Neural progenitor cells derived from embryonic stem cells

• US 20090075373A1
• Filed: 06/13/2008
• Published: 03/19/2009
• Est. Priority Date: 03/14/2000
• Status: Active Grant

• Current Assignee: Benjamin Eithan Reubinoff, Martin Frederick Pera, Tamir Ben-Hur
• Original Assignee: Benjamin Eithan Reubinoff, Martin Frederick Pera, Tamir Ben-Hur
• Inventors: Pera, Martin Frederick, Reubinoff, Benjamin Eithan, Ben-Hur, Tamir
• Granted Patent: US 8,137,969 B2
• Time in Patent Office: Days
• US Class Current: 435/366
• 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: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: C12N 2500/32 Amino acids
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/135 Platelet-derived growth factor [PDGF]
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• 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 2501/39 Steroid hormones
• 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 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0619 Neurons
• CPC Class Codes: C12N 5/0622 Glial cells, e.g. astrocytes, oligodendrocytes; Schwann cells
• CPC Class Codes: C12N 5/0623 : Stem cells

Abstract

The present invention relates to undifferentiated human embryonic stem cells, methods of cultivation and propagation and production of differentiated cells. In particular it relates to the production of human ES cells capable of yielding somatic differentiated cells in vitro, as well as committed progenitor cells such as neural progenitor cells capable of giving rise to mature somatic cells including neural cells and/or glial cells and uses thereof.

This invention provides methods that generate in vitro and in vivo models of controlled differentiation of ES cells towards the neural lineage. The model, and cells that are generated along the pathway of neural differentiation may be used for: the study of the cellular and molecular biology of human neural development, discovery of genes, growth factors, and differentiation factors that play a role in neural differentiation and regeneration, drug discovery and the development of screening assays for teratogenic, toxic and neuroprotective effects.

Claims

1. A method of generating neurospheres comprising:

(a) culturing undifferentiated pluripotent hES cells for at least two weeks under conditions that promote somatic differentiation without the generation of embryoid bodies to generate differentiating hES cells; and

(b) reculturing said differentiating hES cells in serum free medium, thereby generating neurospheres.

2. The method of claim 1, wherein said hES cells are obtained from a blastocyst stage of development of an in vitro fertilized human embryo.

3. The method of claim 1, wherein the neurospheres express a marker selected from the group consisting of N-CAM, A2B5, nestin and vimentin.

4. The method of claim 1, wherein the neurospheres do not express Oct-4.

5. The method of claim 1, wherein said culturing is continuous.

6. A method of generating neurospheres comprising:

(a) culturing undifferentiated pluripotent hES cells for at least two weeks under adherent conditions to generate differentiating hES cells; and

(b) reculturing said differentiating hES cells in serum free medium, thereby generating neurospheres.

7. The method of claim 6, wherein said hES cells are obtained from a blastocyst stage of development of an in vitro fertilized human embryo.

8. The method of claim 6, wherein the neurospheres express a marker selected from the group consisting of N-CAM, A2B5, nestin and vimentin.

9. The method of claim 6, wherein the neurospheres do not express Oct-4.

10. The method of claim 6, wherein said culturing is continuous.

11. The method of claim 6, wherein said culturing under adherent conditions comprises culturing on feeder cells.