Methods for differentiating dopaminergic neurons from stem cells

Abstract

This disclosure provides methods for differentiating pluripotent stem cells, including induced pluripotent stem cells, into lineage-specific floor plate midbrain progenitor cells, determined dopaminergic neuron progenitor cells, fate-determined dopaminergic neuron progenitor cells, and / or dopaminergic neuron cells. Also provided are the use of the composition, such as for treating neurodegenerative diseases and conditions, including Parkinson's disease, as well as products and kits for such use.

Claims

1. A method for differentiating pluripotent stem cells into dopaminergic neuronal progenitor cells, wherein the method is a) Performing a first incubation, which includes culturing pluripotent stem cells non-adherently in a first culture vessel under conditions that produce cell spheroids, wherein the first incubation is 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 (day 0) in the absence of x) an activator of sonic hedgehog (SHH) signaling and y) an inhibitor of glycogen synthase kinase 3β (GSK3β) signaling, ii) Performing a first incubation, which begins on the second day (first day) of the first incubation, and includes exposing the pluripotent stem cells to at least one activator of sonic hedgehog (SHH) signaling and at least one inhibitor of glycogen synthase kinase 3β (GSK3)β) signaling, (b) A method comprising carrying out a second incubation, which further includes adhering the spheroid cells in a second culture vessel under conditions that differentiate the cells into dopaminergic neuronal progenitor cells.

2. The method according to claim 1, wherein the dopaminergic neuron progenitor cell is a determined dopaminergic neuron progenitor cell.

3. The method according to claim 1, wherein the pluripotent stem cells are induced pluripotent stem cells.

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

5. The method according to claim 1, further comprising exposing the pluripotent stem cells to a ROCK inhibitor (ROCKi) starting on day 0.

6. The method according to claim 5, wherein the pluripotent stem cells were not exposed to ROCKi before being exposed to the TGF-β / activin-Nodal signaling inhibitor and the bone morphogenetic protein (BMP) signaling inhibitor in the first incubation.

7. The above method provides the pluripotent stem cells, a) Starting on day 0 and continuing until day 4, the TGF-β / activin-Nodal signaling inhibitor, b) Starting on day 0 and continuing until day 10, the BMP signaling inhibitor c) Starting on day 1 and continuing until day 6, the activator of the sonic hedgehog signaling pathway, d) The method according to claim 1, comprising exposure to the glycogen synthase kinase 3β (GSK3β) signaling inhibitor starting on day 1 and continuing until day 12.

8. The method according to claim 1, wherein the BMP signaling inhibitor is LDN193189.

9. The method according to claim 8, wherein the cells are exposed to LDN193189 at concentrations of approximately 10 nM to 500 nM, approximately 20 nM to approximately 400 nM, approximately 50 nM to approximately 200 nM, or approximately 75 nM to approximately 150 nM, or optionally approximately 100 nM.

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

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

12. The method according to claim 1, wherein the activator of the SHH signaling pathway is SHH or palmorfamine.

13. The method according to claim 12, wherein the cells are exposed to SHH at concentrations of approximately 10 ng / mL to 500 ng / mL, approximately 20 ng / mL to approximately 400 ng / mL, approximately 50 ng / mL to approximately 200 ng / mL, or approximately 75 ng / mL to approximately 150 ng / mL, or optionally approximately 100 ng / mL.

14. The method according to claim 12, wherein the cells are exposed to palmorfamine at concentrations of approximately 0.1 μM to approximately 20 μM, approximately 0.5 μM to approximately 10 μM, approximately 1 μM to approximately 5 μM, approximately 1 μM to approximately 3 μM, or approximately 1.5 μM to approximately 2.5 μM, or optionally approximately 2 μM.

