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

• US 7,972,850 B2
• Filed: 11/08/2006
• Issued: 07/05/2011
• 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: Li, Xue-jun, Zhang, Su-Chun
• Primary Examiner(s): Nguyen; Quang
• Application Number:
  US11/594,455
• Publication Number:
  US 20080206865A1
• Time in Patent Office: 1,700 Days
• Field of Search: None
• US Class Current: 435/377
• 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 generating a population of human spinal motor neurons comprising:

(a) culturing human Sox1⁻

, Pax6⁺cells characterized by an early rosette morphology for 4-6 days in the presence of retinoic acid (RA), wherein a population of Hox⁺cells are created;

(b) culturing the cells resulting from step (a) in the presence of RA and sonic hedgehog (SHH); and

(c) culturing the cells resulting from step (b) in neuronal differentiation medium and in the presence of SHH, wherein the resulting cells express HB9, HoxB1, HoxB6, HoxC5, HoxC8, choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) and produce acetylcholine.

2. The method of claim 1 wherein the cells are exposed to SHH between 6-7 days in step (b).

3. A population of human spinal motor neurons generated by the method of claim 1, wherein the spinal motor neurons express HB9, HoxB1, HoxB6, HoxC5, HoxC8, ChAT and VAChT and produce acetylcholine.

4. The method of claim 1 wherein the cells are exposed to SHH between 6-10 days in step (b).

5. The method of claim 1 wherein the cells are exposed to neuronal differentiation medium between 7-21 days in step (c).