Generation of dopaminergic neurons from human nervous system stem cells

• US 6,395,546 B1
• Filed: 02/01/2000
• Issued: 05/28/2002
• Est. Priority Date: 02/01/2000
• Status: Expired due to Term

• Current Assignee: Levesque Biosciences Incorporated
• Original Assignee: Neurogeneration, Inc. (Sanofi )
• Inventors: Zobel, Rita, Levesque, Michel F.
• Primary Examiner(s): Yucel, Remy
• Assistant Examiner(s): LOEB, BRONWEN
• Application Number:
  US09/490,569
• Time in Patent Office: 847 Days
• Field of Search: 435/368,377,455 536/24.5
• 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 2501/01 Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/235 Leukemia inhibitory factor [LIF]
• CPC Class Codes: C12N 2501/385 of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
• CPC Class Codes: C12N 2501/60 Transcription factors
• CPC Class Codes: C12N 5/0619 Neurons

Abstract

The present invention relates to methods for generating dopaminergic neurons in vitro from embryonic and adult central nervous system cells. Specifically, these cells are isolated, cultured in vitro and stimulated to differentiate into dopaminergic neurons by down-regulating COUP-TFI and/or COUP-TFII expression or increasing NOT1 expression. These newly generated dopaminergic neurons may serve as an excellent source for cell replacement therapy in neurological disorders in which the dopaminergic system is compromised.

Claims

1. A method of generating dopaminergic neuronal cells comprising:

obtaining stem cells from mammalian central nervous system tissue;

culturing said stem cells in vitro; and

down-regulating COUP-TFI and/or COUP-TFII expression in said stem cells, thereby generating said dopaminergic neuronal cells.

2. The method of claim 1 wherein said stem cells are cultured in the presence of one or more growth factors, said one or more growth factors being selected from the group consisting of epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and leukemia inhibitory factor (LIF).

3. The method of claim 1 wherein said stem cells are induced to begin differentiating into neuronal cell types by the addition of one or more chemicals to said cultures, said chemicals being selected from the group consisting of all-trans retinoic acid and dibutyryl cAMP.

4. The method of claim 1 wherein said stem cells are obtained from fetal or juvenile central nervous system tissue.

5. The method of claim 1 wherein said stem cells are obtained from adult central nervous system tissue.

6. The method of claim 1 in which COUP-TFI and/or COUP-TFII expression is down-regulated using antisense oligonucleotides directed to COUP-TFI and/or COUP-TFII.

7. The method of claim 1 wherein said stem cells are obtained by:

dissecting central nervous system tissue;

incubating said tissue with trypsin solution;

adding trypsin inhibitor;

mechanically triturating said tissue; and

centrifuging said tissue.

8. The method of claim 1 wherein said stem cells are obtained from human tissue.

9. The method of claim 2 wherein said stem cells are caused to begin differentiating into neuronal cell types by withdrawing said one or more growth factors.