present in the culture media at a concentration of 50 nM.
In an embodiment, the GDNF pathway agonist is selected from the group consisting of GDNF, BT13, BT44, and combinations thereof. In an embodiment, the GDNF pathway agonist is present in the culture media at a concentration within a range of 5-15 ng/ml. In an embodiment, the GDNF pathway agonist is GDNF, which is present in the culture media at a concentration of 10 ng/ml.
In an 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, API-1 (pyrido[2,3-d]pyrimidines), and combinations thereof. In an embodiment, the AKT pathway antagonist is present in the culture media at a concentration within a range of 50-200 nM. In an embodiment, the AKT pathway antagonist is MK2206, which is present in the culture media at a concentration of 125 nM.
In an embodiment, the culture media in step (b) comprises two PKC pathway agonists (e.g., a benzolactam-derived PKC pathway agonist and a phorbol ester PKC pathway agonist). In an embodiment, the two PKC pathway agonists are independently selected from the group consisting of (−)-Indolactam-V, Bryostatin 1, PEP 005, Phorbol 12,13-dibutyrate, Phorbol 12-myristate 13-acetate, TPPB, Okadaic Acid, Prostratin, SC10, 1-Stearoyl-2-arachidonoyl-sn-glycerol, 6-(N-Decylamino)-4-Hydroxymethylindole, and combinations thereof. In an embodiment, one PKC pathway agonist is (−)-Indolactam-V, which is present in the culture media at a concentration within a range of 200-400 nM (e.g., at a concentration of 300 nM). In an embodiment, one PKC pathway agonist is Prostratin, which is present in the culture media at a concentration within a range of 0.75-2.0 μM (e.g., at a concentration of 1.0 μM).
In an 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 an embodiment, the BDNF pathway agonist is present in the culture media at a concentration within a range of 5-15 ng/ml. In an embodiment, the BDNF pathway agonist is BDNF, which is present in the culture media at a concentration of 10 ng/ml.
In an embodiment, the IGF-1 pathway agonist in step (b) is selected from the group consisting of IGF1, IGF1-Ado, X10, mecasermin, IGF2, Insulin, Rg5, IGF-1 30-41, Demethylasterriquinone B1, IGF-1 24-41, and combinations thereof. In an embodiment, the IGF-1 pathway agonist is present in the culture media at a concentration within a range of 5-15 ng/ml. In an embodiment, the IGF-1 pathway agonist is IGF-1, which is present in the culture media at a concentration of 10 ng/ml.
In an embodiment, the ascorbic acid pathway agonist 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 an embodiment, the ascorbic acid pathway agonist is present in the culture media at a concentration within a range of 100-300 μM. In an embodiment, the ascorbic acid pathway agonist is 2-phospho-L-ascorbic acid, which is present in the culture media at a concentration of 200 μM.
In an embodiment, the PPAR-γ pathway agonist is selected from the group consisting of Rosiglitazone, Ciglitazone, Edaglitazone, GW 1929 hydrochloride, Pioglitazone Hydrochloride, Troglitazone, Tesaglitazar, Carnosic Acid, Indomethacin, Pioglitazone Hydrochloride, CAY10506, CAY10599, PAz-PC, Rosiglitazone Maleate, Rosiglitazone Hydrochloride, Rosiglitazone-d3, GW1929, S26948, 9-Nitrooleate, Azelaoyl-PAF, DRF 2519, PGPC, Methyl-8-hydroxy-8-(2-pentyl-oxyphenyl)-oct-5-ynoate, 20-carboxy Arachidonic Acid, 15-deoxy-Delta 12,14-Prostaglandin J2 solution, CAY15073, and combinations thereof. In an embodiment, the PPAR-γ pathway agonist is present in the culture media at a concentration within a range of 250-750 nM. In an embodiment, the PPAR-γ pathway agonist is Rosiglitazone, which is present in the culture media at a concentration of 500 nM.
In an embodiment, the N2 supplement is present in the culture media at a concentration of 1%.
In an embodiment of the method of generating somatostatin+ mature interneurons, the human MGE-NPCs (from which the somatostatin+ mature interneurons are generated) can be obtained by a method comprising:
In an embodiment, the human pluripotent stem cells are induced pluripotent stem cells (iPSCs). In an embodiment, the human pluripotent stem cells are embryonic stem cells.
In another aspect, the disclosure pertains to various culture media for generating human forebrain neural stem or progenitor cells. In an embodiment, the disclosure provides a culture media 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, an SHH pathway agonist and a PKC pathway antagonist. In an embodiment, the disclosure provides a culture media 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 an SHH pathway agonist. In an embodiment, the disclosure provides a culture media for obtaining human ASCL1+ medial ganglionic eminence 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 an embodiment, the disclosure provides a culture media for obtaining human immatured neurons comprising an IGF-1 pathway agonist, a Notch pathway antagonist, at least one FGFR pathway antagonist (e.g., an FGFR1 pathway antagonist and an FGFR4 pathway antagonist), a GDNF pathway agonist, and an AKT pathway antagonist. In an embodiment, the disclosure provides a culture media for obtaining human mature somatostatin+ interneurons comprising at least one PKC pathway agonist (e.g., a benzolactam-derived PKC pathway agonist and a phorbol ester PKC pathway agonist), a BDNF pathway agonist, an IGF-1 pathway agonist, an ascorbic acid pathway agonist, a PPAR-γ pathway agonist, and an N2 supplement.
In another aspect, the disclosure pertains to isolated cell cultures of human forebrain neural stem or progenitor cells. In an embodiment, the disclosure provides an isolated cell culture of human OTX2+ FEZF2+ SIX3+ forebrain neural stem cells (FB-NSCs), the culture comprising human OTX2+ FEZF2+ SIX3+ FB-NPCs cultured in a culture media comprising 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. In an embodiment, the disclosure provides an isolated cell culture of human NKX2-1+ ventral forebrain neural stem cells (VFB-NSCs), the culture comprising human NKX2-1+ VFB-NPCs cultured in a culture media comprising a BMP pathway antagonist, a MEK pathway antagonist, a WNT pathway antagonist and an SHH pathway agonist. In an embodiment, the disclosure provides an isolated cell culture of human ASCL1+ medial ganglionic eminence neural progenitor cells (MGE-NPCs), the culture comprising human ASCL1+ MGE-NPCs cultured in a culture media 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 an embodiment, the disclosure provides an isolated cell culture of human immatured neurons, the culture comprising human immatured neurons cultured in a culture media comprising an IGF-1 pathway agonist, a Notch pathway antagonist, at least one FGFR pathway antagonist (e.g., an FGFR 1 pathway antagonist and an FGFR4 pathway antagonist), a GDNF pathway agonist, and an AKT pathway antagonist. In an embodiment, the disclosure provides an isolated cell culture of human mature somatostatin+ interneurons, the culture comprising human mature somatostatin+ interneurons cultured in a culture media comprising at least one PKC pathway agonist (e.g., a benzolactam-derived PKC pathway agonist and a phorbol ester PKC pathway agonist), a BDNF pathway agonist, an IGF-1 pathway agonist, an ascorbic acid pathway agonist, a PPAR-γ pathway agonist, and an N2 supplement.
In yet another aspect, the disclosure pertains to human forebrain neural stem or progenitor cells generated by the methods of the disclosure. In an embodiment, the disclosure provides human OTX2+ FEZF2+ SIX3+ forebrain neural stem cells (FB-NSCs) generated by a method of the disclosure. In an embodiment, the disclosure provides human NKX2-1+ ventral forebrain neural stem cells (VFB-NSCs) generated by a method of the disclosure. In an embodiment, the disclosure provides human ASCL1+ medial ganglionic eminence neural progenitor cells (MGE-NPCs) generated by a method of the disclosure. In an embodiment, the disclosure provides human immatured neurons generated by a method of the disclosure. In an embodiment, the disclosure provides human somatostatin+ mature interneurons generated by a method of the disclosure.
The methods and compositions of the disclosure are useful in generation of human forebrain neural stem or progenitor cells, human immatured neurons and human somatostatin+ mature interneurons for research or therapeutic purposes, such as in the treatment of neurological disorders (e.g., cognitive or mood disorders associated with reduced levels of somatostatin or decreased numbers of somatostatin+ interneurons).
