Derivation of neural stem cells from embryonic stem cells

• US 8,338,176 B2
• Filed: 07/29/2008
• Issued: 12/25/2012
• Est. Priority Date: 07/30/2007
• Status: Active Grant

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Inventors: Daadi, Marcel M., Steinberg, Gary K.
• Primary Examiner(s): Crouch, Deborah
• Application Number:
  US12/182,050
• Publication Number:
  US 20090035284A1
• Time in Patent Office: 1,610 Days
• Field of Search: None
• US Class Current: 435/377
• CPC Class Codes: A61K 35/30 Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2500/46 Amines, e.g. putrescine
• 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 2501/235 Leukemia inhibitory factor [LIF]
• CPC Class Codes: C12N 2501/392 Sexual steroids
• CPC Class Codes: C12N 2501/91 Heparin
• CPC Class Codes: C12N 2502/08 cells of the nervous system
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2533/32 Polylysine, polyornithine
• CPC Class Codes: C12N 5/0619 Neurons
• CPC Class Codes: C12N 5/0623 Stem cells
• CPC Class Codes: G01N 33/502 for testing non-proliferative effects

Abstract

Provided is a method for the derivation of neural stem cells (NSCs) from embryonic stem cells (ESCs) and the use of the NSCs for treatment of various neural disorders. The NSCs that are derived from the ESCs are tissue-specific multipotent NSCs with a stable growth rate, unlimited self-renewal capacity, and a predictable differentiation profile. Being both non-tumorigenic and engraftable, the NSCs of the present invention have utility in repopulation stroke-damaged tissue. The NSCs of the present invention may be differentiated to produce tyrosine-hydroxylase expressing neurons, which may be used as a source of dopaminergic neurons for subjects suffering from a condition characterized by dopaminergic dysfunction, such as Parkinson'"'"'s disease.

Claims

1. A method of deriving a homogeneous population of human neural stem cells (NSCs) comprising the steps of:

(a) culturing human embryonic stem cells (ESCs) as spheres in serum-free media comprising epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and leukemia inhibitory factor (LIF);

(b) selecting and plating the spheres in a cell culture container in serum-free media comprising EGF, bFGF, and LIF until the spheres attach to the container and the cells form clusters; and

(c) selecting and plating the clusters for a number of passages sufficient to change the adherent properties of the cells, wherein the cells grow as an adherent monolayer of nestin positive, homogenous, self-renewing cells.

16. The method of claim 1, wherein the number of passages sufficient to change the adherent properties of the cells is five or more.

2. A method of deriving a homogeneous population of human neural stem cells (NSCs) from embryonic stem cells (ESCs) comprising the steps of:

(a) culturing an ESC line in serum-free media comprising epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and leukemia inhibitory factor (LIF) until spheres (primary spheres) form in the media;

(b) selecting and plating the primary spheres in a cell culture container in serum-free media comprising EGF, bFGF, and LIF until the spheres attach to the container and the ESCs form clusters;

(c) removing the clusters from the culture container and replanting the clusters as secondary spheres in a container with serum-free media comprising EGF, bFGF, and LIF, wherein the secondary spheres attach to the container; and

(d) repeating step (c) for a number of passages sufficient to change the adherent properties of the cells, wherein the cells grow as an adherent monolayer of nestin positive, homogenous, self-renewing cells.

3. The method of claim 2, wherein step (c) is repeated with subsequent spheres until a population of cells of a desired concentration is obtained.

4. The method of claim 3, wherein the self-renewing cells express a neural progenitor stem cell marker.

5. The method of claim 4, wherein the neural progenitor stem cell marker is nestin.

6. The method of claim 4, wherein the NSCs are induced to differentiate by removal of EGF, bFGF, and LIF from the culture media and plating on a substrate.

7. The method of claim 6, wherein the substrate is coated with poly-L-ornithine (PLO).

8. The method of claim 6, wherein the differentiated NSCs to give rise to neurons, astrocytes, and oligodendrocytes.

9. The method of claim 8, wherein the neurons are identified by expression of a neuronal marker.

10. The method of claim 9, wherein the neuronal marker is Tuj1 (β

-III-tubulin) or MAP-2 (microtubule associated protein

     2).

11. The method of claim 8, wherein the astrocytes are identified by expression of an astrocyte marker.

12. The method of claim 11, wherein the astrocyte marker is GFAP (glial fibrillary acid protein).

13. The method of claim 8, wherein the oligodendrocytes are identified by expression of an oligodendrocyte marker.

14. The method of claim 13, wherein the oligodendrocyte marker is GC (galactocerebrocide) or MBP (myelin basic protein).

15. The method of claim 2, wherein the EGF, bFGF, and LIF are each present in the media in a concentration ranging from 1 μ

g/mL to 1 ng/mL.

17. The method of claim 2, wherein the number of passages sufficient to change the adherent properties of the cells is five or more.