Method of in vitro differentiation of neural stem cells, motor neurons and dopamine neurons from primate embryonic stem cells

• US 20050095706A1
• Filed: 08/27/2004
• Published: 05/05/2005
• Est. Priority Date: 10/03/2001
• Status: Active Grant

• Current Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Original Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Inventors: Thomson, James A., Zhang, Su-Chun, Li, Xue-jun, Duncan, Ian David
• Granted Patent: US 7,588,937 B2
• Time in Patent Office: Days
• US Class Current: 435/368
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/119 Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
• CPC Class Codes: C12N 2501/392 Sexual steroids
• CPC Class Codes: C12N 2501/91 Heparin
• CPC Class Codes: C12N 2502/13 connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0623 Stem cells

Abstract

A method of differentiating embryonic stem cells into neural and motor cells is disclosed. In one embodiment, the invention comprises culturing a population of cells comprising a majority of cells that are characterized by an early rosette morphology and are Sox1⁻/Pax6⁺ in the presence of FGF2, FGF4, FGF8, FGF 9, or RA wherein the cells are characterized by an neural tube-like rosette morphology and are Pax6⁺/Sox1⁺.

Claims

1. A method of creating a population of cells comprising a synchronous population of cells that are cultured from embryonic stem cells, are characterized by an early rosette morphology, and are Sox1⁻

, Pax6⁺, comprising the steps of;

(a) propagating embryonic stem cells into embryoid bodies and

(b) propagating embryoid bodies into a synchronous population of neural stem cells in the form of early rosettes, wherein this propagation is in the presence of FGF2, FGF8, FGF9 or FGF4.

2. The method of claim 1 wherein the time period between step (a) and development of early rosettes is 8-10 days.

3. The method of claim 1 wherein the population of Pax6⁺/Sox1⁻

cells is at least 70% of the total cell population.

4. The method of claim 1 wherein embryoid bodies are cultured for between 4-6 days in the presence of FGF2, FGF8, FGF9 or FGF4.

5. The method of claim 1 wherein the growth factor is FGF-2.

6. A population of cells generated by the method of claim 1.

7. A method of creating a population of synchronized neural stem cells wherein the cells are characterized by a neural tube-like rosette morphology and are Pax6⁺/Sox1⁺, the method comprising the step of culturing cells that are characterized by an early rosette morphology and are Sox1⁻

, Pax6⁺ for 4-6 days in the presence of FGF2, FGF4, FGF8, or RA.

8. The method of claim 7 wherein the exposure is between 4-6 days.

9. A population of cells created by the method of claim 8.

10. The cells of claim 9 wherein the cells were cultured with FGF8 and are EN1⁺.

11. The cells of claim 9 wherein the cells were cultured with FGF2 and are Bf1⁺.

12. The cells of claim 9 wherein the cells were cultured with RA and are Hox⁺.

13. A method of creating a population of midbrain dopamine neurons, comprising the step of culturing the cells of claim 10 to SHH while in the presence of FGF8, wherein the resulting cells express TH, AADC, EN-1, VMAT2 and DAT but do not express DbH and PNMT, and wherein the cells produce dopamine.

14. The method of claim 13 wherein the exposure to SHH is between 6-7 days.

15. A population of cells created by the method of claim 13.

16. A method of isolating a population of spinal motor neurons comprising the step of culturing the cells of claim 12 with SHH, wherein the resulting cells express HB9, HoxB1, HoxB6, HoxC5, HoxC8, ChAT and VAChT and produce acetylcholine.

17. The method of claim 16 wherein the cells are exposed to SHH between 6-7 days.

18. A population of cells created by the method of claim 16.

19. A method of creating a population of forebrain dopamine neurons, comprising the step of culturing the cells of claim 11 with SHH, wherein the resulting cells express TH, AADC, Bf1, Otx2 but do not express DBH and PNMT and wherein the cells produce dopamine.

20. The method of claim 19 wherein the cells are exposed to SHH for 6-7 days.

21. A population of cells created by the method of claim 19.

22. A method of examining a test compound for the ability to perturb neural cell development comprising the step of exposing the test compound to the cells of claim 15 and examining the results of the exposure compared to a control population of cells that has not been exposed to the test compound.

23. A method of examining a test compound for the ability to perturb neural cell development comprising the step of exposing the test compound to the cells of claim 18 and examining the results of the exposure compared to a control population of cells that has not been exposed to the test compound.

24. A method of examining a test compound for the ability to perturb neural cell development comprising the step of exposing the test compound to the cells of claim 21 and examining the results of the exposure compared to a control population of cells that has not been exposed to the test compound.