Other features and advantages of the invention will be apparent from the following detailed description and claims.
Described herein are methodologies and compositions that allow for the generation of forebrain neural progenitors from human pluripotent stem cells, as well as matured somatostatin+ interneurons derived therefrom, under chemically-defined culture conditions using a small molecule based approach. The methods of the disclosure generate medial ganglionic eminence neural progenitors (the precursors to somatostatin+ interneurons) in a three stage protocol in which OTX2+ FEZF2+ SIX3+ forebrain neural stem cells (FB-NSCs) are generated in three days, followed by generation of NKX2-1+ ventral forebrain neural stem cells (VFB-NSCs) by day six of culture, followed by generation of ASCL1+ medial ganglionic eminence neural progenitor cells (MGE-NPCs) by day nine of culture. Thus, the disclosure allows for obtention of MGE-NPCs in a significantly shorter time than prior art protocols using chemically-defined culture conditions. The MGE-NPCs then can be further differentiated according to a two stage protocol in which the MGE-NPCs are first differentiated into immatured neurons in three days (day 12 of culture following the nine day protocol to generate MGE-NPCs), followed by differentiation into mature somatostatin+ interneurons in 14 days (day 26 of culture).
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 for the identification of chemically-defined culture media, comprising agonists and/or antagonists of particular signaling pathways, that is sufficient to generate forebrain neural stem and progenitor cells, including FB-NSCs, VFB-NSCs, and MGE-NPCs, under defined conditions in a short amount of time. The optimized culture media was further validated by a factor criticality analysis, which examined the effects of eliminating individual agonist or antagonist agents, as described in Example 2. Immunohistochemistry further confirmed the phenotype of the cells generated by the MGE-NPC differentiation protocol, as described in Example 3. The MGE-NPCs were then further differentiated into immatured neurons and mature somatostatin+ interneurons in a two-stage culture protocol, as described in Example 4. This latter protocol was further validated by a factor criticality analysis, as described in Example 5. Immunohistochemistry further confirmed the phenotype of the cells generated by the somatostatin+ interneuron differentiation protocol, as described in Example 6.
Various aspects of the invention are described in further detail in the following subsections.
The starting cells used in the cultures of the disclosure are human pluripotent stem cells. As used herein, the term “human pluripotent stem cell” (abbreviated as hPSC) refers to a human stem cell that has the capacity to differentiate into a variety of different cell types. The term “pluripotent” as used herein refers to a cell with the capacity, under different conditions, to differentiate to cell types of characteristic of all three germ cell layers (endoderm, mesoderm and ectoderm). Pluripotent cells are characterized primarily by their ability to differentiate to all three germ layers, for example, using a nude mouse and teratomas formation assay. Pluripotency can be also evidenced by the expression of embryonic stem (ES) cell markers, although the preferred test for pluripotency is the demonstration of the capacity to differentiate into cells of each of the three germ layers.
Human pluripotent stem cells include, for example, induced pluripotent stem cells (iPSC) and human embryonic stem cells, such as ES cell lines. Non-limiting examples of induced pluripotent stem cells (iPSC) 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, J. A. et al. (1998) Science 282:1145-1147). Human pluripotent stem cells (PSCs) express cellular markers that can be used to identify cells as being 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. Since the methods of generating forebrain neural progenitor of the disclosure are used to differentiate (maturate) the starting pluripotent stem cell population, in various embodiments the forebrain neural progenitor cell populations generated by the methods of the 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.
The pluripotent stem cells are subjected to culture conditions, as described herein, that induce cellular differentiation. As used herein, the term “differentiation” refers to the development of a cell from a more primitive stage towards a more mature (i.e. less primitive) cell, typically exhibiting phenotypic features of commitment to a particular cellular lineage.
As used herein, a “neural stem cell” refers to a cell that is more differentiated than a pluripotent stem cell in that it is committed to the neural lineage but still has the capacity to differentiate into different types of cells along the neural lineage.
As used herein a “neural progenitor cell” refers to a cell that is more differentiated than a neural stem cell and that can be further differentiated into a particular type of neural cell.
In embodiments, cells can be identified and characterized based on expression of one or more biomarkers, such as particular biomarkers of neural progenitors or forebrain region-committed neural cells. A “positive” biomarker is one that is expressed on a cell of interest, whereas a “negative” biomarker is one that is not expressed on a cell of interest.
Non-limiting examples of biomarkers whose expression can be assessed in the characterization of cells of interest include genes involved in 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 ventral forebrain including NKX2-1 and absence of PAX6 (i.e., PAX6 as a negative biomarker), which is a dorsal forebrain marker (Stoykova et al. (2000) J. Neurosci. 20:8042-8050), and genes expressed in neuronal progenitor cells in medial ganglionic eminence (MGE) region including ASCL1, LHX6 and DLX1 (Silberberg et al. (2016) Neuron 92:59-74). In embodiments, a FB-NSC is OTX2+ FEZF2+ SIX3+. In embodiments, a VFB—NSC is NKX2-1+. In embodiments, a VFB-NSC is NKX2-1+ and PAX6 negative (PAX6−). In embodiments, an MBE-NPC is ASCL1+. In embodiments, an MBE-NPC is positive for at least two markers selected from ASCL1, LHX6 and DLX1. In embodiments, an MBE-NPC is ASCL1+ LHX6+ DLX1+.
Non-limiting examples of markers whose expression can be evaluated on stage 4 and/or stage 5 cells include: MAP2 (a mature neuron marker; see e.g., Chamak et al. (1987) J. Neurosci. 7:3163-3170); DCX (an immature neuron marker; see e.g., Ayanlaja et al. (2017) Front. Mol. Neurosci. 10:199); TUJ1 (a pan neural marker; see e.g., Memberg and Hall (1995) J. Neurobiol. 27:26-43); NeuN (a neuronal nuclear marker; see e.g., Gusel′Nikova and Korzhevskiy (2015) Acta Naturae 7:42-47); LHX6 (a cortical interneuron marker; see e.g., Cruz-Santos et al. (2022) Cells 11:853; Liodis et al. (2007) J. Neurosci. 27:3078-3089); MASH1 (ASCL1) (a proneuronal transcription factor; see e.g., Fitzgerald et al. (2020) Stem Cells 38:1375-1386; Shi et al. (2016) J. Biol. Chem. 291:13560-13570); SATB1 (a marker of MGE-derived cortical interneurons; see e.g., Denaxa et al. (2012) Cell Reports 2:1351-1362); SOX6 (a marker expressed in post-mitotic progenitors of MGE region; see e.g., Chen (2017) Sci. Reports 7:1-11; Batista-Brito et al. (2009) Neuron 63:466-481); GAD1/2 (GABAergic interneuron markers); GABA (a marker specifically expressed by GABAergic interneurons); Synapsin 1 (a pre-synaptic marker); and Somatostatin (SST; a marker of somatostatin interneurons). Expression of markers of cell types other than interneurons also can be evaluated, non-limiting examples of which include GFAP (a glial marker) and nestin (a neural stem cell marker).
As used herein, expression by a cell of only “low” levels of a biomarker of interest is intended to refer to a level that is at most 20%, and more preferably, less than 20%, less than 15%, less than 10% or less than 5% above background levels (wherein background levels correspond to, for example, the level of expression of a negative control marker that is considered to not be expressed by the cell).
In embodiments, the cells generated by the methods of the disclosure are forebrain neural stem cells (FB-NSCs). As used herein, a “forebrain neural stem cell” or “FB-NSC” refers to a stem cell-derived neural stem cell that expresses at least one biomarker, and preferably two or all three biomarkers, selected from OTX2, FEZF2 and SIX3. An FB-NSC may also express additional biomarkers, including but not limited to beta III-tubulin and/or KI67.
In embodiments, the cells generated by the methods of the disclosure are ventral forebrain neural stem cells (VFB-NSCs), which are more differentiated (more mature) cells than FB-NSCs and committed along the ventral lineage. As used herein, a “ventral forebrain neural stem cell” or “VFB-NSC” refers to a stem cell-derived neural cell that expresses the biomarker NKX2-1. In an embodiment, the VFB-NSC does not express, or only expresses low levels of, the biomarker PAX6. In addition, an VFB NSC may also express additional biomarkers, including but not limited to beta III-tubulin and/or KI67.
