Method for identification of cell growth or differentiation factors

• US 8,143,059 B2
• Filed: 04/28/2003
• Issued: 03/27/2012
• Est. Priority Date: 11/21/1994
• Status: Expired due to Term

• Current Assignee: National Jewish Health
• Original Assignee: National Jewish Health
• Inventors: Keller, Gordon M., Hawley, Robert G., Choi, Kyunghee
• Primary Examiner(s): Lankford, L Blaine
• Application Number:
  US10/425,492
• Publication Number:
  US 20040106195A1
• Time in Patent Office: 3,256 Days
• Field of Search: 435/377, 435/325, 435/4
• US Class Current: 435/377
• CPC Class Codes: A61K 2035/124 the cells being hematopoietic, bone marrow derived or blood cells
• CPC Class Codes: C12N 2500/24 Iron; Fe chelators; Transferrin
• CPC Class Codes: C12N 2501/105 Insulin-like growth factors [IGF]
• CPC Class Codes: C12N 2501/125 Stem cell factor [SCF], c-kit ligand [KL]
• CPC Class Codes: C12N 2501/14 Erythropoietin [EPO]
• CPC Class Codes: C12N 2501/165 Vascular endothelial growth factor [VEGF]
• CPC Class Codes: C12N 2501/22 Colony stimulating factors (G-CSF, GM-CSF)
• CPC Class Codes: C12N 2501/23 Interleukins [IL]
• CPC Class Codes: C12N 2501/235 Leukemia inhibitory factor [LIF]
• CPC Class Codes: C12N 2502/28 Vascular endothelial cells
• CPC Class Codes: C12N 2503/02 Drug screening
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2510/04 Immortalised cells
• CPC Class Codes: C12N 5/0603 Embryonic cells production of embryos, nuclear transfer A01K67/027; Embryoid bodies
• CPC Class Codes: C12N 5/0635 B lymphocytes
• CPC Class Codes: C12N 5/0636 T lymphocytes
• CPC Class Codes: C12N 5/0641 Erythrocytes
• CPC Class Codes: C12N 5/069 Vascular Endothelial cells
• CPC Class Codes: C12N 5/0692 Stem cells; Progenitor cells; Precursor cells

Abstract

The present invention relates to novel immortalized precursor cell populations derived from embryonic stem cell populations and methods to produce such cell populations. Also disclosed is an assay to identify regulatory compounds capable of controlling cell growth for therapeutic and experimental use.

Claims

1. A method to identify at least one compound expressed differentially during differentiation of embryonic cells, comprising:

a. comparing expression of at least one compound expressed by a first pluripotent embryonic stem cell-derived cell population to expression of at least one compound expressed by a control cell population selected from the group consisting of;

an embryonic stem cell population, progeny of the pluripotent embryonic stem cell-derived cell population, a non-embryonic stem cell-derived population, and a second pluripotent embryonic stem cell-derived cell population that is different from the first pluripotent embryonic stem cell-derived cell population; and

b. identifying at least one compound that is expressed at a different level in the control cell population compared with the first pluripotent embryonic stem cell-derived cell population;

whereby a difference in expression level is indicative of a compound that is expressed differentially during differentiation of embryonic cells;

wherein the first pluripotent embryonic stem cell-derived cell population is not totipotent and differentiates into primitive erythroid cells, definitive erythroid cells, macrophages, neutrophils, mast cells, T cells, endothelial cells, B cells, natural killer cells, megakaryocytes, eosinophils, endothelial cell lineages, endoderm cell lineages, mesoderm cell lineages, ectoderm cell lineages and progenitors and progeny thereof, and wherein said first pluripotent embryonic stem cell-derived cell population is derived by culturing an embryonic stem cell population to obtain an embryoid body cell population, followed by culturing said embryoid body cell population under conditions effective to produce said pluripotent cell population, said conditions comprising an embryonic blast cell medium.

2. The method of claim 1, wherein the first pluripotent embryonic stem cell-derived cell population is selected from the group consisting of:

a pluripotent embryonic stem cell-derived embryoid body cell population and a pluripotent embryonic stem cell-derived blast cell population.

