METHODS OF CULTURING EMBRYONIC STEM CELLS AND CONTROLLED DIFFERENTIATION

• US 20090197334A1
• Filed: 01/30/2009
• Published: 08/06/2009
• Est. Priority Date: 06/20/2000
• 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: Pera, Martin Frederick
• Granted Patent: US 9,080,147 B2
• Time in Patent Office: Days
• US Class Current: 435/377
• CPC Class Codes: C12N 2501/13 Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/33 Insulin
• CPC Class Codes: C12N 2502/088 neural stem cells
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0603 Embryonic cells production of embryos, nuclear transfer A01K67/027; Embryoid bodies
• 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

Abstract

The present invention provides a preparation of undifferentiated embryonic stem (ES) cells sustainable for a prolonged period in an undifferentiated state which will undergo stem cell renewal or somatic differentiation. Preferably the cells are capable of somatic differentiation in vitro and are inclined to differentiate away from an extraembryonic lineage. The present invention also provides method of culturing embryonic stem (ES) cells to improve stem cell maintenance and persistence in culture. The method also provides a culture of ES cells prepared by the method as well as differentiated cells derived from the embryonic cells resulting from directed differentiation procedures provided by the present invention.

Claims

1. A method of culturing undifferentiated ES cells, said method including:

obtaining a source of undifferentiated ES cells; and

culturing the ES cells in the presence of an indirect or direct antagonist of a BMP-mediated default pathway of extraembryonic endoderm differentiation.

3. The method according to claim 1 wherein the antagonist is a direct antagonist of the BMP-2 mediated default pathway selected from the group consisting of fetuin, noggin, chordin, gremlin, follistatin, cerberus, amnionless, DAN, or the ectodomain of BMPR1A (a BMP receptor protein) or ligand binding domains from other BMP receptors.

4. The method according to claim 3 wherein the antagonist comprises noggin.

5. The method according to claim 4 wherein the antagonist is a human or mouse noggin.

6. The method according to claim 4 wherein the noggin is a mouse BMP antagonist noggin comprising amino acid residues 20 to 232 of mouse noggin.

7. The method according to claim 4 wherein the noggin is in the range of 100 to 500 ng/ml.

8. The method according to claim 4, wherein the period sufficient to differentiate the ES cell to a progenitor cell is at least 5 days and the noggin is in the range of 100 to 500 ng/ml.

9. The method according to claim 1 wherein the antagonist is an indirect antagonist of the BMP-2 mediated default pathway selected from the group consisting of insulin, insulin analogue, or a cell derived insulin or insulin analogue-induced-factor.

10. The method according to claim 9 further including culturing the embryonic stem cells in the presence of a fibroblast feeder layer.

11. The method according to claim 9 wherein the insulin or insulin analogue is present in the range of 10 ng/ml to 10 μ

g/ml.

12. The method according to claim 9, said method including:

obtaining a source of undifferentiated ES cells; and

culturing the ES cells in the presence of a cell derived insulin or insulin analogue induced factor.

13. The method according to claim 12 wherein the cell derived insulin or insulin analogue induced factor is derived from conditioned medium of another culture of ES cells exposed to insulin or insulin analogue.

14. The method according to claim 13 wherein the other culture of ES cells is exposed to insulin in the range of 10 ng/ml to 10 μ

g/ml.

2. A method of producing a progenitor cell from an ES cell, said method comprising:

obtaining a source of undifferentiated ES cells; and

culturing the ES cells in the presence of an antagonist of a BMP mediated default pathway of extra embryonic endoderm differentiation for a period sufficient to differentiate the ES cell to a progenitor cell.

15. A method of producing a somatic cell from an undifferentiated ES cell, said method including:

obtaining a progenitor cell comprising obtaining a source of undifferentiated ES cells;

culturing the ES cells in the presence of an antagonist of a BMP mediated default pathway of extra embryonic endoderm differentiation for a period sufficient to differentiate the ES cell to a progenitor cell;

culturing the progenitor cell in a progenitor culture medium; and

obtaining a somatic cell from a lineage of the progenitor.

16. The method according to claim 15 wherein the progenitor culture medium is derived from a culture of neural progenitors which can differentiate into neural or glial cells.