Reagents and Methods for Using Human Embryonic Stem Cells to Evaluate Toxicity of Pharmaceutical Compounds and Other Chemicals

• US 20070248947A1
• Filed: 04/10/2007
• Published: 10/25/2007
• Est. Priority Date: 04/10/2006
• Status: Active Grant

• Current Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Original Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Inventors: Cezar, Gabriela
• Granted Patent: US 8,703,483 B2
• Time in Patent Office: Days
• US Class Current: 435/4
• CPC Class Codes: G01N 2500/10 involving cells
• CPC Class Codes: G01N 33/5038 involving detection of metabolites per se
• CPC Class Codes: G01N 33/5073 Stem cells
• CPC Class Codes: G01N 33/5091 for testing the pathological state of an organism

Abstract

The invention provides biomarker profiles of cellular metabolites and methods for screening chemical compounds including pharmaceutical agents, lead and candidate drug compounds and other chemicals using human embryonic stem cells (hESC) or lineage-specific cells produced therefrom. The inventive methods are useful for testing toxicity, particularly developmental toxicity and detecting teratogenic effects of such chemical compounds.

Claims

1. A method for identifying one or a plurality of cellular metabolites having a molecular weight of from about 10 to about 1500 Daltons that is differentially produced in human embryonic stem cells (hESCs) or hESC-derived lineage-specific cells contacted with a test compound, the method comprising the steps of:

a) culturing hESCs or hESC-derived lineage-specific cells in the presence or absence of a test compound;

b) assaying one or a plurality of cellular metabolites having a molecular weight of from about 10 to about 1500 Daltons in hESCs or hESC-derived lineage-specific cells cultured in the presence and absence of the test compound; and

c) identifying at least one metabolite product having a molecular weight of from about 10 to about 1500 Daltons that is differentially produced in the cells in the presence or absence of the test compound.

2. A method according to claim 1, wherein at least one of the cellular metabolites is produced in greater amounts in the presence of the test compound than in the absence of the test compound.

3. A method according to claim 1, wherein at least one of the cellular metabolites is produced in greater amounts in the absence of the test compound than in the presence of the test compound.

4. A method according to claim 1, wherein the cellular metabolites are secreted or excreted from the hESCs or hESC-derived lineage-specific cells.

5. A method according to claim 1, wherein the cellular metabolite is a small molecule product having a molecular weight of from about 100 to about 1000 Daltons.

6. A method according to claim 1, wherein the test compound is a toxic or teratogenic compound.

7. A method according to claim 1, wherein one or a plurality of cellular metabolites is assayed using a physical separation method.

8. A method according to claim 7, wherein the physical separation method is liquid chromatography/electrospray ionization time of flight mass spectrometry.

9. A method according to claim 1, wherein the cellular metabolites are tetrahydrofolate, dihydrofolate or other metabolites in the folate metabolic pathway, glutathione, or oxidized glutathione.

10. A method according to claim 1, wherein the candidate cellular metabolites are kynurenine, 8-methoxykynurenate, N′

-formylkynurenine 7,8-dihydro-7,8-dihydroxykynurenate, 5-Hydroxytryptophan, N-acetyl-D-tryptophan, glutamate, pyroglutamic acid or other metabolites in the tryptophan or glutamate metabolic pathways, histamine, dopamine, 3,4-dihydroxybutyric acid, serotonin, or gamma-aminobutyric acid (GABA).

11. A method according to claim 1, wherein a plurality of cellular metabolites are identified.

12. A method according to claim 10, wherein the plurality of identified cellular metabolites comprise a biomarker profile.

13. A method according to claim 11, wherein one of the cellular metabolites comprising a biomarker profile is kynurenine.

14. A method according to claim 11, wherein the test compound is a toxic or teratogenic compound.

15. A method according to claim 14, wherein the plurality of identified cellular metabolites comprise a biomarker profile characteristic of hESC or hESC-derived lineage-specific cell response to a toxic or teratogenic compound.

42. A method according to claim 4′

, wherein the plurality of identified cellular metabolites comprise a biomarker profile.

43. A method according to claim 42, wherein one of the cellular metabolites comprising a biomarker profile is kynurenine.

