Neural progenitor cells derived from embryonic stem cells

• US 8,460,931 B2
• Filed: 01/31/2012
• Issued: 06/11/2013
• Est. Priority Date: 03/14/2000
• Status: Expired due to Fees

• Current Assignee: Hadasit Medical Research Services and Development Limited, ES Cell International Pte Ltd. (Lineage Cell Therapeutics, Inc.)
• Original Assignee: ES Cell International Pte Ltd. (Lineage Cell Therapeutics, Inc.)
• Inventors: Pera, Martin Federick, Ben-Hur, Tamir, Reubinoff, Benjamin Eithan
• Primary Examiner(s): Ton, Thaian N
• Application Number:
  US13/361,976
• Publication Number:
  US 20120122109A1
• Time in Patent Office: 497 Days
• Field of Search: 435/377, 435/366, 435/368, 435/384
• 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: 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 inducing neural differentiation of embryonic stem (ES) cells comprising:

(a) culturing isolated, undifferentiated, human ES cells in the presence of serum free medium supplemented with growth factors which include epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) to produce neurospheres containing neural progenitor cells, wherein said neurospheres do not express alphafetoprotein; and

(b) culturing said neurospheres under conditions that induce neural differentiation, thereby inducing neural differentiation of the ES cells.

2. The method claim 1, wherein the inducing neural differentiation results in generation of neurons.

3. The method of claim 2, further comprising:

(c) culturing the neural progenitor cells on an adhesive substrate comprising poly-D-lysine in the presence of a serum free media and growth factors; and

(d) inducing the neural progenitor cells to differentiate into neurons by withdrawal of the growth factors, thereby generating neurons.

4. The method of claim 1, wherein the inducing neural differentiation results in generation of oligodendrocytes and/or astrocytes.

5. The method of claim 4 further comprising:

(c) culturing the neural progenitor cells in serum free medium in the presence of PDGF-AA and bFGF; and

(d) plating the neural progenitor cells on an adhesive substrate which comprises poly-D-lysine and fibronectin, in serum free medium without PDGF-AA or bFGF, thereby inducing differentiation of the neural progenitor cells into oligodendrocytes or astrocytes.

6. The method of claim 4 further comprising:

(c) culturing the neural progenitor cells in serum free medium in the presence of PDGF-AA and bFGF;

(d) plating the neural progenitor cells on an adhesive substrate which comprises poly-D-lysine and fibronectin;

(e) culturing the neural progenitor cells in serum free medium in the presence of PDGF-AA, bFGF and T3; and

(f) inducing differentiation of the neural progenitor cells by withdrawing PDGF-AA and bFGF in the medium.

7. The method of claim 1, wherein said ES cells are prepared according to a method comprising:

obtaining an in vitro fertilised human embryo and growing the embryo to a blastocyst stage of development;

removing inner cells mass (ICM) cells from the embryo;

culturing ICM cells under conditions which do not induce extraembryonic differentiation and cell death, and promote proliferation of undifferentiated stem cells; and

recovering stem cells.

8. The method of claim 7, wherein the method for preparing ES cells is further characterized by:

culturing the ICM cells on a fibroblast feeder layer to promote proliferation of embryonic stem cells prior to recovering the stem cells from the feeder layer, wherein the fibroblast feeder cells are arrested in their growth;

replating the stem cells from the fibroblast feeder layer onto another fibroblast feeder layer; and

culturing the stem cells for a period sufficient to promote proliferation of morphologically undifferentiated stem cells.

9. The method of claim 3, further comprising determining an expression of a neuronal cell marker following step (d).

10. The method of claim 3, wherein said adhesive substrate further comprises laminin.

11. The method of claim 10, further comprising culturing said cells in the presence of retinoic acid following step (c).

12. The method of claim 1, wherein said culturing ES cells does not generate embryoid bodies.

13. The method of claim 1, wherein said culturing said ES cells is effected as monolayers or spheres.

14. The method of claim 9, wherein said neuronal cell marker is selected from the group consisting of 200 kDa neurofilament protein, 160 kDa neurofilament protein, MAP2a+b, glutamate, synaptophysin, glutamic acid decarboxylase and .beta.-tubulin.

15. The method of claim 2, wherein said neurons are mature neurons.