Granted Patent B2

US 12,565,639 · App. 18/742,917 · Granted Mar 3, 2026

Inventors: David Tastad (San Diego, CA); Louisa Zebrowski (San Diego, CA); Ai Zhang (San Diego, CA); Branden Clark (San Diego, CA)

Assignee: Aspen Neuroscience, Inc.

C12N5/0618A01N1/125A61K35/30C12N2500/38C12N2500/40C12N2501/13C12N2501/15C12N2501/155C12N2501/16C12N2501/41C12N2501/42C12N2501/727C12N2506/45

Abstract

Claims (41)

1 . A method of differentiating pluripotent stem cells into dopaminergic neuronal progenitor cells, the method comprising:

a) performing a first incubation comprising non-adherently culturing pluripotent stem cells in a first multiwell culture vessel under conditions to produce a cellular spheroid, wherein the first incubation comprises:

(i) exposing the pluripotent stem cells to at least one inhibitor of TGF-β/activin-Nodal signaling and at least one inhibitor of bone morphogenetic protein (BMP) signaling for at least one day, wherein Day 0 is the first day on which the pluripotent stem cells are exposed to the inhibitor of TGF-β/activin-Nodal signaling and the inhibitor of bone morphogenetic protein (BMP) signaling, in the absence of: x) an activator of Sonic Hedgehog (SHH) signaling, and y) an inhibitor of glycogen synthase kinase 3ß (GSK3ß) signaling; and

(ii) starting on Day 1 of the first incubation, wherein Day 1 is one day after the first day that the pluripotent stem cells are first exposed to the inhibitor of TGF-β/activin-Nodal signaling and the inhibitor of bone morphogenetic protein (BMP) signaling, exposing the cells obtained from step (i) to at least one activator of Sonic Hedgehog (SHH) signaling and at least one inhibitor of glycogen synthase kinase 3β (GSK3β) to produce the cellular spheroid; and

b) performing a second incubation comprising adherently culturing cells of the spheroid in a second culture vessel under conditions to further differentiate the cells into dopaminergic neuronal progenitor cells;

wherein the dopaminergic neuronal progenitor cells exhibit increased expression of FOXA2 and CORIN and decreased expression of NKX2.1 and PITX2 compared to cells differentiated in a method that comprises performing the first incubation in adherent culture.

2 . The method of claim 1 , wherein the pluripotent stem cells are induced pluripotent stem cells.

3 . The method of claim 1 , wherein the pluripotent stem cells are autologous to a subject to be treated with the dopaminergic neuronal progenitor cells.

4 . The method of claim 1 , wherein the first incubation further comprises exposing the pluripotent stem cells to a ROCK inhibitor (ROCKi) starting on Day 0.

5 . The method of claim 4 , wherein the pluripotent stem cells were not exposed to a ROCKi prior to exposing the pluripotent stem cells to the inhibitor of TGF-β/activin-Nodal signaling and the inhibitor of bone morphogenetic protein (BMP) signaling in the first incubation.

6 . The method of claim 1 , wherein the method comprises exposing the pluripotent stem cells to:

a) the inhibitor of TGF-β/activin-Nodal signaling beginning on Day 0 and through Day 4;

b) the inhibitor of BMP signaling beginning on Day 0 and through Day 10;

c) the activator of Sonic Hedgehog signaling beginning on Day 1 and through Day 6; and

d) the inhibitor of glycogen synthase kinase 3ß (GSK3B) signaling beginning on Day 1 and through Day 12.

7 . The method of claim 1 , wherein the inhibitor of BMP signaling is LDN193189.

8 . The method of claim 7 , wherein the cells are exposed to LDN193189 at a concentration of between 10 nM and 500 nM, between 20 nM and about 400 nM, between 50 nM and 200 nM, or between 75 nM and 150 nM, optionally 100 nM.

9 . The method of claim 1 , wherein the inhibitor of TGF-β/activin-Nodal signaling is SB431542.

