Kits for neural conversion of human embryonic stem cells

• US 10,287,546 B2
• Filed: 01/31/2014
• Issued: 05/14/2019
• Est. Priority Date: 02/17/2009
• Status: Active Grant

• Current Assignee: Memorial Sloan-Kettering Cancer Center
• Original Assignee: Memorial Sloan-Kettering Cancer Center
• Inventors: Chambers, Stuart, Studer, Lorenz
• Primary Examiner(s): Gamett, Daniel C
• Application Number:
  US14/169,286
• Publication Number:
  US 20170159012A1
• Time in Patent Office: 1,929 Days
• Field of Search: None
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/41 Hedgehog proteins; Cyclopamine (inhibitor)
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/60 Transcription factors
• CPC Class Codes: C12N 2501/998 Proteins not provided for elsewhere
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2506/45 from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 5/0619 Neurons

Abstract

The present invention relates generally to the field of cell biology of stem cells, more specifically the directed differentiation of pluripotent or multipotent stem cells, including human embryonic stem cells (hESC), somatic stem cells, and induced human pluripotent stem cells (hiPSC) using novel culture conditions. Specifically, methods are provided for obtaining neural tissue, floor plate cells, and placode including induction of neural plate development in hESCs for obtaining midbrain dopamine (DA) neurons, motorneurons, and sensory neurons. Further, neural plate tissue obtained using methods of the present inventions are contemplated for use in co-cultures with other tissues as inducers for shifting differentiation pathways, i.e. patterning.

Claims

1. A kit, comprising:

a first inhibitor of Small Mothers Against Decapentaplegic (SMAD) protein signaling (“

first SMAD inhibitor”

) that comprises an inhibitor of bone morphogenetic protein (BMP) signaling,

a second inhibitor of Small Mothers Against Decapentaplegic (SMAD) protein signaling (“

second SMAD inhibitor”

) that comprises an inhibitor of TGFβ

/Activin-Nodal signaling, and

an activator of Sonic Hedgehog (SHH) signaling that comprises a SHH protein.

2. The kit of claim 1, further comprising instructions for inducing differentiation of pluripotent stem cells.

3. The kit of claim 2, wherein said instructions comprise exposing a plurality of pluripotent stem cells to said first and second SMAD inhibitors to produce a cell population comprising at least about 10% differentiated cells expressing CDX2, wherein said exposing to said first SMAD inhibitor is discontinued after about one day from the initial exposure of said pluripotent stem cells to said first SMAD inhibitor.

4. The kit of claim 3, wherein said differentiated cells are trophectodermal precursors.

5. The kit of claim 2, wherein said instructions comprise exposing a plurality of pluripotent stem cells to the first and second SMAD inhibitors to produce a cell population comprising at least about 10% differentiated cells expressing SIX1,wherein said exposing to said first SMAD inhibitor is discontinued after about two days or about three days from the initial exposure of said pluripotent stem cells to said first SMAD inhibitor.

6. The kit of claim 5, wherein said differentiated cells are placodal precursors.

7. The kit of claim 2, wherein said instructions comprise exposing a plurality of pluripotent stem cells to said first and second SMAD inhibitors to produce a cell population comprising at least about 10% differentiated cells expressing paired box homeotic gene-6 (PAX6).

8. The kit of claim 7, wherein said exposing comprises exposing said pluripotent stem cells to said first and second SMAD inhibitors for at least about 10 days.

9. The kit of claim 8, wherein said exposing comprises exposing said pluripotent stem cells to said first and second SMAD inhibitors for about 10days or about 11 days.

10. The kit of claim 1, wherein said SHH protein comprises a modified recombinant protein at least about 95% identical to a mouse Sonic Hedgehog N-terminal fragment.

