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

• US 9,080,151 B2
• Filed: 05/06/2011
• Issued: 07/14/2015
• 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:
  US13/068,285
• Publication Number:
  US 20110275152A1
• Time in Patent Office: 1,530 Days
• Field of Search: 435/377, 435/366
• US Class Current: 1/1
• 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 forebrain dopamine neurons, comprising the steps of:

(a) providing a synchronous population of human Sox1−

, Pax6+ cells characterized by an early rosette morphology;

(b) culturing the human Sox1−

, Pax6+ cells of step (a) in the presence of fibroblast growth factor 2 (FGF2) for 4-6 days, wherein a population of cells expressing brain factor 1 (Bf1) and orthodenticle homeobox2 (Otx2) is generated;

(c) culturing the Bf1+ cells resulting from step (b) in the presence of sonic hedgehog (SHH) and FGF8 for 6-7 days; and

(d) culturing the cells resulting from step (c) in a neuronal differentiation medium to obtain a cell population in which about one third of the differentiated cells are human forebrain dopamine-producing neurons that express tyrosine hydroxylase (TH), aromatic acid decarboxylase (AADC), Bf1, Otx2, but do not express dopamine beta-hydroxylase (Dβ

H) and phenylethanolamine N-methyltransferase (PNMT).

2. The method of claim 1 wherein FGF2 is present at a concentration of 10-20 ng/ml in step (b).

3. The method of claim 1 wherein SHH is present at a concentration of 50-250 ng/ml in step (c).

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