EMBRYONIC STEM CELLS AND NEURAL PROGENITOR CELLS DERIVED THEREFROM

• US 20090258421A1
• Filed: 02/06/2009
• Published: 10/15/2009
• Est. Priority Date: 03/14/2000
• Status: Active Grant

• Current Assignee: ES Cell International Pte Ltd. (Lineage Cell Therapeutics, Inc.)
• Original Assignee: ES Cell International Pte Ltd. (Lineage Cell Therapeutics, Inc.)
• Inventors: REUBINOFF, Benjamin Eithan, PERA, Martin Frederick, BEN-HUR, Tamir
• Granted Patent: US 7,947,498 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 provides 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, and 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. The invention also provides methods that generate in vitro and in vivo models of controlled differentiation of ES cells towards the neural lineage. The model, and the 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, for the discovery of genes, growth factors, and differentiation factors that play a role in neural differentiation and regeneration, for drug discovery and for the development of screening assays for teratogenic, toxic and neuroprotective effects.

Claims

1. A method of producing human neural progenitor cells from human embryonic stem (hES) cells in vitro, said method comprising:

(a) culturing undifferentiated pluripotent hES cells for 2-3 weeks under adherent conditions so as to generate differentiating cells, wherein said culturing does not induce extra embryonic differentiation or cell death; and

(b) culturing said differentiating cells in the presence of serum free medium supplemented with growth factors which include epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), thereby obtaining neural progenitor cells, wherein said neural progenitor cells are capable of further differentiation into neurons, into oligodendrocytes, and into astrocytes, and wherein said neural progenitor cells express polysialated N-CAM, nestin, vimentin and the transcription factor Pax-6.

2. The method of claim 1, wherein said culturing undifferentiated pluripotent hES cells for 2-3 weeks is effected on fibroblast feeder cells.

3. The method of claim 1 wherein said undifferentiated pluripotent hES 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.

4. The method of claim 3, wherein the method for preparing said undifferentiated pluripotent hES 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.

5. A method of inducing differentiation of neural progenitors into neurons, the method comprising:

(a) generating neural progenitor cells according to claim 1,

(b) culturing the neural progenitor cells on an adhesive substrate in the presence of a serum free media and growth factors; and

(c) inducing the neural progenitor cells to differentiate into neurons by withdrawal of the growth factors.

6. The method of claim 5, further comprising determining an expression of a neuronal cell marker following step (c).

7. The method of claim 5, wherein said adhesive substrate comprises poly-D-lysine and laminin.

8. The method of claim 7, further comprising culturing said cells in the presence of retinoic acid following step (b).

9. A method of inducing differentiation of neural progenitors into oligodendrocytes or astrocytres, the method comprising:

(a) generating neural progenitor cells according to claim 1;

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

(c) inducing differentiation of the neural progenitor cells by withdrawing said PDGF-AA and bFGF from the medium.

10. The method of claim 1, wherein said culturing said differentiating hES cells eliminates non-neural cells.

11. The method of claim 1, wherein said differentiating cells are cultured as monolayers or spheres.

12. The method of claim 1, further comprising selecting from said differentiating cells, cells destined to give rise to neural progenitor cells following step (b).

13. The method of claim 6, 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 β

-tubulin.

14. The method of claim 1, wherein said culturing undifferentiated pluripotent hES cells does not generate embryoid bodies.

15. The method of claim 1, wherein said culturing undifferentiated pluripotent hES cells is continuous.

16. The method according to claim 5 wherein said neurons are mature neurons.

17. A method of producing human neural progenitor cells from human embryonic stem (hES) cells in vitro, the method comprising:

culturing undifferentiated pluripotent hES cells so as to generate differentiating hES cells and not generate embryoid bodies, wherein said culturing does not induce extra embryonic differentiation or cell death; and

culturing said differentiating cells in the presence of serum free medium supplemented with growth factors which include epidermal growth factor (EGF)

and basic fibroblast growth factor (bFGF), thereby obtaining neural progenitor cells, wherein said neural progenitor cells are capable of further differentiation into neurons, into oligodendrocytes, and into astrocytes, and wherein said neural progenitor cells express polysialated N-CAM, nestin, vimentin and the transcription factor Pax-6.

18. A method of inducing differentiation of neural progenitors into neurons, said method comprising:

(a) generating neural progenitor cells according to claim 17;

(b) culturing the neural progenitor cells on an adhesive substrate in the presence of a serum free media and growth factors; and

(c) inducing the neural progenitor cells to differentiate into neurons by withdrawal of the growth factors.

19. The method of claim 18, wherein said adhesive substrate comprises poly-D-lysine and laminin.

20. The method of claim 18, further comprising determining an expression of a neuronal cell marker following step (c).