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

• US 8,153,424 B2
• Filed: 10/31/2007
• Issued: 04/10/2012
• Est. Priority Date: 10/03/2001
• Status: Expired due to Fees

• 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
• Primary Examiner(s): Nguyen, Quang
• Application Number:
  US11/932,582
• Publication Number:
  US 20080227137A1
• Time in Patent Office: 1,623 Days
• Field of Search: None
• US Class Current: 435/366
• 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 human ventral spinal progenitor cells, the method comprising the steps of:

(a) obtaining a synchronous population of cells that are cultured from human 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 in adherent culture, wherein the resulting cells are positive for Hoxb4 and are negative for at least one of Otx2 or Bf1;

(c) expanding the resulting cells of step (b) in a suspension culture with a sufficient amount of retinoic acid; and

(d) culturing the cells of step (c) in a suspension culture with a sufficient amount of retinoic acid and an agent selected from the group consisting of sonic hedgehog and purmorphamine, wherein at least 80% of the resulting cells are Olig2⁺, Nkx2.2⁺, Irx3⁺ and Pax7⁻

.

2. The method of claim 1, wherein the time period that the synchronous population of cells recited in step (a) are cultured from embryonic stem cells 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 (d) 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 synchronous cell population of step (a).

6. The method of claim 1, wherein the cells of step (c) are expanded for 6-8 days prior to step (d).

7. A population of human ventral spinal progenitor cells created by the method of claim 1, wherein at least 80% within the population are human ventral spinal progenitor cells and are Hox4⁺, HoxC8⁺, Olig2⁺, Nkx2.2⁺, Irx3⁺, Otx2⁻

, Pax7⁻

and Phox2b⁻

.

8. The method of claim 1, wherein purmorphamine is present at a concentration of at least 0.5 μ

M.

9. The method of claim 1, wherein purmorphamine is present at a concentration of at least 1 μ

M.

10. The method of claim 1, wherein the cells are cultured in a suspension culture with a sufficient amount of retinoic acid and purmorphamine in step (d).