IP Library › Granted Patent US 12,729,368
Granted Patent B2
US 12,729,368 · App. 18/207,363 · Granted Sep 8, 2026

Methods and compositions for generating oligodendrocyte progenitor cells

Inventor: Nooshin Amini (Cleveland, OH)
Assignee: Trailhead Biosystems Inc.
C12N5/0622C12N2501/105C12N2501/115C12N2501/135C12N2501/155C12N2501/16C12N2501/33C12N2501/38C12N2501/385C12N2501/395C12N2501/405C12N2501/41C12N2501/415C12N2501/42C12N2501/48C12N2501/60C12N2501/727C12N2506/08C12N2506/45
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Quick Facts
Patent No.
US 12,729,368
App. No.
18/207,363
Granted
Sep 8, 2026
Kind
B2
Abstract

Methods for generating pre-oligodendrocyte progenitor cells (pre-OPCs), oligodendrocyte progenitor cells (OPCs) and pre-myelinating oligodendrocytes (preOLs) from human pluripotent stem cells are provided using chemically-defined culture media that allow for generation of pre-OPCs in as little as three days, SOX10+ OLIG2+ NKX2-2+ OPCs in as little as twelve days and CD9+A2B5+O4+CNPase+ preOLs in as little as eighteen days. Two alternative culture protocols for generating OPCs are provided. Culture media, isolated cell populations and kits are also provided.

Claims (36)

1 . A method of generating CD9+A2B5+O4+CNPase+ pre-myelinating oligodendrocytes (preOLs) comprising culturing SOX10+ OLIG2+ NKX2-2+ oligodendrocyte progenitor cells (OPCs) OPCs in a culture medium media comprising an insulin-like growth factor 1 receptor (IGF1R) pathway agonist, a tropomyosin-related kinase C (TrkC) pathway agonist, a platelet-derived growth factor receptor (PDGFR) pathway agonist, a thyroid hormone receptor agonist, and an insulin receptor agonist such that CD9+A2B5+O4+CNPase+ preOLs are generated.

2 . The method of claim 1 , wherein the OPCs are cultured in the culture medium for six days.

3 . The method of claim 1 , wherein the IGF1R pathway agonist is selected from the group consisting of IGF-1, IGF-2, insulin, Rg5, IGF-1 30-41, Demethylasterriquinone B1, IGF1-Ado, X10, mecasermin, and combinations thereof.

4 . The method of claim 1 , wherein the TrkC pathway agonist is selected from the group consisting of neurotrophin-3 (NT-3), peptidomimetics based on b-turns of NT-3, LM22B 10, GNF 5837, and combinations thereof.

5 . The method of claim 1 , wherein the PDGFR pathway agonist is PDGF-AA.

6 . The method of claim 1 , wherein the thyroid hormone receptor agonist is selected from the group consisting of T3, T4, Resmetirom, TRb agonist 3 (Compound 3), Sobetirome, Tiratricol, and combinations thereof.

7 . The method of claim 1 , wherein the insulin receptor agonist is selected from the group consisting of insulin, IGF-1, IGF-2, Demethylasterriquinone B1, MK-5160, MK-1092, and combinations thereof.

8 . A method of generating CD9+A2B5+O4+CNPase+ preOLs comprising:

(a) culturing human OLIG2+ pre-oligodendrocyte progenitor cells (pre-OPCs) in a culture medium comprising a fibroblast growth factor receptor (FGFR) pathway agonist, a mammalian target of rapamycin (mTOR) pathway antagonist, a sonic hedgehog (SHH) pathway agonist, an Akt pathway antagonist, and an Akt pathway agonist on days 0-3 to obtain a population of cells;

(b) culturing the population of cells from step (a) in a culture medium comprising an FGFR pathway agonist, a PDGFR pathway agonist, an Akt pathway antagonist, and a retinoic acid (RA) pathway agonist on days 3-9, such that SOX10+ OLIG2+ NKX2-2+ OPCs are generated; and

(c) culturing the population of cells from step (b) in a culture medium comprising an IGF1R pathway agonist, a TrkC pathway agonist, a PDGFR pathway agonist, a thyroid hormone receptor agonist, and an insulin receptor agonist on days 9-15 such that CD9+A2B5+O4+CNPase+ preOLs are generated.

9 . The method of claim 8 , wherein the human OLIG2+ pre-OPCs are obtained by culturing human pluripotent stem cells in a culture medium comprising a RA pathway agonist, an Akt pathway agonist, an mTOR pathway agonist, a WNT pathway antagonist, an SHH pathway agonist, a bone morphogenetic protein (BMP) pathway antagonist, and a protein kinase C (PKC) pathway antagonist on days −3-0.

10 . The method of claim 8 , wherein the FGFR pathway agonist is FGF2, SUN11602, or combinations thereof.

11 . The method of claim 8 , wherein the mTOR pathway antagonist is selected from the group consisting of AZD 3147, Dactolisib, Rapamycin, Everolimus, AZD 8055, Temsirolimus, PI-103, NU7441, BC-LI-0186, eCF 309, ETP 45658, Niclosamide, Omipalisib, PF 04691502, PF 05212384, Torin1, Torin 2, WYE 687, XL 388, STK16-IN-1, PP 242, Torkinib, Ridaforolimus, Sapanisertib, Voxtalisib, and combinations thereof.

