Method of nociceptor differentiation of human embryonic stem cells and uses thereof

Patent metadata

Field	Value
Publication number	US-9453198-B2
Application number	US-201113697274-A
Country	US
Kind code	B2
Filing date	May 19, 2011
Priority date	May 25, 2010
Publication date	Sep 27, 2016
Grant date	Sep 27, 2016

Abstract

The present invention relates to the field of stem cell biology, in particular the linage specific differentiation of pluripotent or multipotent stem cells, which can include, but is not limited to, human embryonic stem cells (hESC), human induced pluripotent stem cells (hiPSC), somatic stem cells, cancer stem cells, or any other cell capable of lineage specific differentiation. Specifically described are methods to direct the lineage specific differentiation of hESC and/or hiPSC to nociceptors (i.e. nociceptor cells) using novel culture conditions. The nociceptors made using the methods of the present invention are further contemplated for various uses including, but limited to, use in in vitro drug discovery assays, pain research, and as a therapeutic to reverse disease of, or damage to, the peripheral nervous system (PNS). Further, compositions and methods are provided for producing melanocytes from human pluripotent stem cells for use in disease modeling.

First claim

The invention claimed is:

1. A kit comprising a first signaling inhibitor, a second signaling inhibitor and a third signaling inhibitor, wherein said first inhibitor is capable of lowering transforming growth factor beta (TGFβ)/Activin-Nodal signaling, said second inhibitor is capable of lowering Small Mothers Against Decapentaplegic (SMAD) signaling and said third inhibitor is capable of lowering glycogen synthase kinase 3β (GSK3β) for activation of wingless (Wnt) signaling, further comprising instructions for inducing directed differentiation of a pluripotent stem cell into a differentiated neural crest lineage cell or a neuronal lineage cell, in vitro, comprising (i) contacting said stem cell with said first and said second inhibitors for up to 96 hours and (ii) contacting said stem cell with said third inhibitor for up to 240 hours.

2. The kit of claim 1 , wherein said first inhibitor is SB431542.

3. The kit of claim 1 , wherein said second inhibitor is LDN193189.

4. The kit of claim 1 , wherein said third inhibitor is CHIR99021.

5. A kit comprising a first signaling inhibitor, a second signaling inhibitor, and a third signaling inhibitor, wherein said first inhibitor is capable of lowering transforming growth factor beta (TGFβ)/Activin-Nodal signaling, said second inhibitor is capable of lowering Small Mothers Against Decapentaplegic (SMAD) signaling, and said third inhibitor is capable of lowering glycogen synthase kinase 3β (GSK3β) for activation of wingless (Wnt) signaling, further comprising a fourth inhibitor that lowers fibroblast growth factor (FGF) receptor family signaling, wherein said FGF receptor family signaling comprising vascular endothelial growth factor (VEGF) receptor signaling, fibroblast growth factor (FGF) receptor signaling, and platelet-derived growth factor (PDGF) tyrosine kinase receptor signaling.

6. The kit of claim 5 , wherein said fourth inhibitor is SU5402.

7. A kit comprising a first signaling inhibitor, a second signaling inhibitor and a third signaling inhibitor, wherein said first inhibitor is capable of lowering transforming growth factor beta (TGFβ)/Activin-Nodal signaling, said second inhibitor is capable of lowering Small Mothers Against Decapentaplegic (SMAD) signaling and said third inhibitor is capable of lowering glycogen synthase kinase 3β (GSK3β) for activation of wingless (Wnt) signaling, further comprising an inhibitor capable of lowering Notch signaling.

8. The kit of claim 7 , wherein said inhibitor that is capable of lowering Notch signaling is selected from the group consisting of N—[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPI) and (N—[N-(3,5-difluorophenacetyl)-L-alanyl]-(S)-phenylglycine-4-(4-(8 biotinamido)octylamino)benzoyl)benzyl)methylamide).

9. The kit of claim 1 , further comprising, one or more antibody used for the detection of expression of one or more protein(s) selected from the group consisting of nestin, OCT4, PAX6, TUJ1, SOX10, NTRK1, ISL1, POU4F1 (BRN3A), NEUROG2, NEUROG1, MAP2, OTX2, DLK1, DKK1, CUZD1, MSX1, ID2, AP2B, ETS1, FOXD3, NGN1, DCX, TUBB3, SYT4, STMN2, INA, GAP43, TAC1, VGLUT2, SLC15A3, and TRPV1.

