Clonal cultures of primate embryonic stem cells

• US 7,217,569 B2
• Filed: 05/06/2003
• Issued: 05/15/2007
• Est. Priority Date: 03/09/2000
• Status: Expired due to Term

• Current Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Original Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Inventors: Thomson, James A
• Primary Examiner(s): Crouch; Deborah
• Application Number:
  US10/430,497
• Publication Number:
  US 20030190748A1
• Time in Patent Office: 1,470 Days
• Field of Search: 800/10, 435/325, 435/366, 435/375, 435/377
• US Class Current: 435/377
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• 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

Disclosed herein are methods for culturing primate embryonic stem cells. These cells are cultured on a prolonged and stable basis in the presence of exogenously supplied fibroblast growth factor and in the absence of animal serum. Preferably there is also a fibroblast feeder layer. Also disclosed is a culture media containing fibroblast feeder layer and the fibroblast growth factor.

Claims

1. A method of establishing a clonal human embryonic stem cell line capable of sustaining a phenotype of normal embryonic stem cells following prolonged in vitro culture, the method comprising the steps of

providing a culture of undifferentiated human embryonic stem cells;

removing an individual undifferentiated human embryonic stem cell in a separate culture which includes at least about 4 ng/ml of exogenously supplied human fibroblast growth factor and which is essentially free of mammalian serum; and

culturing the individual undifferentiated cell in a serum-free medium which includes at least about 4 ng/ml of exogenously supplied human fibroblast growth factor such that the cell expands into a clonal culture of undifferentiated human embryonic stem cells maintaining the ability to differentiate into endoderm, mesoderm and ectoderm after at least six months of culture.

2. The method of claim 1 wherein the clonal culture of human embryonic stem cells is maintained for over twelve months.

3. The method of claim 1 wherein in the culturing step the serum-free medium contains collagenase.

4. The method of claim 3 wherein the collagenase is collagenase type IV.

5. The method of claim 3 wherein the concentration of collagenase is 1 mg/ml.

6. A method of establishing a clonal human embryonic stem cell line capable of sustaining a phenotype of normal embryonic stem cells following prolonged in vitro culture, the method comprising the steps of

providing a culture of undifferentiated human embryonic stem cells;

removing an individual undifferentiated human embryonic stem cell from the culture and placing the individual cell in a separate culture which includes at least about 4 ng/ml of basic human fibroblast growth factor and mouse embryonic feeder cells but is essentially free of mammalian serum; and

culturing the cell in the separate culture such that the cell expands into a clonal culture of undifferentiated human embryonic stem cells which can maintain the ability to differentiate into endoderm, mesoderm and ectoderm after at least eight months of culture.