In embodiments, the cells generated by the methods of the disclosure are medial ganglionic eminence neural progenitor cells (MGE-NPCs). As used herein, a “medial ganglionic eminence neural progenitor cell” or “MGE-NPC” refers to a stem cell-derived neural cell that expresses at least one biomarker, and preferably two or all three biomarkers, selected from ASCL1, LHX6 and DLX1. An MGE-NPC may also express additional biomarkers, including but not limited to beta III-tubulin and/or KI67.
The committed MGE-NPCs generated by the methods of the disclosure can be further cultured in vitro to generate mature somatostatin+ interneurons, e.g., according to the culture protocols described herein. As used herein, a mature somatostatin+ neuron refers to a neuronally-derived cell that expresses the biomarker somatostatin (SST) and may also express other biomarkers. In an embodiment, a stage 4 or stage 5 interneuron expresses one or more markers selected from the group consisting of MAP2, DCX, TUJ1, NeuN, LHX6, MASH1, SATB1, SOX6, GAD1/2 and GABA, Synapsin 1 and Somatostatin (SST). In an embodiment, a stage 4 interneuron expresses one or more markers selected from the group consisting of MAP2, DCX, TUJ1, NeuN, LHX6, MASH1, SATB1, SOX6, GAD1/2 and GABA. In an embodiment, a stage 4 interneuron lacks expression of GFAP (a glial marker). In an embodiment, a stage 4 interneuron lacks expression of Nestin (a neural stem cell marker). In an embodiment, a stage 5 mature interneuron expresses one or more markers selected from the group consisting of MAP2, DCX, TUJ1, NeuN, LHX6, MASH1, GAD1/2, GABA, Synapsin 1 and Somatostatin (SST). In an embodiment, a stage 5 interneuron lacks expression of GFAP (a glial marker).
II. Culture Media Components The methods of the disclosure for generating FB-NSC, VFB-NSC and MGE-NPCs, as well as immatured neurons and somatostatin+ interneurons from the MGE-NPCs, comprise culturing human pluripotent stem cells in culture media comprising specific agonist and/or antagonists of cellular signaling pathways. In various embodiments, the culture media lacks serum, lacks exogenously added growth factors, lacks animal products, is serum-free, is xeno-free and/or is feeder layer free. In various embodiments, the culture media lacks a SMAD2/3 inhibitor or antagonist, lacks a dual SMAD inhibitor or antagonist, or lacks a TGFβ pathway antagonist.
As described in Example 1, a culture media comprising 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 was sufficient to generate OTX2+ FEZF2+ SIX3+ forebrain neural stem cells (FB-NSCs) in as little as three days (referred to herein as “stage 1” of the differentiation protocol). Further differentiation of the FB-NSCs in a culture media comprising a BMP pathway antagonist, a MEK pathway antagonist, a WNT pathway antagonist and an SHH pathway agonist was sufficient to generate NKX2-1+ VFB-NSCs in another three days (referred to herein as “stage 2”). Still further differentiation of the VFB-NSCs in a culture media 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 was sufficient to generate ASCL1+ medial ganglionic eminence neural progenitor cells (MGE-NPCs) in another three days (referred to herein as “stage 3”), for an overall three-stage nine day protocol for generating MGE-NPCs. The MGE-NPCs can be further differentiated into immatured neurons by further culture for three days in a culture media comprising an IGF-1 pathway agonist, a Notch pathway antagonist, at least one FGFR pathway antagonist (e.g., an FGFR1 pathway antagonist and an FGFR4 pathway antagonist), a GDNF pathway agonist, and an AKT pathway antagonist (referred to herein as “stage 4”). Finally, the immatured neurons can be further differentiated into mature somatostatin+ interneurons by further culture for two weeks (14 days) in a culture media comprising at least one PKC pathway agonist (e.g., a benzolactam-derived PKC pathway agonist and a phorbol ester PKC pathway agonist), a BDNF pathway agonist, an IGF-1 pathway agonist, an ascorbic acid pathway agonist, a PPAR-γ pathway agonist, and an N2 supplement (referred to herein as “stage 5”).
As used herein, an “agonist” of a cellular signaling pathway is intended to refer to an agent that stimulates (upregulates) the cellular signaling pathway. Stimulation of the cellular signaling pathway can be initiated extracellularly, for example by use of an agonist that activates a cell surface receptor involved in the signaling pathway (e.g., the agonist can be a receptor ligand). Additionally, or alternatively, stimulation of cellular signaling can be initiated intracellularly, for example by use of a small molecule agonist that interacts intracellularly with a component(s) of the signaling pathway.
As used herein, an “antagonist” of a cellular signaling pathway is intended to refer to an agent that inhibits (downregulates) the cellular signaling pathway. Inhibition of the cellular signaling pathway can be initiated extracellularly, for example by use of an antagonist that blocks a cell surface receptor involved in the signaling pathway. Additionally, or alternatively, inhibition of cellular signaling can be initiated intracellularly, for example by use of a small molecule antagonist that interacts intracellularly with a component(s) of the signaling pathway.
Agonists and antagonists used in the methods of the disclosure are known in the art and commercially available. They are used in the culture media at a concentration effective to achieve the desired outcome, e.g., generation of forebrain neural stem or progenitor cells expressing forebrain markers of interest. Non-limiting examples of suitable agonist and antagonists agents, and effective concentration ranges, are described further below.
Antagonists of the BMP (bone morphogenetic protein) pathway include agents, molecules, compounds, or substances capable of inhibiting (downregulating) the BMP signaling pathway, which biologically is activated by binding of BMP to a BMP receptor, which are activin receptor-like kinases (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 culture media at a concentration within a 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, which is present in the culture media at a concentration within a range of 100-500 nM, 100-400 nM, 150-350 nM or 200-300 nM. In one embodiment, the BMP pathway antagonist is LDN193189, which is present in the culture media at a concentration of 275 nM in step (a) (stage 1) and 250 nM in step (b) (stage 2).
Antagonists of the MEK pathway include agents, molecules, compounds, or substances capable of inhibiting (downregulating) the signaling pathway of one or more of the 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 culture media at a concentration within a 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, which is present in the culture media at a concentration within a 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, which is present in the culture media at a concentration of 110 nM in step (a) (stage 1) and 100 nM in step (b) (stage 2).
Antagonists of the WNT pathway include agents, molecules, compounds, or substances capable of inhibiting (downregulating) the canonical Wnt/β-catenin signaling pathway, which biologically is activated by binding of a Wnt-protein ligand to a Frizzled family receptor. 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 culture media at a concentration within a 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, which is present in the culture media at a concentration of 10-500 nM, 50-250 nM or 50-150 nM. In one embodiment, the WNT pathway antagonist is XAV939, which is present in the culture media at a concentration of 110 nM in step (a) (stage 1) and 100 nM in step (b) (stage 2).
Agonists of the SHH (sonic hedgehog) pathway include agents, molecules, compounds, or substances capable of stimulating (activating) signaling through the SHH pathway, which biologically involves 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 culture media at a concentration within a 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, which is present in the culture media 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, which is present in the culture media at a concentration of 550 nM in step (a) (stage 1) and 500 nM in steps (b) and (c) (stages 2 and 3).
Antagonists of the AKT pathway include agents, molecules, compounds, or substances capable of inhibiting (downregulating) the signaling pathway of one or more of the serine/threonine kinase AKT family members, which include AKT1 (also designated PKB or RacPK), AKT2 (also designated PKBβ or RacPK-β) and AKT 3 (also designated PKBγ or thyoma viral proto-oncogene 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, Miransertib (ab235550), Borussertib, Cerisertib, API-1 (pyrido[2,3-d]pyrimidines), and combinations thereof. In one embodiment, the AKT pathway antagonist is present in the culture media at a concentration within a 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, which is present in the culture media at a concentration within a range of 25-300 nM, 50-200 nM, 75-200 nM or 125-150 nM. In one embodiment, the AKT pathway antagonist is MK2206, which is present in the culture media at a concentration of 138 nM in step (a) (stage 1). In one embodiment, the AKT pathway antagonist is MK2206, which is present in the culture media at a concentration of 125 nM in step (d) (i.e., stage 4). In one embodiment, the AKT pathway antagonist is MK2206 in steps (a) and (d) (i.e., stages 1 and 4), such as at a concentration of 138 nM in step (a) (stage 1) and at a concentration of 125 nM in step (d) (i.e., stage 4).
