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

• US 7,588,937 B2
• Filed: 08/27/2004
• Issued: 09/15/2009
• 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
• Primary Examiner(s): Nguyen; Quang
• Application Number:
  US10/928,805
• Publication Number:
  US 20050095706A1
• Time in Patent Office: 1,845 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 obtaining a population of midbrain dopamine neurons, comprising:

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

, Pax6⁺;

(b) culturing the cells obtained in (a) in a neural basal medium with fibroblast growth factor-8 (FGF8), wherein resulting cells are Pax6⁺, Sox1⁺ and engrailed 1 positive (EN-1+) neuroepithelial cells;

(c) expanding the cells from step (b) in a neural basal medium with sonic hedgehog (SHH) and FGF8, wherein resulting cells are dopaminergic neural precursors; and

(d) culturing the cells from step (c) in a neural basal differentiation medium without SHH and FGF8 until resulting cells express tyrosine hydroxylase (TH), aromatic acid decarboxylase (AADC), EN-1, vesicular monoamine transporter 2 (VMAT2) and dopamine transporter (DAT) but do not express dopamine beta hydroxylase (DbH) and phenylethanolamine N-methyltransferase (PNMT), and produce dopamine.

2. The method of claim 1, wherein the exposure to SHH is between 6-7 days.