Methods of culturing embryonic stem cells and controlled differentiation

• US 7,112,437 B2
• Filed: 06/20/2001
• Issued: 09/26/2006
• Est. Priority Date: 06/20/2000
• Status: Expired due to Term

• Current Assignee: National University Hospital (Government of Singapore), Hadasit Medical Research Services and Development Limited, Hadassah University Hospital (Hadassah, The Women's Zionist Organization of America, Inc.), ES Cell International Pte Ltd. (Lineage Cell Therapeutics, Inc.)
• Original Assignee: ES Cell International Pte Ltd. (Lineage Cell Therapeutics, Inc.)
• Inventors: Pera, Martin Frederick
• Primary Examiner(s): WOITACH, JOSEPH T
• Application Number:
  US09/885,679
• Publication Number:
  US 20020022267A1
• Time in Patent Office: 1,924 Days
• Field of Search: 435/325, 435/1.1, 435/405, 800/3, 800/4
• US Class Current: 435/325
• 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 producing a human neural progenitor cell from a human ES cell, said method comprising:

obtaining a source of an undifferentiated human ES cell; and

culturing the ES cell in the presence of an antagonist of a BMP mediated default pathway of extra embryonic endoderm differentiation to differentiate the ES cell to a progenitor cell, wherein said progenitor cell lacks at least one marker of said undifferentiated ES cell; and

culturing the progenitor cell in a neural progenitor cell culture medium to obtain a neural progenitor cell.

2. The method of claim 1 wherein the source of said undifferentiated human ES cell is selected from the group consisting of an embryo, a blastocyst, and a culture of undifferentiated embryonic stem cells.

3. The method of claim 2 wherein said antagonist is noggin.

4. The method of claim 3 wherein said noggin is a human or mouse noggin.

5. The method of claim 3 wherein said noggin is a mouse BMP antagonist noggin comprising amino acid residues 20 to 232 of mouse noggin.

6. The method of claim 3 wherein said noggin is in the range of 100 to 500 ng/ml.

7. The method of any one of claims 1 to 6 wherein the ES cell is differentiated to said progenitor cell is by culturing the ES cell in the presence of noggin for at least 5 days, wherein the noggin is in the range of 100 to 500 ng/ml.

8. The method of claim 1 wherein said at least one marker of said undifferentiated ES cell is October-4 or cripto.

9. A method of producing a human progenitor cell from a human ES cell, said method comprising:

obtaining a source of an undifferentiated human ES cell; and

culturing the ES cell in the presence of an antagonist of a BMP mediated default pathway of extra embryonic endoderm differentiation under conditions sufficient to differentiate the ES cell to a progenitor cell, wherein said progenitor cell lacks at least one marker of said undifferentiated ES cell, lacks a marker of neuroectoderm, and is capable of differentiating into a neural progenitor cell.

10. The method of claim 9 wherein said antagonist is noggin.

11. The method of claim 10, wherein the ES cell is cultured in the presence of noggin for at least 5 days.

12. The method of claim 10 wherein said noggin is a human or mouse noggin.

13. The method of claim 10 wherein said noggin is comprises amino acid residues 20 to 232 of mouse noggin.

14. The method of claim 10 wherein said noggin is in the range of 100 to 500 ng/ml.

15. The method of claim 9 wherein the source of said undifferentiated human ES cell is selected from the group consisting of an embryo, a blastocyst, and a culture of undifferentiated embryonic stem cells.

16. The method of claim 9, wherein said at least one marker of said undifferentiated ES cell is October-4 or cripto.

17. The method of claim 9, wherein said marker of neuroectoderm is nestin or Pax 6.

18. The method of claim 9, wherein said progenitor cell is unreactive with any one of the antibodies selected from the group consisting of PHM4 recognising MHC Class 1 surface molecules, anti-desmin, UJ13A reactive with polysialylated N-CAM, Cam 5.2 reactive with low molecular weight cytokeratins, AMF reactive with vimentin intermediate filaments, antibody to 160 kDa neurofilament protein, GCTM-2 reactive with a proteoglycan present on the surface of ES cells, TG42.1 reactive with a 25 kDa protein which copurifies with the proteoglycan recognised by GCTM-2 and is found on stem cells and other cell types, and monoclonal antibody GCTM-5 reactive with a molecule present on a small proportion of cells in spontaneously differentiating human ES cell cultures.

19. The method of claim 9, wherein said progenitor cell, upon further culturing in a neural progenitor culture medium, differentiates into said neural progenitor cell.