10. The kit of claim 1 , further comprising, one or more pair of PCR primers for the detection of mRNA expression of one or more gene(s) selected from the group consisting of nestin, OCT4, PAX6, TUJ1, SOX10, NTRK1, ISL1, POU4F1 (BRN3A), NEUROG2, NEUROG1, MAP2, OTX2, DLK1, DKK1, CUZD1, MSX1, ID2, AP2B, ETS1, FOXD3, NGN1, DCX, TUBB3, SYT4, STMN2, INA, GAP43, TAC1, VGLUT2, SLC15A3, and TRPV1.

11. The kit of claim 1 , further comprising, one or more antibody used for the detection of expression of one or more protein(s) selected from the group consisting of Protachykinin-1 (TAC1), vesicular glutamate transporter 2 (VGLUT2) and solute carrier family 15, member 3 (SLC15A3).

12. The kit of claim 1 , further comprising, one or more pair of PCR primers for the detection of mRNA expression of one or more gene(s) selected from the group consisting of Protachykinin-1 (TAC1), vesicular glutamate transporter 2 (VGLUT2) and solute carrier family 15, member 3 (SLC15A3).

13. The kit of claim 1 , further comprising, instructions comprising steps for adding the first and second inhibitor two days before adding the third inhibitor.

14. The kit of claim 1 , further comprising, instructions comprising steps for making neural stem cell precursors and making nociceptor cells.

15. The kit of claim 1 , further comprising a human pluripotent stem cell.

16. The kit of claim 15 , wherein said human pluripotent stem cell is a human embryonic stem cell.

17. The kit of claim 15 , wherein said human pluripotent stem cell is a human induced pluripotent stem cell.

18. The kit of claim 13 , wherein said human pluripotent stem cell is a transgenic human pluripotent stem cell (hPSC) containing a bacterial artificial chromosome (BAC) comprising a SOX10 gene having an inserted reporter sequence encoding a green fluorescent protein.

19. A method for inducing directed differentiation of a pluripotent stem cell, comprising
a) providing, in vitro:
i) a cell culture comprising human pluripotent stem cells; and
ii) a first signaling inhibitor, a second signaling inhibitor and a third signaling inhibitor, wherein said first inhibitor is capable of lowering transforming growth factor beta (TGFβ)/Activin-Nodal signaling, said second inhibitor is capable of lowering Small Mothers Against Decapentaplegic (SMAD) signaling and said third inhibitor is capable of lowering glycogen synthase kinase 3β (GSK3β) for activation of wingless (Wnt) signaling;

b) contacting said stem cell with said first and said second inhibitors for up to 96 hours; and
c) contacting said stem cell with said third inhibitor for up to 240 hours;
wherein a differentiated neural crest lineage cell or a neuronal lineage cell is produced.

20. The method of claim 19 , wherein said first inhibitor is SB431542.

21. The method of claim 19 , wherein said second inhibitor is LDN193189.

22. The method of claim 19 , wherein said third inhibitor is CHIR99021.

23. The method of claim 19 , further comprising contacting said stem cell with a fourth inhibitor that lowers fibroblast growth factor (FGF) receptor family signaling, wherein said FGF receptor family signaling comprising vascular endothelial growth factor (VEGF) receptor signaling, fibroblast growth factor (FGF) receptor signaling, and platelet-derived growth factor (PDGF) tyrosine kinase receptor signaling.

24. The method of claim 23 , wherein said fourth inhibitor is SU5402.

25. The method of claim 23 , further comprising contacting said stem cell with a fifth inhibitor capable of lowering Notch signaling, wherein a neural crest lineage cell is produced.

26. The method of claim 25 , wherein said fifth inhibitor is selected from the group consisting of N—[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) and (N—[N-(3,5-difluorophenacetyl)-L-alanyl]-(S)-phenylglycine-4-(4-(8 biotinamido)octylamino)benzoyl)benzyl)methylamide).

27. The method of claim 19 , further comprising contacting said stem cell with a fourth inhibitor and a fifth inhibitor, wherein said fourth inhibitor is SU5402 and said fifth inhibitor is N—[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) for directed differentiated of a neural crest lineage cell into a peptidergic nociceptor cell.

Assignees

• Studer Lorenz
• Chambers Stuart M
• Qi Yuchen
• Mica Yvonne Marissa
• Memorial Sloan Kettering Cancer Center

Inventors

• Studer Lorenz
• Chambers Stuart M
• Qi Yuchen
• Mica Yvonne Marissa

Classifications

• A61P43/00

  Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 · CPC title

• A61P25/02

  for peripheral neuropathies · CPC title

• C12N2506/02

  from embryonic cells · CPC title

• C12N5/062 Primary

  Sensory transducers, e.g. photoreceptors; Sensory neurons, e.g. for hearing, taste, smell, pH, touch, temperature, pain · CPC title

• C12N2501/15

  Transforming growth factor beta (TGF-β) · CPC title