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

• US 20080206865A1
• Filed: 11/08/2006
• Published: 08/28/2008
• 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: Thomson, James A., Zhang, Su-Chun, Li, Xue-jun, Duncan, Ian David
• Granted Patent: US 7,972,850 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 isolating a population of spinal motor neurons comprising:

culturing Sox1⁻

, Pax6⁺ cells characterized by an early rosette morphology for 4-6 days in the presence of FGF2, FGF4, FGF8, or RA with SHH, wherein a population of Hox⁺ cells are created; and

culturing the cells with RA;

wherein the resulting cells express HB9, HoxB1, HoxB6, HoxC5, HoxC8, ChAT and VAChT and produce acetylcholine.

2. The method of claim 1 wherein the cells are exposed to SHH between 6-7 days.

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