Differentiation of human pluripotent stem cells to multipotent neural crest cells

• US 10,053,667 B2
• Filed: 01/19/2016
• Issued: 08/21/2018
• Est. Priority Date: 12/31/2010
• Status: Active Grant

• Current Assignee: The University of Georgia Research Foundation, Inc.
• Original Assignee: The University of Georgia Research Foundation, Inc.
• Inventors: Dalton, Stephen, Menendez, Laura M.
• Primary Examiner(s): Leavitt, Maria G
• Application Number:
  US15/000,382
• Publication Number:
  US 20160237405A1
• Time in Patent Office: 945 Days
• Field of Search: 424 937
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• 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 5/0623 Stem cells

Abstract

The present invention relates to the differentiation of human pluripotent cells, including human pluripotent stems cells to produce a self-renewing multipotent neural crest cell population in a single step method without the requirement of isolation of intermediate cells and without appreciable contamination (in certain preferred instances, virtually none) with Pax6+ neural progenitor cells in the population of p75+ Hnk1+ Ap2+ multipotent neural crest-like cells. The multipotent neural crest cell population obtained can be clonally amplified and maintained for >25 passages (>100 days) while retaining the capacity to differentiate into peripheral neurons, smooth muscle cells and mesenchymal precursor cells.

Claims

1. A method of producing p75+ Hnk1+ Ap2+ multipotent neural crest-like cells from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) comprising differentiating said stem cells in a differentiation medium in the absence of feeder cells consisting essentially of an effective amount of a glycogen synthase kinase (GSK) inhibitor in combination with an effective amount of an Activin A inhibitor to produce a population of cells comprising p75+ Hnk1+ Ap2+ multipotent neural crest-like stem cells and PAX6+ neural progenitor cells, wherein said GSK inhibitor is (2′

Z,3′

E)-6-Bromoindimbin-3′

-oxime (BIO) and said Activin A inhibitor is SB 431542 and wherein said PAX6+ neural progenitor cells produced comprise no more than 10% of the total population of p75+ Hnk1+ Ap2+ multipotent neural crest-like cells and PAX6+ neural progenitor cells; and

optionally isolating said neural p75+ Hnk1+ Ap2+ multipotent neural crest-like cells from said population of cells.

2. The method according to claim 1 wherein hESCs are differentiated into said neural p75+ Hnk1+ Ap2+ multipotent neural crest-like cells.

3. The method according to claim 1 wherein hiPSCs are differentiated into said neural p75+ Hnk1+ Ap2+ multipotent neural crest-like cells.

4. The method according to claim 1 wherein said neural p75+ Hnk1+ Ap2+ multipotent neural crest-like cells are isolated from said population of cells.

5. The method according to claim 2 wherein said neural p75+ Hnk1+ Ap2+ multipotent neural crest-like cells are isolated from said population of cells.

6. The method according to claim 3 wherein said neural p75+ Hnk1+ Ap2+ multipotent neural crest-like cells are isolated from said population of cells.

7. The method according to claim 1 wherein said differentiation medium further includes an effective amount of at least one bone morphogenic inhibitor selected from the group consisting of noggin, chordin, sclerostin, follistatin, gremlin, dorsomorphorin and connective tissue growth factor (CTFG).

8. The method according to claim 2 wherein said differentiation medium further includes an effective amount of at least one hone morphogenic inhibitor selected from the group consisting of noggin, chordin, sclerostin, follistatin, gremlin, dorsomorphorin and connective tissue growth factor (CTFG).

9. The method according to claim 3 wherein said differentiation medium further includes an effective amount of at least one bone morphogenic inhibitor selected from the group consisting of noggin, chordin, sclerostin, follistatin, gremlin, dorsomorphorin and connective tissue growth factor (CTFG).

10. The method according to claim 4 wherein said differentiation medium further includes an effective amount of at least one bone morphogenic inhibitor selected from the group consisting of noggin, chordin, sclerostin, follistatin, gremlin, dorsomorphorin and connective tissue growth factor (CTFG).

11. The method according to claim 5 wherein said differentiation medium further includes an effective amount of at least one bone morphogenic inhibitor selected from the group consisting of noggin, chordin, sclerostin, follistatin, gremlin, dorsomorphorin and connective tissue growth factor (CTFG).

12. The method according to claim 6 wherein said differentiation medium further includes an effective amount of at least one bone morphogenic inhibitor selected from the group consisting of noggin, chordin, sclerostin, follistatin, gremlin, dorsomorphorin and connective tissue growth factor (CFTG).

13. A method of producing p75+ Hnk1+ Ap2+ multipotent neural crest-like cells from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) comprising differentiating said stem cells in a differentiation medium in the absence of feeder cells consisting essentially of an effective amount of a Wnt protein in combination with an effective amount of an Activin A inhibitor to produce a population of cells comprising p75+ Hnk1+ Ap2+ multipotent neural crest-like stem cells and PAX6+ neural progenitor cells, wherein said Wnt protein is Wnt3a and said Activin A inhibitor is SB 431542 and wherein said PAX6+ neural progenitor cells produced comprise no more than 10% of the total population of p75+ Hnk1+ Ap2+ multipotent neural crest-like cells and PAX6+ neural progenitor cells;

and optionally isolating said neural p75+ Hnk1+ Ap2+ multipotent neural crest-like cells from said population of cells.

14. The method according to claim 13 wherein hESCs are differentiated into said neural p75+ Hnk1+ Ap2+ multipotent neural crest-like cells.

15. The method according to claim 13 wherein hiPSCs are differentiated into said neural p75+ Hnk1+ Ap2+ multipotent neural crest-like cells.

16. The method according to claim 13 wherein said neural p75+ Hnk1+ Ap2+ multipotent neural crest-like cells are isolated from said population of cells.

17. The method according to claim 14 wherein said neural p75+ Hnk1+ Ap2+ multipotent neural crest-like cells are isolated from said population of cells.

18. The method according to claim 15 wherein said neural p75+ Hnk1+ Ap2+ multipotent neural crest-like cells are isolated from said population of cells.

19. The method according to claim 13 wherein said differentiation medium further includes an effective amount of at least one bone morphogenic inhibitor selected from the group consisting of noggin, chordin, sclerostin, follistatin, gremlin, dorsomorphorin and connective tissue growth factor (CTFG).

20. The method according to claim 14 wherein said differentiation medium further includes an effective amount of at least one bone morphogenic inhibitor selected from the group consisting of noggin, chordin, sclerostin, follistatin, gremlin, dorsomorphorin and connective tissue growth factor (CTFG).

21. The method according to claim 15 wherein said differentiation medium further includes an effective amount of at least one bone morphogenic inhibitor selected from the group consisting of noggin, chordin, sclerostin, follistatin, gremlin, dorsomorphorin and connective tissue growth factor (CTFG).

22. The method according to claim 16 wherein said differentiation medium further includes an effective amount of at least one bone morphogenic inhibitor selected from the group consisting of noggin, chordin, sclerostin, follistatin, gremlin, dorsomorphorin and connective tissue growth factor (CTFG).

23. The method according to claim 17 wherein said differentiation medium further includes an effective amount of at least one bone morphogenic inhibitor selected from the group consisting of noggin, chordin, sclerostin, follistatin, gremlin, dorsomorphorin and connective tissue growth factor (CTFG).

24. The method according to claim 18 wherein said differentiation medium further includes an effective amount of at least one bone morphogenic inhibitor selected from the group consisting of noggin, chordin, sclerostin, follistatin, gremlin, dorsomorphorin and connective tissue growth factor (CTFG).