Marker system for preparing and characterizing high-quality human embryonic stem cells

• US 20040180347A1
• Filed: 03/13/2003
• Published: 09/16/2004
• Est. Priority Date: 03/13/2003
• Status: Active Grant

• Current Assignee: Asterias Biotherapeutics Incorporated (Lineage Cell Therapeutics, Inc.)
• Original Assignee: Geron Corporation
• Inventors: Gold, Joseph D., Mandalam, Ramkumar, Brandenberger, Ralph, Irving, John M., Stanton, Lawrence W., Mok, Michael
• Granted Patent: US 7,153,650 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: G01N 33/5005 involving human or animal cells immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025

Abstract

This disclosure provides a system for qualifying embryonic stem cells intended for human therapy. A large-scale sequencing project has identified important markers that are characteristic of undifferentiated pluripotent cells. Combinations of these markers can be used to validate the self-renewing capacity of ES cells, and their ability to differentiate into tissue types suitable for regenerative medicine. The marker system of this invention has been used to screen feeder cells, media additives, and culture conditions that promote proliferation of stem cells without differentiation. A culture system optimized by following these markers is suitable for rapid expansion of undifferentiated cells from existing lines, or the derivation of new lines that are equally apposite for clinical use.

Claims

1. A method for assessing a culture of undifferentiated human embryonic stem (hES) cells or their progeny, comprising detecting or measuring:

a marker from the following list;

Cripto, gastrin-releasing peptide (GRP) receptor, and podocalyxin-like protein; and

either hTERT and/or Oct 3/4, or a second marker from the list.

2. The method of claim 1, comprising detecting or measuring at least two markers from the list.

3. The method of claim 1, comprising detecting or measuring at least two markers from the list, and detecting or measuring hTERT and/or Oct 3/4.

4. The method of claim 1, comprising detecting or measuring Cripto, gastrin-releasing peptide (GRP) receptor podocalyxin-like protein, hTERT, and Oct 3/4.

5. The method of claim 1, wherein expression of the marker(s) is detected or measured at the mRNA level by PCR amplification

6. The method of claim 1, wherein expression of the marker(s) is detected or measured at the protein level by antibody assay.

7. The method of claim 1, comprising quantifying the proportion of undifferentiated hES cells or differentiated cells in the culture from said marker expression.

8. The method of claim 1, comprising assessing the ability of a culture system or component thereof to maintain hES cells in an undifferentiated state from said marker expression.

9. The method of claim 8, comprising assessing the ability of a soluble factor to maintain hES cells in an undifferentiated state from said marker expression.

10. The method of claim 8, comprising assessing the ability of a culture medium to maintain hES cells in an undifferentiated state from said marker expression.

11. The method of claim 8, comprising assessing the ability of a preparation of feeder cells to maintain hES cells in an undifferentiated state from said marker expression.

12. The method of claim 1, comprising assessing the ability of a culture system or component thereof to cause differentiation of hES cells into a culture of lineage-restricted precursor cells and/or terminally differentiated cells.

13. The method of claim 1, comprising assessing the suitability of an hES cell culture for preparing cells for human administration.

14. The method of claim 1, wherein the level of the marker is determined to be at least 100-fold higher than the level of the marker in BJ fibroblasts.

15. The method of claim 1, wherein the level of the marker is determined to be no less than 100-fold lower than the level of the marker in hES cells, cultured on an extracellular matrix in medium conditioned with mouse embryonic fibroblasts and containing 4 ng/mL basic fibroblast growth factor.

16. The method of claim 1, further comprising modifying the culture conditions so as to cause the hES cells to increase expression of the marker detected or measured in the culture.