10 . The method of claim 9 , wherein the cells are exposed to SB431542 at a concentration of between 1 μM and 20 μM, between 5 μM and 15 μM, or between 8 μM and 12 μM, optionally 10 μM.

11 . The method of claim 1 , wherein the activator of SHH signaling is SHH or purmorphamine.

12 . The method of claim 11 , wherein the cells are exposed to SHH at a concentration of between 10 ng/mL and 500 ng/mL, between 20 ng/ml and 400 ng/mL, between 50 ng/mL and 200 ng/mL, or between 75 ng/mL and about 150 ng/mL, about 100 ng/mL.

13 . The method of claim 11 , wherein the cells are exposed to purmorphamine at a concentration of between 0.1 μM and 20 μM, between 0.5 μM and 10 μM, between 1 μM and about 5 μM, between 1 μM and 3 μM, or between 1.5 μM and 2.5 μM, optionally at 2 μM.

14 . The method of claim 1 , wherein the inhibitor of GSK3β signaling is CHIR99021.

15 . The method of claim 14 , wherein the cells are exposed to CHIR99021 at a concentration of between 0.1 μM and 5 μM, between 0.5 μM and 4 μM, between 0.5 UM and 2 μM, optionally 1 μM; and on each of Days 2 through 12, the cells are exposed to CHIR99021 at a concentration of between 0.1 μM and 5 μM, between 0.5 μM and 4 μM, or between 1 μM and 3 μM, optionally 2 μM.

16 . The method of claim 1 , wherein the first incubation comprises a media exchange on one or more of Days 1 through 6.

17 . The method of claim 16 , wherein the first incubation comprises a media exchange on each of Days 1 through 6.

18 . The method of claim 1 , wherein the second incubation begins on Day 7.

19 . The method of claim 1 , wherein the cells of the spheroid are disassociated to produce a cell suspension prior to the second incubation, and cells of the cell suspension are adherently cultured in the second culture vessel.

20 . The method of claim 1 , wherein the second incubation comprises exposing the cells of the spheroid to an inhibitor of bone morphogenetic protein (BMP) signaling and an inhibitor of GSK3β signaling.

21 . The method of claim 20 , wherein the second incubation further comprises exposing the cells to (i) brain-derived neurotrophic factor (BDNF); (ii) ascorbic acid; (iii) glial cell-derived neurotrophic factor (GDNF); (iv) dibutyryl cyclic AMP (dbcAMP); (v) transforming growth factor beta-3 (TGFβ3); and (vi) an inhibitor of Notch signaling.

22 . The method of claim 1 , further comprising harvesting the dopaminergic neuronal progenitor cells.

23 . The method of claim 22 , wherein the dopaminergic neuronal progenitor cells are harvested on Day 14 or later.

24 . The method of claim 22 , further comprising formulating the harvested dopaminergic neuronal progenitor cells with a cryoprotectant.

25 . The method of claim 24 , further comprising cryopreserving the formulated harvested dopaminergic neuronal progenitor cells.

26 . The method of claim 1 , wherein each well of the first multiwell culture vessel comprises a plurality of microwells.

27 . The method of claim 1 , wherein the dopaminergic neuronal progenitor cells:

a) express Forkhead Box A2 (FOXA2) at greater than 100 transcripts per million (TPM) in bulk RNA sequencing (RNAseq) analysis;

b) express Corin, serine peptidase (CORIN) at greater than 300 TPM in bulk RNAseq analysis;

c) express Paired Like Homeodomain 2 (PITX2) at less than 50 TPM in bulk RNAseq analysis; and

d) express NK2 Homeobox 1 (NKX2.1) at less than 10 TPM in bulk RNAseq analysis.

28 . The method of claim 1 , wherein the dopaminergic neuronal progenitor cells produce significantly less serotonin compared to cells differentiated in a method that comprises performing the first incubation in adherent culture.

Assignments (1)

Continuity (2)

References Cited (135)