Cultivation of Primate Embryonic Cells

• US 20090023208A1
• Filed: 09/29/2008
• Published: 01/22/2009
• Est. Priority Date: 03/09/2000
• Status: Abandoned Application

• Current Assignee: James A. Thomson, Mark Levenstein
• Original Assignee: James A. Thomson, Mark Levenstein
• Inventors: Thomson, James A., Levenstein, Mark
• Application Number:
  US12/240,640
• Publication Number:
  US 20090023208A1
• Time in Patent Office: Days
• US Class Current: 435/366
• 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 the stem cells in a culture essentially free of mammalian fetal serum and in a stem cell culture medium including amino acids, vitamins, salts, minerals, transferrin or a transferrin substitute, insulin or an insulin substitute, albumin, and a fibroblast growth factor that is supplied from a source other than just a feeder layer and is present in a concentration that is at least as high as the maintenance concentration, the medium capable of supporting the culture and proliferation of human undifferentiated proliferating euploid embryonic stem cells for at least six passages, without the need for feeder cells or for exposure of the medium to feeder cells.

2. The method of claim 1, wherein the culture is essentially free of any animal serum.

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

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

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

culturing the 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 essentially free of mammalian fetal serum and containing at least about 100 ng/ml of a fibroblast growth factor capable of activating a fibroblast growth factor signaling receptor, wherein the growth factor is supplied from a source other than just a feeder layer, the medium supporting the proliferation of stem cells in an undifferentiated state without feeder cells or conditioned medium.

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

7. The method of claim 5 wherein the FGF is selected from the group consisting of FGF2, FGF4, FGF9, FGF17 and FGF18.

8. A culture of human embryonic stem cells comprising:

human embryonic stem cells; 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 essentially free of mammalian fetal serum and containing at least a maintenance concentration of a fibroblast growth factor capable of activating a fibroblast growth factor signaling receptor, the medium capable of culturing 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 is capable of maintaining the stem cells in an undifferentiated state indefinitely with normal karyotype.

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

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

human embryonic stem cells 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 essentially free of mammalian fetal serum and containing at least a maintenance concentration of a fibroblast growth factor selected from the group consisting of FGF2, FGF4, FGF9, FGF17, and FGF18, the culture being independent of feeder cells while the cells remain euploid and in an undifferentiated state.

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