Antagonists of the PKC (protein kinase C) pathway include agents, molecules, compounds, or substances capable of inhibiting (downregulating) a PKC signaling pathway, which biologically is mediated by a PKC family member. The PKC family of serine/threonine kinases comprises fifteen isozymes, including the “classical” PKC subcategory, which contain the isoforms α, β1, β2 and γ. In one embodiment, the PKC pathway antagonist inhibits the activity of at least one (and in other embodiments, at least two or three) PKC enzyme 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 culture media at a concentration within a 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, which is present in the culture media 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, which is present in the culture media at a concentration of 110 nM in step (a) (stage 1).
Antagonists of the TAK1 (also known as MAP3K7) pathway include agents, molecules, compounds, or substances capable of inhibiting (downregulating) signaling through TAK1 (MAP3K7). 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 culture media at a concentration within a range of 1-5 μM, 1-3 μM or 1.5-2.5 μM. In one embodiment, the TAK1 pathway antagonist is Takinib. In one embodiment, the TAK1 pathway antagonist is Takinib, which is present in the culture media at a concentration of 1-5 μM, 1-3 μM or 1.5-2.5 μM. In one embodiment, the TAK1 pathway antagonist is Takinib, which is present in the culture media in step (c) of the method (i.e., stage 3) at a concentration of 2 μM.
Antagonists of the TGFβ (transforming growth factor beta) pathway include agents, molecules, compounds, or substances capable of inhibiting (downregulating) signaling through a TGFβ receptor family member, a family of serine/threonine kinase receptors. 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 culture media at a concentration within a 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, which is present in the culture media 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, which is present in the culture media in step (c) of the method (i.e., stage 3) at a concentration of 500 nM.
Antagonists of the TRK pathway include agents, molecules, compounds, or substances capable of inhibiting (downregulating) signaling through TrkA, TrkB and/or TrkC tyrosine kinase. In one embodiment, the TRK pathway antagonist is selected from the group consisting of GNF-5837, BMS-754807, UNC2020, Taletrectinib, Altiratinib, Selitrectinib, PF 06273340, and combinations thereof. In one embodiment, the TRK pathway antagonist is present in the culture media at a concentration within a 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 A GNF-5837, which is present in the culture media 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, which is present in the culture media in step (c) of the method (i.e., stage 3) at a concentration of 50 nM.
Antagonists of the Notch pathway include agents, molecules, compounds, or substances capable of inhibiting (downregulating) signaling through or activity of the Notch transcription factor. 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, Bruceine D, BMS 299897; Compound E; DBZ; L-685458; LY 450139; MRK 560; PF 3084014 Hydrobromide; Begacestat; JLK6; L-685458; LY 3039478; and combinations thereof. In one embodiment, the Notch pathway antagonist is present in the culture media at a concentration within a 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, which is present in the culture media at a concentration of 25-200 nM, 50-150 nM or 75-125 nM. In one embodiment, the Notch pathway antagonist is GSI-XX, which is present in the culture media in step (c) of the method (i.e., stage 3) at a concentration of 100 nM. In one embodiment, the Notch pathway antagonist is GSI-XX, which is present in the culture media in step (d) of the method (i.e., stage 4) at a concentration of 100 nM. In one embodiment, the Notch pathway antagonist is GSI-XX, which is present in the culture media in steps (c) and (d) of the method (i.e., stages 3 and 4) at a concentration of 100 nM.
Agonists of the IGF1 (insulin-like growth factor 1) pathway 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, IGF2; Insulin; Rg5; IGF-1 30-41; Demethylasterriquinone B1; IGF-1 24-41, and combinations thereof. In one embodiment, the IGF1 pathway agonist is present in the culture media at a concentration within a 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, which is present in the culture media 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, which is present in the culture media 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 media in step (d) of the method (i.e., stage 4) at a concentration of 10 ng/ml. In one embodiment, the IGF1 pathway agonist is IGF1, which is present in the culture media in step (e) of the method (i.e., stage 5) at a concentration of 10 ng/ml. In one embodiment, the IGF1 pathway agonist is IGF1, which is present in the culture media at a concentration of 10 ng/ml in steps (c), (d) and (e) (i.e., stages 3, 4 and 5) of the method.
Antagonists of the FGFR (fibroblast growth factor receptor) pathway include agents, molecules, compounds, or substances capable of inhibiting (downregulating) signaling through or activity of FGFR. In one embodiment, the FGFR pathway antagonist is selected from the group consisting of PD173074; BLU9931; FIIN 1 Hydrochloride; PD161570; SU5402; SU6668; AP24534; PD166866; AZD4547; BGJ398, BLU-554; H3B-6527; FGF401; and combinations thereof. In one embodiment, the FGFR antagonist is a pan-FGFR antagonist. In one embodiment, the FGFR antagonist is an FGFR1 antagonist. In one embodiment, the FGFR1 antagonist is selected from the group consisting of PD173074, PD161570, SU5402, SU6668, AP24534, PD166866, and combinations thereof. In one embodiment, the FGFR antagonist is an FGFR4 antagonist. In one embodiment, the FGFR4 antagonist is selected from the group consisting of BLU9931, BLU-554, H3B-6527, FGF401, and combinations thereof. In one embodiment, the FGFR pathway antagonist is present in the culture media at a concentration within a range of 10-100 nM, 25-75 nM or 40-60 nM or at a concentration of 50 nM. In one embodiment, the FGFR pathway antagonist is PD173074. In one embodiment, the FGFR pathway antagonist is PD173074, which is present in the culture media at a concentration of within a range of 10-100 nM, 25-75 nM or 40-60 nM or at a concentration of 50 nM. In one embodiment, the FGFR pathway antagonist is BLU9931. In one embodiment, the FGFR pathway antagonist is BLU9931, which is present in the culture media at a concentration of within a range of 10-100 nM, 25-75 nM or 40-60 nM or at a concentration of 50 nM. In one embodiment, in the stage 4 (step (d)) media, two FGFR antagonists are used, such as an FGFR1 antagonist (e.g., PD173074, e.g., at 50 nM) and an FGFR4 antagonist (e.g., BLU9931, e.g., at 50 nM).
Agonists of the glial cell line-derived neurotrophic factor (GDNF) pathway include agents, molecules, compounds, or substances capable of stimulating (upregulating) 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 culture media at a concentration within a range of 1-100 ng/ml, 5-15 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, which is present in the culture media at a concentration of 1-100 ng/ml, 5-15 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 culture media in stage 4 (step (d)) of the method at a concentration of 10 ng/ml.
Agonists of the PKC pathway include agents, molecules, compounds, or substances capable of stimulating (activating) signaling through the PKC pathway. In one embodiment, the PKC pathway agonist is selected from the group consisting of (−)-Indolactam-V, Bryostatin 1, PEP 005, Phorbol 12,13-dibutyrate, Phorbol 12-myristate 13-acetate, TPPB; Okadaic Acid, Prostratin, SC10, 1-Stearoyl-2-arachidonoyl-sn-glycerol, 6-(N-Decylamino)-4-Hydroxymethylindole, and combinations thereof. In an embodiment, the PKC pathway agonist is a benzolactam-derived PKC pathway agonist. In an embodiment, the PKC pathway agonist is a phorbol ester PKC agonist. In one embodiment, the PKC pathway agonist is (−)-Indolactam-V. In one embodiment, the PKC pathway agonist is (−)-Indolactam-V, which is present in the culture media at a concentration within a range of 100-500 nM, 200-400 nM or 250-350 nM or at a concentration of 300 nM. In one embodiment, the PKC pathway agonist is Prostratin. In one embodiment, the PKC pathway agonist is Prostratin, which is present in the culture media at a concentration within a range of 0.5-2.5 μM, 0.75-2.0 μM or 1-1.5 μM or at a concentration of 1 μM. In one embodiment, in the stage 5 (step (e)) media, two PKC pathway agonists are used, such as (−)-Indolactam-V (e.g., at 300 nM) and Prostratin (e.g., at 1 μM).