3. The method of claim 1, wherein the first pluripotent embryonic stem cell-derived cell population is produced by culturing an embryonic stem cell population from about 1 day to about 7 days in the absence of leukocyte inhibitory factor and in an embryoid body cell medium comprising platelet-poor fetal bovine serum or normal fetal bovine serum selected for its ability to stimulate differentiation of EB cells and for not having inhibitors of ES cell differentiation.

4. The method of claim 1, wherein the control cell population is an embryonic stem cell population.

5. The method of claim 1, wherein the compound comprises a cellular marker that defines one or more populations of embryonic cells.

6. The method of claim 1, wherein the compound regulates a cell function selected from the group consisting of:

maintaining the survival of a cell, inducing the propagation of a cell, and stimulating the differentiation of a cell.

7. The method of claim 1, wherein the compound is selected from the group consisting of:

a cell surface molecule, a secreted molecule, a cytoplasmic signal transduction molecule, and a nucleic acid binding protein.

8. The method of claim 1, wherein the compound is selected from the group consisting of:

a cell surface receptor, a membrane-bound ligand, an adhesion protein, a carbohydrate moiety, a cytoplasmic signal transduction protein, a growth factor, and a transcription factor.

9. The method of claim 1, wherein the compound is selected from the group consisting of:

a protein, a nucleic acid molecule, a lipid and a carbohydrate.

10. The method of claim 1, wherein the compound is a protein.

11. The method of claim 10, wherein the expression of at least one compound is detected by a method selected from the group consisting of:

a) detecting binding of an antibody of known specificity to a protein produced by the first pluripotent embryonic stem cell-derived cell population as compared to the control cell population;

b) detecting binding of a protein of known binding specificity to a protein produced by the first pluripotent embryonic stem cell-derived cell population as compared to the control cell population;

c) detecting an enzymatic activity in the first pluripotent embryonic stem cell-derived cell population as compared to the control cell population, wherein the presence of the enzymatic activity indicates the presence of a specific enzyme produced by the first pluripotent embryonic stem cell-derived cell population;

d) detecting a biological activity of a known protein in the first pluripotent embryonic stem cell-derived cell population as compared to the control cell population, wherein the biological activity is selected from the group consisting of;

cell receptor function, cell adhesion, signal transduction, transcription, cellular growth and proliferation, cell survival and cellular differentiation, wherein the presence of the biological activity indicates the presence of a specific protein produced by the first pluripotent embryonic stem cell-derived cell population; and

,

e) detecting a biological activity of a protein produced by the first pluripotent embryonic stem cell-derived cell population as compared to the control cell population and isolating the protein from the first pluripotent embryonic stem cell-derived cell population by protein separation techniques, wherein the biological activity is a marker indicating isolation of the protein, and wherein the biological activity is selected from the group consisting of;

cell receptor function, cell adhesion, signal transduction, transcription, cellular growth and proliferation, cell survival and cellular differentiation.

12. The method of claim 10, wherein the expression of at least one compound is detected by detecting binding of an antibody of known specificity to a protein produced by the first pluripotent embryonic stem cell-derived cell population as compared to the control cell population by a method selected from the group consisting of an immunoblot assay, an immunoprecipitation assay, an enzyme immunoassay, a radioimmunoassay, an immunofluorescent antibody assay, flow cytometry and immunoelectron microscopy.

13. The method of claim 10, wherein the expression of at least one compound is detected by detecting binding of a protein of known binding specificity to a protein produced by the first pluripotent embryonic stem cell-derived cell population as compared to the control cell population, wherein the protein of known binding specificity is selected from the group consisting of a ligand and a receptor.

14. The method of claim 10, wherein the expression of at least one compound is detected by detecting in the first pluripotent embryonic stem cell-derived cell population as compared to the control cell population, enzymatic activity associated with a signal transduction protein.

15. The method of claim 14, wherein the enzymatic activity associated with a signal transduction protein is selected from the group consisting of kinase activity, phosphatase activity and phospholipase activity.

16. The method of claim 10, wherein the expression of at least one compound is detected by detecting a biological activity of a protein produced by the first pluripotent embryonic stem cell-derived cell population as compared to the control cell population and isolating the protein from the first pluripotent embryonic stem cell-derived cell population by protein separation techniques selected from the group consisting of gel electrophoresis and chromatography, wherein the biological activity is a marker indicating isolation of the protein, and wherein the biological activity is selected from the group consisting of:

cell receptor function, cell adhesion, signal transduction, transcription, cellular growth and proliferation, cell survival and cellular differentiation.

