Cultivation of human embryonic stem cells in the absence of feeder cells or without conditioned medium

• US 7,439,064 B2
• Filed: 03/11/2005
• Issued: 10/21/2008
• Est. Priority Date: 03/09/2000
• Status: Expired due to Fees

• Current Assignee: WiCell Research Institute, Inc.
• Original Assignee: WiCell Research Institute, Inc.
• Inventors: Thomson, James A., Levenstein, Mark
• Primary Examiner(s): Crouch; Deborah
• Application Number:
  US11/078,737
• Publication Number:
  US 20050244962A1
• Time in Patent Office: 1,320 Days
• Field of Search: 435/366, 435/377, 435/383
• US Class Current: 435/377
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2500/44 Thiols, e.g. mercaptoethanol
• 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

The invention relates to methods for culturing human embryonic stem cells by culturing the stem cells in an environment essentially free of mammalian fetal serum and in a stem cell culture medium including amino acids, vitamins, salts, minerals, transferring, insulin, albumin, and a fibroblast growth factor that is supplied from a source other than just a feeder layer the medium. Also disclosed are compositions capable of supporting the culture and proliferation of human embryonic stem cells without the need for feeder cells or for exposure of the medium to feeder cells.

Claims

1. A method of culturing human embryonic stem cells, comprising:

culturing human embryonic stem cells on a matrix in a culture medium free of serum and in a stem cell culture medium containing amino acids, vitamins, salts, minerals, transferrin or a transferrin substitute, insulin or an insulin substitute, albumin, and a fibroblast growth factor supplied from a source other than a feeder layer, the fibroblast growth factor present in a concentration at least as high as a maintenance concentration, wherein the medium supports the culture and proliferation of undifferentiated proliferating euploid human embryonic stem cells for at least six passages without feeder cells or conditioned medium.

2. The method of claim 1 wherein the FGF is selected from FGF2, FGF4, FGF9, FGF17 and FGF18.

3. The method of claim 1 wherein the FGF is FGF2 which is present in the medium at 100 ng/ml.

4. A method of culturing human embryonic stem cells in defined media without serum and without feeder cells on a matrix, the method comprising:

culturing human embryonic stem cells in a culture medium containing albumin, amino acids, vitamins, minerals, at least one transferrin or transferrin substitute, at least one insulin or insulin substitute, the culture medium free of serum and containing at least about 100 ng/ml of a fibroblast growth factor, and culturing without feeder cells or conditioned medium, wherein the medium supports proliferation of the human embryonic stem cells in an undifferentiated state.

5. The method of claim 4, wherein said culturing step includes the embryonic stem cells proliferating in culture for over one month while maintaining the potential of the embryonic stem cells to differentiate into derivatives of endoderm, mesoderm, and ectoderm tissues, and while maintaining the karyotype of the embryonic stem cells.

6. The method of claim 4 wherein the FGF is selected from FGF2, FGF4, FGF9, FGF17 and FGF18.

7. A culture of human embryonic stem cells comprising:

human embryonic stem cells, a matrix; and

a stem cell medium containing albumin, amino acids, vitamins, minerals, at least one transferrin or transferrin substitute, at least one insulin or insulin substitute, the culture medium free of serum and containing a fibroblast growth factor supplied from a source other than a feeder layer, the fibroblast growth factor present in a concentration at least as high as a maintenance concentration, wherein the medium supports the culture of the embryonic stem cells indefinitely in the absence of serum and in the absence of feeder cells and also in the absence of medium exposed to feeder cells,

wherein the culture maintains the embryonic stem cells in an undifferentiated state indefinitely with normal karyotype.

8. The culture of claim 7 wherein the fibroblast growth factor is FGF2 which is present in the medium in a concentration of at least about 100 ng/ml.

9. A culture of feeder independent human embryonic stem cells comprising

human embryonic stem cells on a matrix in a stem cell culture medium, the stem cell culture medium comprising albumin, amino acids, vitamins, minerals, at least one transferrin or transferrin substitute, at least one insulin or insulin substitute, the culture medium free of serum and containing a fibroblast growth factor present in a concentration at least as high as a maintenance concentration, wherein the fibroblast growth factor is selected from FGF2, FGF4, FGF9, FGF17, and FGF18, wherein the culture is independent of feeder cells while the human embryonic stem cells remain euploid and in an undifferentiated state.

10. A culture as claimed in claim 9 wherein the fibroblast growth factor is present at a concentration of at least about 100 ng/ml.