11. The kit of claim 2, wherein said instructions comprise exposing a plurality of pluripotent stem cells to said first and second SMAD inhibitors and exposing said cells to the activator of SHH signaling to produce a cell population comprising at least about 10% differentiated cells expressing one or more marker selected from the group consisting of forkhead box protein A2 (FOXA2), SSH, Netrin-1, and F-Spondin.

12. The kit of claim 11, wherein said instructions comprise initially exposing said cells to said activator of SHH signaling within about 60 hours from the initial exposure of the pluripotent stem cells to said first and second SMAD inhibitors.

13. The kit of claim 11, wherein said differentiated cells are neural floor plate cells.

14. The kit of claim 13, wherein said differentiated cells are anterior floor plate cells.

15. The kit of claim 11, wherein said instructions comprise initially exposing said cells to said activator of SHH signaling no later than about 7 days from the initial exposure of said pluripotent stem cells to said first SMAD inhibitor.

16. The kit of claim 15, wherein said instructions comprise initially exposing said cells to said activator of SHH signaling about one day from the initial exposure of said pluripotent stem cells to said first SMAD inhibitor.

17. The kit of claim 1, further comprising one or more caudalizing factor.

18. The kit of claim 17, wherein said one or more caudalizing factor is selected from the group consisting of wingless-type MMTV integration site family member 1 (Wnt-I), Fibroblast Growth Factor 8 (FGF8) and Retinoic Acid (RA).

19. The kit of claim 17, further comprising instructions for inducing differentiation of pluripotent stem cells, wherein said instructions comprise exposing a plurality of pluripotent stem cells to said first and second SMAD inhibitors, and exposing said cells to said activator of SHH signaling and the one or more caudalizing factor to produce a cell population comprising at least about 10% differentiated cells expressing one or more marker selected from the group consisting of serine peptidase (CORIN), LIM homeobox transcription factor 1, beta (LMX1B), EN1, neurogenin 2 (NGN2), and nephroblastoma overexpressed gene (NOV).

20. The kit of claim 19, wherein said differentiated cells are posterior floor plate cells, dopaminergic progenitors, or a combination thereof.

21. The kit of claim 19, wherein said differentiated cells are midbrain floor plate cells.

22. The kit of claim 17, further comprising instructions for inducing differentiation of pluripotent stem cells, wherein said instructions comprise exposing a plurality of pluripotent stem cells to said first and second SMAD inhibitors, and exposing said cells to said activator of SHH signaling and said one or more caudalizing factor to produce a cell population comprising at least about 10% differentiated cells expressing Tyrosine hydroxylase (TH).

23. The kit of claim 22, wherein said differentiated cells are dopaminergic cells.

24. The kit of claim 22, further comprising brain-derived neurotrophic factor (BDNF), ascorbic acid, or a combination thereof, and wherein said caudalizing factor is FGF8.

25. The kit of claim 17, further comprising instructions for inducing differentiation of pluripotent stem cells, wherein said instructions comprise exposing a plurality of pluripotent stem cells to said first and second SMAD inhibitors, and exposing said cells to said activator of SHE signaling and said one or more caudalizing factor to produce a cell population comprising at least about 10% differentiated cells expressing one or more marker selected from the group consisting of ISL1 and HB9.

26. The kit of claim 25, wherein said differentiated cells are motor neuronal cells.

27. The kit of claim 25, further comprising BDNF, ascorbic acid, or a combination thereof, and wherein said caudalizing factor is RA.

28. The kit of claim 1, wherein said inhibitor of TGFβ

/Activin-Nodal signaling comprises 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl] benzamide (SB431542).

29. The kit of claim 1, wherein said inhibitor of BMP signaling is selected from the group consisting of Noggin, a disulfide-linked homodimer of Noggin, Dorsomorphin, LDN-193189, and mixtures thereof.

30. The kit of claim 29, wherein said inhibitor of BMP signaling is LDN-193189.

31. The kit of claim 1, wherein said SHH protein is selected from the group consisting of Sonic hedgehog (SHH) and Sonic C25II.