Protein factors supporting undifferentiated growth of human embryonic stem cells

• US 20060263880A1
• Filed: 05/16/2006
• Published: 11/23/2006
• Est. Priority Date: 05/19/2005
• Status: Abandoned Application

• Current Assignee: Oakland University
• Original Assignee: Oakland University
• Inventors: Chaudhry, G. Rasul
• Application Number:
  US11/435,204
• Publication Number:
  US 20060263880A1
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: C12N 2501/70 Enzymes
• CPC Class Codes: C12N 2502/13 connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
• 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

Factors which are secreted by mouse embryonic fibroblasts which promote maintenance and support the growth of human embryonic stem (hES) cells are provided. The factors can be isolated by chromatographic techniques. The genes, cDNA and amino acid sequences for the factors are disclosed herein and identified. The cDNA of one factor encodes a deduced nitroreductase family containing protein with 285 amino acids and a mass of approximately 33 kD. This factor is identified as iodotyrosine dehalogenase. A second factor supporting undifferentiated hES cell growth is a 453 amino acid encoded by an aldehyde dehydrogenase gene. The use of recombinant factors can eliminate the need for an MEF feeder layer for growing hES cells without differentiation.

Claims

1. A medium for culturing stem cells while maintaining the stem cells in an undifferentiated state comprising one or more polypeptides homologous to iodotyrosine dehalogenase or aldehyde dehydrogenase.

2. The medium of claim 1 wherein the one or more polypeptides are recombinant.

3. A method of culturing stem cells while maintaining the stem cells in an undifferentiated state comprising culturing the stem cells in a medium comprising one or more polypeptides homologous to iodotyrosine dehalogenase or aldehyde dehydrogenase.

4. The method of claim 3 wherein the stem cells are human stem cells.

5. The method of claim 4 wherein the human stem cells are human embryonic stem cells.

6. The method of claim 3 wherein the one or more polypeptides are recombinant.

7. A method of culturing stem cells while maintaining the stem cells in an undifferentiated state comprising culturing the stem cells in a medium comprising one or more polypeptides having an amino acid sequence essentially identical to the amino acid sequences set forth in SEQ ID NO:

1 or SEQ ID NO;

4.

8. The method of claim 7 wherein the stem cells are human stem cells.

9. The method of claim 8 wherein the human stem cells are human embryonic stem cells.

10. The method of claim 7 wherein the one or more polypeptides are recombinant.

11. A method of preparing a partially purified conditioned medium for culturing stem cells while maintaining the cells in an undifferentiated state comprising:

(a) providing a medium;

(b) conditioning the medium by culturing fibroblasts in the medium;

(c) precipitating the conditioned medium with a first salt solution to provide a first set of fractions;

(d) selecting a fraction of the first set of fractions of the precipitated conditioned medium;

(e) applying the selected fraction to a cation-exchange resin;

(f) eluting a second set of fractions with a second salt solution; and

(g) selecting a fraction of the second set of fractions as the partially purified conditioned medium for culturing stem cells.

12. The method of claim 11 wherein the fibroblast are mouse embryonic fibroblasts.

13. The method of claim 11 wherein the second salt solution is a 500 mM potassium chloride solution.

14. A partially purified conditioned medium for culturing stem cells while maintaining the cells in an undifferentiated state prepared by the method comprising:

providing a medium;

conditioning the medium by culturing fibroblasts in the medium;

precipitating the conditioned medium with a first salt solution to provide a first set of fractions;

selecting a fraction of the first set of fractions of the precipitated conditioned medium;

applying the selected fraction to a cation-exchange resin;

eluting a second set of fractions with a second salt solution; and

selecting a fraction of the second set of fractions as the partially purified conditioned medium for culturing stem cells.

15. The method of claim 14 wherein the fibroblast are mouse embryonic fibroblasts.

16. The method of claim 14 wherein the second salt solution is a 500 mM potassium chloride solution.

17. A method of culturing stem cells while maintaining the cells in an undifferentiated state comprising:

(a) providing stem cells for culturing in an undifferentiated state;

(b) providing partially purified conditioned medium for culturing stem cells while maintaining the cells in an undifferentiated state prepared by the method comprising;

providing a medium;

conditioning the medium by culturing fibroblasts in the medium;

precipitating the conditioned medium with a salt to provide a first set of fractions;

selecting one fraction of the first set of fractions of the precipitated conditioned medium;

applying the selected fraction to a cation-exchange resin;

eluting a second set of fractions; and

selecting one of the second set of fractions as the partially purified conditioned medium to a culture of stem cells; and

(c) culturing the stem cells in a medium comprising a fresh culture medium and the partially purified conditioned medium.