12 . The method of claim 8 , wherein the SHH pathway agonist is selected from the group consisting of Purmorphamine, GSA 10, SHH, SAG, and combinations thereof.

13 . The method of claim 8 , wherein 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, Akti1/2, CCT128930, A 674563, PHT 427, Miltefosine, AT 13148, ML 9, BAY 1125976, Oridonin, TIC10, Pectolinarin, Akti IV, 10-DEBC, API-1, SC 66, FPA 124, API-2, Urolithin A, and combinations thereof.

14 . The method of claim 8 , wherein the Akt pathway agonist is selected from the group consisting of Sc79, Demethyl-Coclaurine, LM22B-10, YS-49, YS-49 monohydrate, Demethylasterriquinone B1, Recilisib, N-Oleyol glycine, NSC45586 sodium, Periplocin, CHPG sodium salt, Bilobalide, 6-hydroxyflavone, Musk ketone, SEW2871, 8-Prenylnaringenin, Razuprotafib, and combinations thereof.

15 . The method of claim 8 , wherein the PDGFR pathway agonist is PDGF-AA.

16 . The method of claim 8 , wherein the RA pathway agonist is selected from the group consisting of TTNPB, AM 580, CD 1530, CD 2314, CD 437, Ch 55, BMS 753, BMS 961, Tazarotene, Isotretinoin, Tretinoin, Tamibarotene, ATRA, AC 261066, AC 55649, retinoic acid (RA), Sr11237, adapalene, EC23, 9-cis retinoic acid, 13-cis retinoic acid, 4-oxo retinoic acid, and All-trans Retinoic Acid (ATRA), and combinations thereof.

17 . The method of claim 8 , wherein the culture medium in step (b) further comprises:

(a) a Wnt pathway antagonist; and

(b) a Notch pathway inhibitor.

18 . The method of claim 17 , wherein:

(a) 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; and/or

(b) the Notch pathway inhibitor is gamma secretase inhibitor-XX.

19 . A method of generating human CD9+A2B5+O4+CNPase+ preOLs comprising:

(a) culturing human OLIG2+ pre-OPCs in a culture medium comprising an FGFR pathway agonist, an mTOR pathway antagonist, an SHH pathway agonist, and a WNT pathway agonist on days 0-6 to obtain a population of cells; and

(b) culturing the population of cells from step (a) in a culture medium comprising an FGFR pathway agonist, an IGF-1 pathway agonist, and an RA pathway agonist on days 6-9, such that SOX10+ OLIG2+ NKX2-2+ OPCs are generated; and

(c) culturing the population of cells from step (b) in a culture medium comprising an IGF1R pathway agonist, a TrkC pathway agonist, a PDGFR pathway agonist, a thyroid hormone receptor agonist, and an insulin receptor agonist on days 9-15 such that CD9+A2B5+O4+CNPase+ preOLs are generated.

20 . The method of claim 19 , wherein the human OLIG2+ pre-OPCs are obtained by culturing human pluripotent stem cells in a culture medium comprising a RA pathway agonist, an Akt pathway agonist, an mTOR pathway agonist, a WNT pathway antagonist, an SHH pathway agonist, a BMP pathway antagonist, and a PKC pathway antagonist on days −3-0.

21 . The method of claim 19 , wherein the FGFR pathway agonist is FGF2, SUN11602, or combinations thereof.

22 . The method of claim 19 , wherein the mTOR pathway antagonist is selected from the group consisting of AZD 3147, Dactolisib, Rapamycin, Everolimus, AZD 8055, Temsirolimus, PI-103, NU7441, BC-LI-0186, eCF 309, ETP 45658, Niclosamide, Omipalisib, PF 04691502, PF 05212384, Torin1, Torin 2, WYE 687, XL 388, STK16-IN-1, PP 242, Torkinib, Ridaforolimus, Sapanisertib, Voxtalisib, and combinations thereof.

23 . The method of claim 19 , wherein the SHH pathway agonist is selected from the group consisting of Purmorphamine, GSA 10, SHH, SAG, and combinations thereof.

24 . The method of claim 19 , wherein the WNT pathway agonist is selected from the group consisting of CHIR99021, CHIR98014, SB 216763, SB 415286, LY2090314, 3F8, A 1070722, AR-A 014418, BIO, BIO-acetoxime, AZD1080, WNT3A, alsterpaullone, indirubin-3-oxime, 1-azakenpaullone, kenpaullone, TC-G 24, TDZD 8, TWS 119, NP 031112, AT 7519, KY 19382, AZD2858, and combinations thereof.

25 . The method of claim 19 , wherein the IGF-1 pathway agonist is selected from the group consisting of IGF-1, IGF-2, insulin, Rg5, IGF-1 30-41, Demethylasterriquinone B1, IGF1-Ado, X10, mecasermin, and combinations thereof.

26 . The method of claim 19 , wherein the RA pathway antagonist is selected from the group consisting of AGN193109, BMS 195614, CD 2665, ER 50891, LE 135, LY 2955303, MM11253, and combinations thereof.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jun 12, 2023
From: AMINI, NOOSHIN
To: TRAILHEAD BIOSYSTEMS INC.
Reel/Frame 063921/0622 →
Continuity (3)
Provisional Application 63400222 · Aug 23, 2022
Provisional Application 63396073 · Aug 8, 2022
Related Publication 20240043798A1 · Feb 8, 2024
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