Embryonic stem cells capable of differentiating into desired cell lines

• US 20020187550A1
• Filed: 08/08/2002
• Published: 12/12/2002
• Est. Priority Date: 11/03/1993
• Status: Abandoned Application

• Current Assignee: Diacrin, Inc. (Precigen, Inc.)
• Original Assignee: Diacrin, Inc. (Precigen, Inc.)
• Inventors: Dinsmore, Jonathan H., Ratliff, Judson
• Application Number:
  US10/214,903
• Publication Number:
  US 20020187550A1
• Time in Patent Office: Days
• US Class Current: 435/377
• CPC Class Codes: C07K 14/475 Growth factors; Growth regulators
• CPC Class Codes: C07K 14/715 for cytokines; for lymphokines; for interferons
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2500/36 Lipids
• CPC Class Codes: C12N 2500/84 from mammals
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• CPC Class Codes: C12N 2501/18 Liver cell growth factor (LCGF, Gly-His-Lys)
• CPC Class Codes: C12N 2501/36 Somatostatin
• CPC Class Codes: C12N 2501/39 Steroid hormones
• CPC Class Codes: C12N 2501/395 Thyroid hormones
• CPC Class Codes: C12N 2501/91 Heparin
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 5/0068 General culture methods using substrates for specific animal cell type C12N5/06
• CPC Class Codes: C12N 5/0619 Neurons
• CPC Class Codes: G01N 33/5005 involving human or animal cells immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025

Abstract

An embryonic stem cell which may be induced to differentiate homogeneously into a desired primary cell line. The embryonic stem cell may be engineered with DNA, which encodes a protein or polypeptide which promotes differentiation of the stem cell into a specific cell line, such as, for example, a neuronal cell line, a muscle cell line, or a hematopoietic cell line. The DNA may encode a transcription factor found in the particular cell line. In another alternative, a desired cell line is produced from embryonic stem cells by culturing embryonic stem cells under conditions which provide for a three-dimensional network of embryonic stem cells, and then stimulating embryonic stem cells with an agent, such as retinoic acid, or dimethylsulfoxide, which promotes differentiation of the embryonic stem cells into the desired cell line, such as, for example, a neuronal cell line, or a muscle cell line.

Claims

1. A method of producing a desired cell line from embryonic stem cells, comprising:

(a) culturing embryonic stem cells under conditions which promote growth of said cells at an optimal growth rate; and

(b) culturing said embryonic stem cells under conditions which promote growth of said cells at a rate which is less than that of said optimal growth rate, and in the presence of an agent which promotes differentiation of said embryonic stem cells into said desired cell line.

2. The method of claim 1 wherein, in step (a), said embryonic stem cells are cultured in the presence of a medium including leukemia inhibitory factor, CNTF, or IL-6;

and serum selected from the group consisting of (i) horse serum at a concentration of from about 5% by volume to about 30% by volume; and

(ii) fetal bovine serum at a concentration of from about 15% by volume to about 30% by volume.

3. The method of claim 2 wherein, in step (a), said serum is horse serum at a concentration of about 10% by volume.

4. The method of claim 2 wherein, in step (a), said serum is fetal bovine serum at a concentration of about 15% by volume.

5. The method of claim 1 wherein, in step (a), said embryonic stem cells are cultured in the absence of a feeder layer of cells.

6. The method of claim 1 wherein, in step (b), said agent is selected from the group consisting of retinoic acid and nerve growth factor, and said desired cell line is a neuronal cell line.

7. The method of claim 6 wherein said agent is retinoic acid.

8. The method of claim 7 wherein, in addition to culturing said embryonic stem cells in the presence of said retinoic acid, said embryonic stem cells are grown in the presence of a cytokine.

9. The method of claim 1 wherein, in step (b), said embryonic stem cells are cultured in the presence of fetal bovine serum at a concentration of about 10% by volume.