17. The method of claim 1, wherein the compound is a nucleic acid molecule.

18. The method of claim 17, wherein the expression of at least one compound is detected by detecting expression of an RNA sequence in the first pluripotent embryonic stem cell-derived cell population as compared to expression of the RNA sequence in the control cell population.

19. The method of claim 17, wherein the expression of at least one compound is detected by detecting a nucleic acid sequence in a sample comprising a cDNA product of RNA from the first pluripotent embryonic stem cell-derived cell population as compared to a cDNA product of RNA from the control cell population.

20. The method of claim 17, wherein the expression of at least one compound is detected by detecting a nucleic acid sequence in a sample comprising amplified nucleic acid products of RNA from the first pluripotent embryonic stem cell-derived cell population as compared to amplified nucleic acid products of RNA from the control cell population.

21. The method of claim 17, wherein the expression of at least one compound is detected by sequencing at least one nucleic acid sequence in the first pluripotent embryonic stem cell-derived cell population and in the control cell population.

22. The method of claim 21, further comprising comparing the nucleic acid sequence from the first pluripotent embryonic stem cell-derived cell population with at least one known nucleic acid sequence.

23. The method of claim 17, wherein the expression of at least one compound is detected by performing subtractive hybridization between a nucleic acid sample from the first pluripotent embryonic stem cell-derived cell population and another nucleic acid sample from the control cell population.

24. The method of claim 17, wherein the expression of at least one compound is detected by:

a) contacting a nucleic acid sample from the first pluripotent embryonic stem cell-derived cell population and a nucleic acid sample from the control cell population with a known nucleic acid sequence; and

,

b) detecting whether the known nucleic acid sequence hybridizes to either of the nucleic acid samples.

25. The method of claim 24, wherein the known nucleic acid sequence is selected from the group consisting of a hybridization probe and a polymerase chain reaction (PCR) primer.

26. The method of claim 17, wherein the expression of at least one compound is detected by comparing expression of a nucleic acid from the first pluripotent embryonic stem cell-derived cell population to expression of the nucleic acid in the control cell population, wherein the nucleic acid encodes a protein selected from the group consisting of:

an endothelial cell marker protein, a lymphoid cell marker protein, an epithelial marker protein and a hematopoietic precursor cell marker.

27. The method of claim 17, wherein the expression of at least one compound is detected by comparing expression of a nucleic acid from the first pluripotent embryonic stem cell-derived cell population to expression of the nucleic acid in the control cell population, wherein the nucleic acid encodes a protein selected from the group consisting of:

a stem cell leukemia protein, GATA-1, GATA-2, C-Myb, C-kit ligand, C-fms, Flk-1, beta-globin, betaH1-globin, brachyury, VLA-4 and LFA-1.

28. A method to identify at least one compound expressed differentially during differentiation of embryonic cells, comprising:

a. comparing expression of at least one compound expressed by a first pluripotent embryonic stem cell-derived cell population to expression of at least one compound expressed by a control cell population selected from the group consisting of;

an embryonic stem cell population, progeny of the pluripotent embryonic stem cell-derived cell population, a non-embryonic stem cell-derived population, and a second pluripotent embryonic stem cell-derived cell population that is different from the first pluripotent embryonic stem cell-derived cell population; and

b. identifying at least one compound that is expressed at a different level in the control cell population compared with the first pluripotent embryonic stem cell-derived cell population;

whereby a difference in expression level is indicative of a compound that is expressed differentially during differentiation of embryonic cells;

wherein the first pluripotent embryonic stem cell-derived cell population is not totipotent and differentiates into primitive erythroid cells, definitive erythroid cells, macrophages, neutrophils, mast cells, T cells, endothelial cells, B cells, natural killer cells, megakaryocytes, eosinophils, and progenitors and progeny thereof, and wherein said first pluripotent embryonic stem cell-derived cell population is derived in vitro from a between about 3 and about 4 day embryoid body cell population.