Agonists of the brain-derived neurotrophic factor (BDNF) pathway include agents, molecules, compounds, or substances capable of stimulating (upregulating) 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 dimeric peptide-6 (TDP6), LM22A-4, and combinations thereof. In one embodiment, the BDNF pathway agonist is present in the culture media at a concentration within a range of 1-100 ng/ml, 5-15 ng/ml, 10-25 ng/ml or 12.5-17.5 ng/ml, or at a concentration of 10 ng/ml. In one embodiment, the BDNF pathway agonist is BDNF. In one embodiment, the BDNF pathway agonist is BDNF, which is present in the culture media at a concentration of 1-100 ng/ml, 5-15 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 culture media in stage 5 of the method at a concentration of 10 ng/ml.
The stage 5 media includes ascorbic acid or analog thereof. In one embodiment, ascorbic acid or analog 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, ascorbic acid or analog is present in the culture media at a concentration within a range of 50-500 μM, 100-300 μM or 150-250 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, 100-300 μM or 150-250 μM. In one embodiment, 2-phospho-L-ascorbic acid is used, which is present in the culture media at a concentration of 200 μM in stage 5 of the method.
Agonists of the PPAR-γ (peroxisome proliferator-activated receptor-gamma) pathway include agents, molecules, compounds, or substances capable of stimulating (activating) signaling through the PPAR-γ pathway. In one embodiment, the PPAR-γ pathway agonist is selected from the group consisting of Rosiglitazone; Ciglitazone; Edaglitazone; GW 1929 hydrochloride; Pioglitazone Hydrochloride; Troglitazone; Tesaglitazar; Carnosic Acid; Indomethacin; Pioglitazone Hydrochloride; CAY10506; CAY10599; PAz-PC; Rosiglitazone Maleate; Rosiglitazone Hydrochloride; Rosiglitazone-d3; GW1929; S26948; 9-Nitrooleate; Azelaoyl-PAF; DRF 2519; PGPC; Methyl-8-hydroxy-8-(2-pentyl-oxyphenyl)-oct-5-ynoate; 20-carboxy Arachidonic Acid; 15-deoxy-Delta12,14-Prostaglandin J2 solution; CAY15073; and combinations thereof. In one embodiment, the PPAR-γ pathway agonist is present in the culture media at a concentration within a range of 100-1000 nM, 200-800 nM, 250-750 nM or 500-600 nM. In one embodiment, the PPAR-γ pathway agonist is Rosiglitazone. In one embodiment, the PPAR-γ pathway agonist is Rosiglitazone, which is present in the culture media at a concentration of 100-1000 nM, 200-800 nM, 250-750 nM or 500-600 nM. In one embodiment, the PPAR-γ pathway agonist is Rosiglitazone, which is present in the culture media at a concentration of 500 nM in step (e) (stage 5).
The stage 5 media further includes an N2 supplement. As used herein, an “N2 supplement” refers to a mixture of cell culture supplements for promoting the growth 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 Media Supplement (R&D Systems) and N-2 Supplement (CSH Protocols). An N2 supplement comprises a transferrin family molecule as well as additional growth-promoting molecules. In one embodiment, the N2 supplement comprises holo-transferrin, insulin (recombinant full chain), progesterone, putrescine, selenite, retroprogesterone, medrogestone, norethindrone, chlormadinone acetate, cyproterone acetate, medroxyprogesterone acetate, and megestrol acetate. In one embodiment, the N2 supplement is present in the culture media at a concentration within a range of 0.1%-5%, 0.5%-3% or 1-2% nM. In one embodiment, an N2 supplement is present in the culture media at a concentration of 1% in stage 5 of the method.
When an agonist or antagonist is used in more than one step of the method, in one embodiment it is the same particular agonist or antagonist that is used for each step in which the agent is present in the culture media. In another embodiment, different agonists or antagonists that affect the same signaling pathway are used in different steps of the method. For example, for the BMP antagonist used in steps (a) and (b) (stages 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), (d) and (e) (stages 3, 4 and 5), in one embodiment the same IGF-1 pathway agonist is used in steps (c), (d) and (e). In another embodiment, different IGF-1 pathway agonists are used in steps (c), (d) and (e). Similarly, for the Notch pathway antagonist used in steps (c) and (d) (stages 3 and 4), in one embodiment the same Notch pathway antagonist is used in steps (c) and (d). In another embodiment, different Notch pathway antagonists are used in steps (c) and (d). Similarly, for the AKT pathway antagonist used in steps (a) and (d) (stages 1 and 4), in one embodiment, the same AKT pathway antagonist is used in steps (a) and (d). In another embodiment, different AKT pathway antagonists are used in steps (a) and (d).
When an agonist or antagonist is used in more than one step of the method, in one embodiment it is the same concentration of the same agonist or antagonist that is used for each step in which the agent is present in the culture media. 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) (stages 1 and 2), in one embodiment the same concentration of 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), (d) and (e) (stages 3, 4 and 5), in one embodiment the same concentration of the same IGF-1 agonist is used in steps (c), (d) and (e). In another embodiment, different concentrations of the same IGF-1 agonist are used in steps (c), (d) and (e). Similarly, for the Notch antagonist used in steps (c) and (d) (stages 3 and 4), in one embodiment the same concentration of the same Notch antagonist is used in steps (c) and (d). In another embodiment, different concentrations of the same Notch antagonist are used in steps (c) and (d). Similarly, for the AKT antagonist used in steps (a) and (d) (stages 1 and 4), in one embodiment, the same concentration of the same AKT antagonist is used in steps (a) and (d). In another embodiment, different concentrations of the same AKT antagonists are used in steps (a) and (d).
In combination with the chemically-defined and optimized culture media described in subsection II above, the methods of generating FB-NSCs, VFB-NSCs, MGE-NPCs, immatured neurons and mature somatostatin+ interneurons of the disclosure utilize standard culture conditions established in the art for cell culture. For example, cells can be cultured at 37° C. and 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 adhered to plates, preferably coated with an extracellular matrix material such as vitronectin. In one embodiment, the stem cells are cultured on a vitronectin coated culture surface (e.g., vitronectin coated 96-well plates).
Pluripotent stem cells can be cultured in commercially available media prior to differentiation. For example, stem cells can be cultured for at least one day in Essential 8 Flex media (Thermo Fisher #A2858501) prior to the start of the differentiation protocol. In a non-limiting exemplary embodiment, stem cells are passaged onto vitronectin (Thermo Fisher #A14700) coated 96-well plates at 150,000 cells/cm2 density and cultured for one day in Essential 8 Flex media prior to differentiation.
To begin the differentiation protocol, the media the stem cells are being cultured in is changed to a basal differentiation media that has been supplemented with signaling pathway agonists and/or antagonists as described above in subsection II. A basal differentiation media can include, for example, a commercially-available base supplemented with additional standard culture media components needed to maintain cell viability and growth, but lacking serum (the basal differentiation media is a serum-free media) or any other exogenously-added growth factors, such as FGF2, PDGF or HGF. In a non-limiting exemplary embodiment, a basal differentiation media contains 1× IMDM (Thermo Fisher #12440046), 1× 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 μM, Insulin (Sigma #11376497001) at 0.7 μg/ml and transferrin (Sigma #10652202001) at 15 μg/ml (also referred to herein as “CDM2” media, as used in the exemplary differentiation protocols shown in
The culture media typically is changed regularly to fresh media. For example, in one embodiment, media is changed every 24 hours.
To generate FB-NSCs, VFB-NSCs, MGE-NPCs, immatured neurons and mature somatostatin+ interneurons, the starting stem cells are cultured in the optimized culture media for sufficient time for cellular differentiation and expression of committed FB-NSC-, VFBNSC-, -MGE-NPC-, immatured neuron- or mature somatostatin+ interneuron-associated markers. As described in the Examples, it has been discovered that culture of pluripotent stem cells in a five-stage method, one optimized for generation of FB-NSCs, a second optimized for generation of VFB-NSCs, a third optimized for the generation of MGE-NPCs, a fourth optimized for the generation of immatured neurons and a fifth optimized for the generation of mature somatostatin+ interneurons can lead to the production of MGE-NPCs in as little as nine days of culture and mature somatostatin+ interneurons in as little as 26 days of culture. The culture period for the first stage (leading to FB-NSCs) is days 0-3, the culture period for the second stage (leading to VFB-NSCs) is days 3-6, the culture period for the third stage (leading to MGE-NPCs) is days 6-9, the culture period for the fourth stage (leading to immatured neurons) is days 9-12 and the culture period for the fifth stage (leading to mature somatostatin+ interneurons) is days 12-26.
