METHOD OF IN VITRO DIFFERENTIATION OF NEURAL STEM CELLS, MOTOR NEURONS AND DOPAMINE NEURONS FROM PRIMATE EMBRYONIC STEM CELLS

• US 20080227137A1
• Filed: 10/31/2007
• Published: 09/18/2008
• 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: Zhang, Su-Chun, Li, Xue-Jun
• Granted Patent: US 8,153,424 B2
• Time in Patent Office: Days
• US Class Current: 435/29
• 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 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0623 Stem cells
• CPC Class Codes: G01N 33/5058 Neurological cells
• CPC Class Codes: G01N 33/5073 Stem cells

Abstract

A method of differentiating embryonic stem cells into ventral spinal progenitor 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 retinoic acid, wherein the cells express Hoxb4, but not Otx2 or Bf1.

Claims

1. A method of creating a population of ventral spinal progenitor cells, the method comprising the steps of:

(a) obtaining a synchronous population of cells that are cultured from embryonic stem cells, that are characterized by an early rosette morphology and that are Pax6⁺/Sox1⁻

;

(b) culturing the cells of step (a) with a sufficient amount of retinoic acid, wherein the resulting cells are Hoxb4⁺, Otx2⁻

or Bf1; and

(c) culturing the cells of step (b) in a suspension culture with a sufficient amount of retinoic acid and an agent selected from sonic hedgehog and an activator of sonic hedgehog pathway, wherein the resulting cells are Olig2⁺ and Pax7⁻

.

2. The method of claim 1, wherein step (a) is between 8 days to 10 days.

3. The method of claim 1, wherein step (b) is between 6 days to 8 days.

4. The method of claim 1, wherein step (c) is between 9 days to 14 days.

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

cells is at least 70% of the cell population.

6. The method of claim 1, wherein the cells of step (b) are expanded in a suspension culture for an additional 6-8 days prior to step (c).

7. The method of claim 1, wherein at least 80% of the cell population is ventral spinal progenitor cells.

8. The method of claim 1, wherein the activator of the sonic hedgehog pathway is purmorphamine.

9. A population of cells created by the method of claim 1.

10. A method of creating a population of ventral spinal motor neurons, comprising the step of:

culturing cells of claim 1 in a suspension culture with a sufficient amount of retinoic acid and an agent selected from sonic hedgehog and an activator of sonic hedgehog pathway, wherein the resulting cells express HB9, HoxB4, HoxB6, HoxC5, HoxC8, ChAT and VAChT and produce acetylcholine.

11. The method of claim 10, wherein at least 80% of the cell population is ventral spinal motor neurons.

12. The method of claim 10, wherein the cells are cultured between 3 weeks and 4 weeks.

13. The method of claim 10, wherein the activator of the sonic hedgehog pathway is purmorphamine.

14. A population of cells created by the method of claim 10.

15. A method of examining a test compound for the ability to perturb neural cell development, comprising the steps of:

(a) exposing the test compound to cells selected from the cells of claim 1 and the cells of claim 10; and

(b) examining the results of the exposure compared to a control population of cells that has not been exposed to the test compound.