10. Neuronal cells produced by the method of claim 6.

11. The method of claim 1 wherein, in step (b), said agent is selected from the group consisting of dimethylsulfoxide and hexamethylene bis-acrylamide, and said desired cell line is a muscle cell line.

12. The method of claim 11 wherein said agent is dimethylsulfoxide.

13. The method of claim 11 wherein said agent is hexamethylene bis-acrylamide.

14. The method of claim 11 wherein, in addition to culturing said embryonic stem cells in the presence of an agent selected from the group consisting of dimethylsulfoxide and hexamethylene bis-acrylamide, said embryonic stem cells are grown in the presence of a cytokine.

15. Muscle cells produced by the method of claim 11.

17. The cell of claim 16 wherein said DNA which encodes a protein or polypeptide which promotes differentiation of said cell into a specific cell line is DNA encoding a transcription factor present in said specific cell line to promote differentiation of said cell into said specific cell line.

18. The cell of claim 17 wherein said DNA encoding a transcription factor is DNA encoding a transcription factor present in neuronal cells, and said specific cell line is a neuronal cell line.

19. The cell of claim 17 wherein said DNA encoding a transcription factor present in a specific cell line is DNA encoding a transcription factor present in muscle cells, and said specific cell line is a muscle cell line.

20. The cell of claim 17 wherein said DNA encoding a transcription factor is DNA encoding a transcription factor present in hematopoietic cells, and the specific cell line is a hematopoietic cell line.

21. Neuronal cells produced from the cell of claim 18.

22. Muscle cells produced from the cell of claim 19.

23. Hematopoietic cells produced from the cell of claim 20.

25. The method of claim 24 wherein said DNA which encodes a protein or polypeptide which promotes differentiation of said embryonic stem cells into a specific cell line is DNA encoding a transcription factor present in neuronal cells and said agent is retinoic acid.

26. Neuronal cells produced by the method of claim 25.

27. The method of claim 24 wherein said DNA which encodes a protein or polypeptide which promotes differentiation of said embryonic stem cells into a specific cell line is DNA encoding a transcription factor present in muscle cells and said agent is selected from the group consisting of dimethylsulfoxide and hexamethylene bis-acrylamide.

28. Muscle cells produced by the method of claim 27.

16. An embryonic stem cell, said embryonic stem cell having been engineered with DNA which encodes a protein or polypeptide which promotes differentiation of said cell into a specific cell line.

24. A method of producing a desired cell line from embryonic stem cells, comprising:

engineering said embryonic stem cells with DNA which encodes a protein or polypeptide which promotes differentiation of said embryonic stem cells into a specific cell line; and

stimulating said embryonic stem cells with an agent which promotes differentiation of said embryonic stem cells into said desired cell line.

29. A method of screening embryonic stem cells for proteins which induce differentiation of embryonic stem cells into a desired cell line comprising:

(a) obtaining RNA from a desired cell or tissue;

(b) constructing cDNA libraries from said RNA obtained from said desired cell or tissue;

(c) cloning such cDNA libraries into expression vectors;

(d) transfecting said vectors into eukaryotic cells;

(e) contacting embryonic stem cells with culture supernatant from said transfected eukaryotic cells to determine if proteins contained in said culture supernatant induce differentiation of said embryonic stem cells into a desired cell line;

(f) obtaining cDNA clones from those cells which induce differentiation of embryonic stem cells into a desired cell line;

(g) transfecting said cDNA clones from said cells which induce differentiation of embryonic stem cells into a desired cell line into eukaryotic cells;

(h) contacting embryonic stem cells with culture supernatant obtained from the eukaryotic cells of step (g);

(i) obtaining cDNA clones from those cells which induce differentiation of embryonic stem cells into a desired cell line;

(j) transfecting said cDNA clones from step (i) into embryonic stem cells; and

(k) determining at least one cDNA clone which induces differentiation of embryonic stem cells into a desired cell line.