Accordingly, in the first stage of the method, which generates FB-NSCs, also referred to herein as “step (a)” or “stage 1”, pluripotent stem cells are cultured in the stage 1-optimized culture media on days 0-3, 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 at least 72 hours, or for 60 hours, or for 64 hours, or for 68 hours, or for 70 hours or for 72 hours.
Accordingly, in the second stage of the method, which generates VFB-NSCs, also referred to herein as “step (b)” or “stage 2”, the FB-NSCs generated in step (a) are further cultured in the stage 2-optimized culture media on days 4-6, 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 for 64 hours, or for 68 hours, or for 70 hours or for 72 hours.
Accordingly, in the third stage of the method, which generates MGE-NPCs, also referred to herein as “step (c)” or “stage 3”, the VFB-NSCs generated in step (b) are further cultured in the stage 3-optimized culture media on days 6-9, 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 for 64 hours, or for 68 hours, or for 70 hours or for 72 hours.
Accordingly, in the fourth stage of the method, which generates immatured neurons, also referred to herein as “step (d)” or “stage 4”, the MGE-NPCs generated in step (c) are further cultured in the stage 4-optimized culture media on days 9-12, 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 for 64 hours, or for 68 hours, or for 70 hours or for 72 hours.
Accordingly, in the fifth stage of the method, which generates mature somatostatin+ interneurons, also referred to herein as “step (e)” or “stage 5”, the immatured neurons generated in step (d) are further cultured in the stage 5-optimized culture media on days 12-26, or starting on day 12 and continuing through day 26, or starting on day 12 and continuing in culture for sufficient time to generate somatostatin+ mature interneurons (e.g., 14 days, or two weeks, of culture in the stage 5 media).
The methods and compositions of the disclosure for generating FB-NSCs, VFB-NSCs, MGE-NPCs, immatured neurons and mature somatotatin+ 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 in the study of forebrain neural progenitor development and biology, including differentiation into GABAergic interneurons, to assist in the understanding and potential treatment of neurodegenerative and psychiatric diseases and disorders involving dysfunction of GABAergic interneurons. For example, FB-NSCs, VFB-NSCs, MBE-NPCs, immatured neurons and mature somatostatin+ interneurons generated using the methods of the disclosure can be further purified according to methods established in the art using agents that bind to surface markers expressed on the cells. Accordingly, in one embodiment, the disclosure provides a method of isolating FB-NSCs, VFB-NSCs, MGE-NPCs, immatured neurons or mature somatostatin+ interneurons, the method comprising:
In one embodiment, the binding agent is an antibody, e.g., a monoclonal antibody (mAb) that binds to the cell surface marker. Cells that bind the antibody can be isolated by methods known in the art, including but not limited to fluorescent activated cell-sorting (FACS) and magnetic activated cell sorting (MACS).
Progenitors of the forebrain neural lineage also are contemplated for use in the treatment of neurodegenerative or psychiatric diseases and disorders, through delivery of the cells to a subject having the disease or disorder, including any such disease or disorder involving dysfunction of GABAergic interneurons. Transplantation of embryonic medial ganglionic eminence (MGE) cells into the adult brain has been shown to result in dispersal and migration of the transplanted cells and differentiation into neurons expressing GABA (Alvarez-Dolado et al. (2006) J. Neurosci. 26:7380-7389). Accordingly, in one embodiment, forebrain neural lineage progenitors are used in the treatment of epilepsy. Transplantation of precursors of GABAergic interneurons into the postnatal neocortex of mice has been shown to reduce epileptic seizures (Baraban et al. (2009) Proc. Natl. Acad. Sci. USA 106:15472-15477). The use of neural progenitors 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. Moreover, changes in the number, density and/or activity of somatostatin+ interneurons, or in the levels of somatostatin, are found throughout many neuropsychiatric and neurological conditions, both in patients and in animal models (see e.g., Liguz-Lecznar et al. (2022) Biomolecules 12:312). For example, reduced somatostatin expression or decreased numbers of somatostatin+ interneurons is observed in many neurological disorders, non-limiting examples of which include Alzheimer's, Parkinson's and Huntington's diseases, schizophrenia and depression.
Accordingly, the forebrain lineage cells of the disclosure also can be used in the treatment of disorders associated with reduced levels of somatostatin and/or decreased numbers of somatostatin+ interneurons that may benefit from restoration of GABAergic interneuron function, including but not limited to Alzheimer's disease, Parkinson's disease, Huntington's disease, schizophrenia, and depression.
The cells of the disclosure also are useful in the screening of potential drugs or for the development of novel cell therapies for the treatment of diseases or disorders involving dysfunction of GABAergic interneurons.
In other aspects, the disclosure provides compositions related to the methods of generating FB-NSCs, VFB-NSCs, MGE-NPCs, immatured neurons and mature somatostatin+ interneurons, including culture media and cell cultures, as well as isolated progenitor cells and cell populations.
In one aspect, the disclosure provides a culture media 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, an SHH pathway agonist and a PKC pathway antagonist. Suitable agents, and concentrations therefor, include those described in subsection II.
In one aspect, the disclosure provides a culture media 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 an SHH pathway agonist. Suitable agents, and concentrations therefor, include those described in subsection II.
In one aspect, the disclosure provides a culture media for obtaining human ASCL1+ medial ganglionic eminence 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. Suitable agents, and concentrations therefor, include those described in subsection II.
In one aspect, the disclosure provides a culture media for obtaining human immatured neurons from MGE-NPCs, the media comprising an IGF-1 pathway agonist, a Notch pathway antagonist, at least one FGFR pathway antagonist (e.g., an FGFR1 pathway antagonist and an FGFR4 pathway antagonist), a GDNF pathway agonist, and an AKT pathway antagonist. Suitable agents, and concentrations therefor, include those described in subsection II.
In one aspect, the disclosure provides a culture media for obtaining human mature somatostatin+ interneurons, the media comprising at least one PKC pathway agonist (e.g., a benzolactam-derived PKC pathway agonist and a phorbol ester PKC pathway agonist), a BDNF pathway agonist, an IGF-1 pathway agonist, an ascorbic acid pathway agonist, a PPAR-γ pathway agonist, and an N2 supplement. Suitable agents, and concentrations therefor, include those described in subsection II.
In one aspect, the disclosure provides an isolated cell culture of human OTX2+ FEZF2+ SIX3+ forebrain neural stem cells (FB-NSCs), the culture comprising human OTX2+ FEZF2+ SIX3+ FB-NPCs cultured in a culture media comprising 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. Suitable agents, and concentrations therefor, include those described in subsection II.
In one aspect, the disclosure provides an isolated cell culture of human NKX2-1+ ventral forebrain neural stem cells (VFB-NSCs), the culture comprising human NKX2-1+ VFB-NPCs cultured in a culture media comprising a BMP pathway antagonist, a MEK pathway antagonist, a WNT pathway antagonist and an SHH pathway agonist. Suitable agents, and concentrations therefor, include those described in subsection II.
In one aspect, the disclosure provides an isolated cell culture of human ASCL1+ medial ganglionic eminence neural progenitor cells (MGE-NPCs), the culture comprising human ASCL1+ MGE-NPCs cultured in a culture media 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. Suitable agents, and concentrations therefor, include those described in subsection II.
In one aspect, the disclosure provides an isolated cell culture of human immatured neurons, the culture comprising human immatured neurons cultured in a culture media comprising an IGF-1 pathway agonist, a Notch pathway antagonist, at least one FGFR pathway antagonist (e.g., an FGFR1 pathway antagonist and an FGFR4 pathway antagonist), a GDNF pathway agonist, and an AKT pathway antagonist. Suitable agents, and concentrations therefor, include those described in subsection II.
In one aspect, the disclosure provides an isolated cell culture of human mature somatostatin+ interneurons, the culture comprising human mature somatostatin+ interneurons cultured in a culture media comprising at least one PKC pathway agonist (e.g., a benzolactam-derived PKC pathway agonist and a phorbol ester PKC pathway agonist), a BDNF pathway agonist, an IGF-1 pathway agonist, an ascorbic acid pathway agonist, a PPAR-γ pathway agonist, and an N2 supplement. Suitable agents, and concentrations therefor, include those described in subsection II.
