Feeder independent extended culture of embryonic stem cells

• US 7,514,260 B2
• Filed: 05/20/2005
• Issued: 04/07/2009
• Est. Priority Date: 05/21/2004
• Status: Active Grant

• Current Assignee: WiCell Research Institute, Inc.
• Original Assignee: WiCell Research Institute, Inc.
• Inventors: Thomson, James A., Xu, Ren-He
• Primary Examiner(s): Ton; Thaian N
• Application Number:
  US11/134,564
• Publication Number:
  US 20060014279A1
• Time in Patent Office: 1,418 Days
• Field of Search: 435/383, 435/366
• US Class Current: 435/383
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• 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

Previous methods for culturing human embryonic stem cells have required either fibroblast feeder cells or a medium which has been exposed to fibroblast feeder cells in order to maintain the stem cells in an undifferentiated state. It has now been found that if an antagonist of bone morphogenic protein is added to the medium in which the stem cells are cultured, together with fibroblast growth factor, the stem cells will remain undifferentiated indefinitely, even without feeder cells or conditioned medium.

Claims

1. A method for culturing human embryonic stem cells in a feeder independent culture, the method comprising the step of

culturing the human embryonic stem cells in a medium including salts, vitamins, amino acids, glucose, a fibroblast growth factor and a bone morphogenetic protein antagonist in the presence of an extracellular matrix in sufficient amount to maintain the stem cells in an undifferentiated state wherein the bone morphogenetic protein antagonist is noggin.

2. The method of claim 1 wherein the medium includes thefibroblast growth factor in a concentration of at least 4 ng/ml.

3. The method of claim 1 wherein the fibroblast growth factor isbFGF at a concentration of 40 ng/ml.

4. In a method of culturing human embryonic stem cells in a medium including salts, vitamins, amino acids, and a fibroblast growth factor with extracellular matrix, the improvement comprising adding to the medium an amount of an antagonist of bone morphogenetic protein signaling in an amount sufficient to maintain the cells in an undifferentiated state without exposure of the stem cells or the medium to feeder cells wherein the bone morphogenetic protein antagonist is noggin.

5. An in vitro cell culture comprising in a culture vessel:

human embryonic stem cells;

an extracellular matrix; and

a culture medium, the culture medium comprising salts, vitamins, amino acids, glucose, a fibroblast growth factor, and a bone morphogenetic protein signaling antagonist wherein the bone morphogenetic protein antagonist is noggin in sufficient amount to maintain the stem cells in an undifferentiated feeder independent state, the medium being free of feeder cells and never having been exposed to feeder cells.

6. The cell culture as claimed in claim 5 wherein the medium includes the fibroblast growth factor in a concentration of at least 4 ng/ml of bFGF.

7. The cell culture as claimed in claim 5 wherein the medium further comprises proteins selected from the group consisting of albumin, insulin and transferrin.

8. The cell culture as claimed in claim 7 wherein the proteins are human.

9. The cell culture as claimed in claim 7 wherein the proteins are recombinant proteins.