15. The method according to claim 1, wherein the inhibitor of GSK3β signaling is CHIR99021.

16. The method according to claim 15, wherein the cells are exposed to CHIR99021 at concentrations of approximately 0.1 μM to approximately 5 μM, approximately 0.5 μM to approximately 4 μM, approximately 0.5 μM to approximately 2 μM, and optionally approximately 1 μM, and on each of days 2 to 12, the cells are exposed to CHIR99021 at concentrations of approximately 0.1 μM to approximately 5 μM, approximately 0.5 μM to approximately 4 μM, or approximately 1 μM to approximately 3 μM, and optionally approximately 2 μM.

17. The method according to claim 1, wherein the first incubation includes changing the culture medium on one or more days between day 1 and day 6.

18. The method according to claim 17, wherein the first incubation includes changing the culture medium on each of days 1 to 6.

19. The method according to claim 1, wherein the second incubation begins on the seventh day or approximately the seventh day.

20. The method according to claim 1, wherein the cells of the spheroid are dissociated before the second incubation to produce a cell suspension, and the cells of the cell suspension are cultured adherently in the second culture vessel.

21. The method according to 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.

22. The method according to claim 21, further comprising 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) (collectively, "BAGCT"), and (vi) an inhibitor of Notch signaling.

23. The method according to claim 1, further comprising collecting the dopaminergic neuronal progenitor cells.

24. The method according to claim 23, wherein the dopaminergic neuronal progenitor cells are collected from day 14 onward.

25. The method according to claim 23, further comprising formulating the collected dopaminergic neuronal progenitor cells together with a cryoprotectant.

26. The method according to claim 25, further comprising cryopreserving the formulated, collected dopaminergic neuronal progenitor cells.

27. a) Performing a first incubation, which includes culturing pluripotent stem cells non-adherently in a first culture vessel under conditions that produce cell spheroids, wherein the first incubation is 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 (day 0) in the absence of x) an activator of sonic hedgehog (SHH) signaling and y) an inhibitor of glycogen synthase kinase 3β (GSK3β) signaling, ii) Performing a first incubation, which includes exposing the pluripotent stem cells to at least one activator of sonic hedgehog (SHH) signaling and at least one inhibitor of glycogen synthase kinase 3β (GSK3)β) signaling, starting on the second day (first day) of the first incubation. (b) A therapeutic composition comprising dopaminergic neuronal progenitor cells produced by a method comprising carrying out a second incubation, which further includes adhering the spheroid cells in a second culture vessel under conditions that differentiate the cells into dopaminergic neuronal progenitor cells.

28. The therapeutic composition, compared to neuronal cells produced using an adherent culture differentiation method, a) To express a higher level of FOXA2, b) Expressing a lower level of PAX6, c) The predicted graft size after transplantation is larger. d) Higher predicted dopamine production levels after transplantation. e) To produce less serotonin, f) Containing a higher percentage of living cells, g) To express a higher level of CORIN, h) Expressing PITX2 at a lower level than, and The therapeutic composition according to claim 27, comprising dopaminergic neuronal progenitor cells exhibiting one or more characteristics selected from the group consisting of i) expressing a lower level of NKX2.

1.

29. The therapeutic composition according to claim 28, wherein the therapeutic composition comprises dopaminergic neuronal progenitor cells that exhibit three or more characteristics selected from the group compared to neuronal cells produced using an adherent culture differentiation method.

30. The therapeutic composition is a) Serotonin production at levels below 2 nM increases less than 2 times when stimulated with KCl compared to the unstimulated baseline. b) More than 90% of the dopaminergic neuronal progenitor cells in the composition are viable. c) FOXA2 expression at over 100 TPM in bulk RNA seq analysis, d) CORIN is expressed at over 300 TPM in bulk RNA seq analysis. e) Expressing PITX2 at less than 50 TPM in bulk RNA seq analysis, f) Expressing NKX2.1 at less than 10 TPM in bulk RNA seq analysis, g) More than 90% of FOXA2-positive cells, and The therapeutic composition according to claim 27, comprising dopaminergic neuronal progenitor cells exhibiting one or more characteristics selected from the group consisting of having a GraftTest™ score of at least 1500.

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