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

• US 7,153,650 B2
• Filed: 03/13/2003
• Issued: 12/26/2006
• Est. Priority Date: 03/13/2003
• Status: Expired due to Fees

• 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
• Primary Examiner(s): Nguyen, Quang
• Application Number:
  US10/389,431
• Publication Number:
  US 20040180347A1
• Time in Patent Office: 1,384 Days
• Field of Search: None
• US Class Current: 435/4
• 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 the differentiation of a population of human embryonic stem cells in vitro, comprising measuring the gastrin-releasing peptide (GRP) receptor expression level in the population of human embryonic stem cells;

wherein a decrease in the GRP receptor expression level relative to the GRP receptor expression level measured in the population at an earlier time point indicates that the population is differentiating.

2. The method of claim 1, wherein the GRP receptor expression level is measured at the mRNA level.

3. The method of claim 2, wherein the GRP receptor expression level is measured at the mRNA level by PCR amplification.

4. The method of claim 1, wherein the GRP receptor expression level is measured at the protein level.

5. The method of claim 4, wherein the GRP receptor expression level is measured at the protein level by antibody assay.

6. The method of claim 1, wherein the GRP receptor expression level is measured using flow cytometry.