In one aspect, the disclosure provides human OTX2+ FEZF2+ SIX3+ forebrain neural stem cells (FB-NSCs) generated by a method of the disclosure (i.e., step (a) or stage 1 of the culture protocol).
In one aspect, the disclosure provides human NKX2-1+ ventral forebrain neural stem cells (VFB-NSCs) generated by a method of the disclosure (i.e., steps (a) and (b), or stages 1 and 2 of the culture protocol).
In one aspect, the disclosure provides human ASCL1+ medial ganglionic eminence neural progenitor cells (MGE-NPCs) generated by a method of the disclosure (i.e., steps (a), (b) and (c), or stages 1, 2 and 3, of the culture protocol).
In one aspect, the disclosure provides human immatured neurons generated by a method of the disclosure (i.e., steps (a), (b), (c), and (d) or stages 1, 2, 3, and 4 of the culture protocol, or by use of stage 4 of the culture protocol when starting from MGE-NPCs).
In one aspect, the disclosure provides human matured somatostatin+ interneurons generated by a method of the disclosure (i.e., steps (a), (b), (c), (d) and (e) or stages 1, 2, 3, 4 and 5 of the culture protocol, or by use of stages 4 and 5 of the culture protocol when starting from MGE-NPCs).
The present invention is further illustrated by the following examples, which should not be construed as further limiting. The contents of figures and all references, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.
A three-stage recipe for generation of medial ganglionic eminence-derived neural progenitors was developed that can guide human pluripotent stem cells to progenitors expressing NKX2-1 and ASCL1 after 9 days in culture. These cells can be further differentiated to mature somatostatin+ interneurons.
This example utilizes a method of High-Dimensional Design of Experiments (HD-DoE), as previously described in Bukys et al. (2020) Iscience 23:101346. The method employs computerized design geometries to simultaneously test multiple process inputs and offers mathematical modeling of a deep effector/response space. The method allows for finding combinatorial signaling inputs that control a complex process, such as during cell differentiation. It allows testing of multiple plausible critical process parameters, as such parameters impact output responses, such as gene expression. Because gene expression provides hallmark features of the phenotype of, for example, a human cell, the method can be applied to identify, and understand, which signaling pathways control cell fate. In the current example, the HD-DOE method was applied with the intent to find conditions for induction of midbrain neural progenitor-expressed genes, directly from the pluripotent stem cell state.
To develop the recipe for each stage, the impact of agonists and antagonists of multiple signaling pathways (herein called effectors) on the expression of two sets of 53 pre-selected genes, after a 3-day treatment, has been tested and modeled. These effectors are small molecules or proteins that are commonly used during stepwise differentiation of stem cells to specific fates. Choice of the effectors were based on current literature on neural induction in forebrain region of developing brain and differentiation of stem cells to neural progenitors.
To test the effectors, experiments with at least 8 factors were designed that can assess the response of cells to 48 or more different combinations of effectors in a range of concentrations. To analyze the models, we focused on expression of genes involved in 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 telencephalon and gene expressed in ventral forebrain including NKX2-1 and absence of PAX6 which is a dorsal forebrain marker (Stoykova et al. (2000) J. Neurosci. 20:8042-8050). The impact of each effector on gene expression level is defined by a parameter called factor contribution that is calculated for each effector during the modeling.
To identify the recipe for stage 1 of differentiation, 96 different combinations of effectors generated using Design-of-Experiments compression through D-optimality were robotically prepared. The effector combinations were prepared in a basal media and were subsequently added to the cells, which were then allowed to differentiate. Three days later RNA extraction was performed, and gene expression was obtained using quantitative PCR analysis. The data were normalized and modeled using partial least squares regression analysis to the effector design, resulting in the generation of gene-specific models, which after model tuning for maximal Q2 predictive power, provided explanation of the effectors ability to control the expression of individual genes, combinatorically, and individually. Solutions within the tested space could then be explored to address desirability.
Optimizing for maximal expression of SIX3 at 3300 and FEZF2 1355 expression values led to robust solutions. This 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
The model was also optimized for maximum expression of FEZF2 separately at 3576 expression value to find any additional factors that can boost expression of forebrain genes. Within the specifications of attaining 80% maximal expression of FEZF2, this complex media composition had a Cpk value (process capability index) of 0.61, with a corresponding to a 3.3% risk of failure. As shown in
To find the set of factors that have the potential to optimize all 3 genes in this experiment, a new assessment was done through dynamic profile analysis with focus on maximal expression of OTX2, FEZF2 and SIX3. The results are shown in
To further enhance the conditions for forebrain differentiation from pluripotency, an additional HD-DoE experiment was performed. Additional gene regulatory models were obtained that were used for preparation of the differentiation protocol. The factors in this experiment included LDN193189, PD173074, BLU9931, Purmorphamine, SC79, MK2206, ZM336372, PD0325901, CHIR99021, XAV939, UCLA-GP130, Tofacitinib and GO6983. As shown in
Considering both HD-DoE experiments, conditions that maximize differentiation of cells to the forebrain region with neural stem cell identity as such relate to robust and elevated 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.
To further guide the differentiation of forebrain-committed neural stem cells to ventral forebrain neural stem cells at stage 2, a HD-DoE experiment was performed for 3 days after termination of stage 1 treatment. At this time, we focused on maximum expression of NKX2-1 that is expressed in ventral forebrain region and minimum expression of PAX6 that is expressed in dorsal forebrain region. This 12-factor experiment included LDN193189, BMP7, PD0325901, MK2206, A8301, XAV939, CHIR99021, Purmorphamine, SANT-1, AGN193109, TTNPB and GSI-XX. As shown in
Considering the model at different optimization settings for maximum differentiation of cells toward ventral forebrain, the recipe for stage 2 of differentiation included LDN193189, PD0325901, XAV939 and Purmorphamine. A representative recipe for Stage 2 differentiation is summarized below in Table 2.
To further guide the cells to medial ganglionic eminence progenitor fate, an additional HD-DoE experiment was performed for 3 days on cells that were treated with stage 1 and stage 2 media for total of 6 days prior to 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 maximum expression of genes expressed in neuronal progenitor cells in medial ganglionic eminence (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, which is an inhibitor of TAK1 pathway, had the highest factor contributions, 18.1, 14.1, 12.7 and 10.1 respectively. GNF5837 and IGF-1 also had positive impact on its expression with factor contributions of 9.4 and 7. MHY 1485 also had positive contribution however the factor was below 2 (
Dynamic profile analysis was used to find common factors with positive impact on expression of ASCL1, LHX6 and DLX1. As shown in
To assess the impact of the elimination of each validated factor, dynamic profile analysis was used and compared the expression level of genes of interest in absence of each finalized factor while others are present. Since expression levels of genes of interest reveal whether the desired outcome is reachable, this factor criticality analysis revealed the extent of importance of each input effector.
In the stage 1 recipe, each of the six finalized factors were removed individually while the other five factors were present and expression levels of forebrain genes was assessed compared to the presence of all six factors together. The results are summarized in
In the stage 2 recipe, each of the four finalized factors were removed while the other three factors remained present and expression levels of NKX2-1, PAX6 and NKX2-2 were assessed compared to presence of all four factors. The results are summarized in
In the stage 3 recipe, each of the six finalized factors were removed while the other five factors remained present and the expression levels of ASCL1, DLX1 and LHX6 were assessed compared to the presence of all six factors. The results are summarized in
To validate the developed recipes described in example 1, cells were treated with stage 1, stage 2 and stage 3 differentiation media, and immunocytochemistry was used to assess biomarkers of ventral forebrain region and neural progenitors at the end of each stage. Biomarkers included SIX3, OTX2, beta III-tubulin a pan neuronal marker, NKX2-1, PAX6, KI67 a proliferation marker, DLX5 a neuronal progenitor marker expressed in LGE and MGE region of forebrain (Wang et al. (2010) J. Neurosci. 30:5334-5345), LHX6, OLIG2 an oligodendrocyte marker, MASH1 (ASCL1), GFAP a glial marker, DCX a marker specifying immature neurons, SOX6 a marker expressed in post-mitotic progenitors of MGE region (Batista-Brito et al. (2009) Neuron 63:466-481) and GABA a marker specifically expressed by GABAergic interneurons. The results are shown in
Immunocytochemistry images confirmed the expression of SIX3 and OTX2 by the end of stage 1 in more than 90% of the cells and PAX6 and OLIG2 were not detected. KI67 was also detected in most of the cells, which confirmed cells are proliferative at this point (
The detection of ventral forebrain neural progenitor markers and the absence of dorsal forebrain markers in differentiated cells after 9 days confirmed the efficiency and robustness of the generated recipes for a 3-stage differentiation protocol for human induced pluripotent stem cell-derived MGE-committed neural progenitors.
Two-stage recipes were developed to induce the medial ganglionic eminence (MGE)-derived neural progenitors (obtained, e.g., as described in Example 1) to differentiate to mature somatostatin interneurons expressing somatostatin and GABA after 17 days in cell culture.
To develop the recipe for each stage, the impact of agonists and antagonists of multiple signaling pathways, herein called effectors, on the expression of two sets of 53 pre-selected genes after a 3-day cell culture treatment, has been evaluated and modeled by using HD-DoE (as described in Example 1). These effectors are small chemical compounds or proteins. They are commonly used during the differentiation of stem cells to specific cell linage. The choice of the effectors was based on the current literature on forebrain neural induction and differentiation of neural progenitor cells to mature neurons.
To evaluate the effectors on SST differentiation, experiments with 8 or 12 factors were designed to assess the responses of cells to 48 or more combinations of effectors in a range of concentrations. To analyze the models, we focused on expression of genes involved in development and patterning of the forebrain interneurons maturation including LHX6 (Liodis et al. (2007) J. Neurosci. 27:3078-3089; Yuan et al. (2018) Elife 7: e37382), SOX6 (Azim et al. (2009) Nature Neurosci. 12:1238-1247; Batista-Brito et al. (2009) Neuron 63:466-481), DLX5 (de Lombares et al. (2019) Aging (Albany NY) 11:6638; Wang et al. (2010) J. Neurosci. 30:5334-5345) and ASCL1 (Barretto et al. (2020) J. Neurosci. Meth. 334:108548; Shi et al. (2016) J. Biol. Chem. 291:13560-13570). At later stages we focused on interneuron maturation genes including somatostatin and absence of Parvalbumin (Horn and Nicoll (2018) Proc. Natl. Acad. Sci. 115:589-594). The impacts of each effector on gene expression level are defined by a parameter called factor contribution that is calculated for each effector during the modeling.
To identify the recipe for differentiation at stage 4, at least 48 different combinations of effectors were robotically prepared by the Design-of-Experiments compression through D-optimality. The effector combinations were prepared in a basal media and were subsequently added to the cells, which were then allowed to differentiate. Three days later, RNA extraction was performed, and gene expression levels were examined using quantitative PCR analysis. The data was normalized and modeled using partial least squares regression analysis to the effector design, resulting in the generation of gene-specific models, which after model tuning for maximal predictive power, provided explanation of the effectors ability to control the expression of individual genes, combinatorically, and individually. Solutions within the tested space could then be explored to address desirability.
As shown in
Valproic acid is a Histone deacetylase inhibitor, MHY1458 is an mTOR activator, LDN193189 is a BMP inhibitor, Substance P is a member of the tachykinin neuropeptide family, CAMP is an activator of the CREB and PKA pathways, PD0325901 is a MEK inhibitor and SB431542 is a TGF-β Receptor Inhibitor. Each of these factors had a significant negative effect on expression of SOX6, with factor contribution of 4.7, 9.5, 9.8, 5.1, 10.9, 5.1, 6.2, respectively. Within the specifications of attaining 80% maximal expression of SOX6, this complex media composition had a Cpk value (process capability index) of 0.4, with a corresponding to a 11% risk of failure.
To find the set of factors that have the potential to optimize SOX6 in this experiment, a new assessment was done through dynamic profile analysis with focus on maximal expression of SOX6. The results are shown in
These experiments led to the validated stage 4 recipe, as shown below in Table 4.
To further guide the differentiation of forebrain-committed neural progenitor cells to somatostatin interneuron cells at stage 5, HD-DoE experiments were performed for 3 days after termination of stage 4 treatment. At this time, we focused on maximum expression of SST that was expressed in Somatostatin (+) interneurons and minimum expression of Parvalbumin that is another type of interneurons. This 8-factor experiment included JQ1, (−)-Indolactam-V, Oleic acid, BDNF, IGF-1, 2-phospho-L-Ascorbic acid, Albumax, MK2206. When the model was maximized for expression of Somatostatin, six effectors were identified that could increase its expression levels, including (−)-Indolactam-V, Oleic acid, BDNF, 2-phospho-L-ascorbic acid, Albumax, and MK2206, as shown in
To investigate the effects of other factors on differentiation of forebrain-committed neural progenitor cells to Somatostatin interneuron cells, an additional HD-DoE experiment was performed for 3 days on cells that were treated with stage 1 to stage 4 medium for a total of 12 days prior to the new experiment. This experiment, the results of which are shown in
Dynamic profile analysis was used to find common factors with positive impact on expression of Somatostatin, the results of which are shown in
In another HD-DoE experiment, the results of which are shown in
Dynamic profile analysis was used to find common factors with positive impact on expression of Somatostatin, the results of which are shown in
To assess the impact of elimination of each validated factor, dynamic profile analysis was again used to compare the expression levels of genes of interest in the absence of each finalized factor while the others were present in the media. Since the expression level of genes of interest revealed whether the desired outcome was reachable, this factor criticality analysis revealed the extent of importance of each input effector.
In the stage 4 recipe, each of the five finalized factors was removed while the other four factors remained present in the media and the expression level of forebrain genes was assessed compared to the presence of all 6 factors. The results are shown in
In the stage 5 recipe, each of seven finalized factors was removed while the other six factors remained present in the media and the expression levels of SST and PVALB were assessed compared to the presence of all seven factors. In the HD-DoE experiment for which the results are shown in
To validate the stage 4 and stage 5 recipes developed in Example 4, cells were treated with stage 1, stage 2, stage 3, stage 4 or stage 5 differentiation medium, and immunofluorescence was used to assess expression of biomarkers of forebrain neurons and neural progenitors at the end of each stage. Biomarkers included MAP2, GABA, GAD1/2, NeuN, SATB1, DCX, TUJ1, GFAP, LHX6, Nestin, Mash1, and SOX6 (see e.g., Batista-Brito et al. (2009) Neuron 63:466-481; Chamak et al. (1987) J. Neurosci. 7:3163-3170; Denaxa et al. (2012) Cell Reports 2:1351-1362; Liodis et al. (2007) J. Neurosci. 27:3078-3089; Shi et al. (2016) J. Biol. Chem. 291:13560-13570). MAP2 is mature neuron marker. DCX is immature neuron marker. TUJ1 is a pan neural marker. LHX6 is a cortical interneuron marker (Cruz-Santos et al. (2022) Cells 11:853; Liodis et al. (2007) J. Neurosci. 27:3078-3089). MASH1 (ASCL1) (Fitzgerald et al., 2020; Shi et al., 2016). GFAP is a glial marker. SOX6 is a marker expressed in post-mitotic progenitors of MGE region (Chen (2017) Sci. Reports 7:1-11; Batista-Brito et al. (2009) Neuron 63:466-481). SATB1 has been reported to increase SST expression in interneurons (Denaxa et al. (2012) Cell Reports 2:1351-1362). GABA is a marker specifically expressed by GABAergic interneurons.
As shown in
As shown in
In summary, detection of mature neuronal markers and interneuron markers in differentiated cells at 27 days, confirmed the efficiency and robustness of the stage 4 and stage 5 recipes for the 2-stage differentiation protocol for MGE-committed neural progenitors to differentiated Somatostatin (+) interneurons.
Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
This application claims priority to U.S. Provisional Application No. 63/539,151, filed Sep. 19, 2023, the entire contents of which is hereby incorporated by reference.
This invention was made with government support under Grant Number: W911NF-17-3-0003 awarded by the U.S. ARMY ACC-AGP-RTP. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63539151 | Sep 2023 | US |