44. A method according to claim 43, wherein the test compound is a toxic or teratogenic compound.

49. A method of claim 1, further comprising the step of comparing at least one metabolite product having a molecular weight of from about 10 to about 1500 Daltons that is differentially produced in the cells in the presence or absence of the test compound with a biomarker profile comprising one or a plurality of cellular metabolites having a molecular weight of from about 10 to about 1500 Daltons that are differentially produced in human embryonic stem cells (hESCs) or hESC-derived lineage-specific cells contacted with a toxic compound or compounds.

52. The method of claim 1, wherein at least two cellular metabolites having a molecular weight of from about 10 to about 1500 Daltons that are differentially produced in the cells in the presence or absence of the test compound are detected.

16. A method for screening a test compound to identify an effect of the compound on human embryonic stem cells (hESCs) or hESC-derived lineage-specific cells contacted with the test compound, the method comprising the steps of:

a) culturing hESCs or hESC-derived lineage-specific cells in the presence or absence of a test compound

b) assaying the hESCs or hESC-derived lineage-specific cells cultured in the presence and absence of the test compound for production of one or a plurality of cellular metabolites having a molecular weight of from about 10 to about 1500 Daltons; and

c) identifying test compounds that produce at least one cellular metabolite having a molecular weight of from about 10 to about 1500 Daltons that is differentially produced in the cells in the presence or absence of the test compound.

17. A method according to claim 16, wherein at least one of the cellular metabolites is produced in greater amounts in the presence of the test compound than in the absence of the test compound.

18. A method according to claim 16, wherein at least one of the cellular metabolites is produced in greater amounts in the absence of the test compound than in the presence of the test compound.

19. A method according to claim 16, wherein the cellular metabolites are secreted or excreted from the hESCs or hESC-derived lineage-specific cells.

20. A method according to claim 16, wherein at least one of the cellular metabolites is a small molecule metabolite having a molecular weight of from about 100 to about 1000 Daltons.

21. A method according to claim 16, wherein the test compound is a toxic or teratogenic compound.

22. A method according to claim 16, wherein the plurality of cellular metabolites are assayed using a physical separation method.

23. A method according to claim 22, wherein the physical separation method is liquid chromatography/electrospray ionization time of flight mass spectrometry.

24. A method according to claim 16, wherein the candidate cellular metabolites are tetrahydrofolate, dihydrofolate or other metabolites in the folate metabolic pathway, glutathione, or oxidized glutathione.

25. A method according to claim 16, wherein the candidate cellular metabolites are kynurenine, 8-methoxykynurenate, N′

-formylkynurenine 7,8-dihydro-7,8-dihydroxykynurenate, 5-Hydroxytryptophan, N-acetyl-D-tryptophan, glutamate, pyroglutamic acid or other metabolites in the tryptophan or glutamate metabolic pathways, histamine, dopamine, serotonin, gamma-aminobutyric acid (GABA) or other butyric acid species.

26. A method according to claim 16 wherein a plurality of cellular metabolites are identified.

27. A method according to claim 26, wherein the plurality of identified cellular metabolites comprise a biomarker profile.

28. A method according to claim 27, wherein one of the cellular metabolites comprising a biomarker profile is kynurenine.

29. A method according to claim 27, wherein the test compound is a toxic or teratogenic compound.

30. A method according to claim 29, wherein the plurality of identified cellular metabolites comprise a biomarker profile characteristic of hESC response to a toxic or teratogenic compound.

50. A method of claim 16, further comprising the step of comparing at least one metabolite product having a molecular weight of from about 10 to about 1500 Daltons that is differentially produced in the cells in the presence or absence of the test compound with a biomarker profile comprising one or a plurality of cellular metabolites having a molecular weight of from about 10 to about 1500 Daltons that are differentially produced in human embryonic stem cells (hESCs) or hESC-derived lineage-specific cells contacted with a toxic compound or compounds.

53. The method of claim 16, wherein at least two cellular metabolites having a molecular weight of from about 10 to about 1500 Daltons that are differentially produced in the cells in the presence or absence of the test compound are detected.

31. A method for assaying a test compound for toxicity or teratogenicity to human embryonic stem cells (hESCs) or hESC-derived lineage-specific cells contacted with the test compound, the method comprising the steps of:

a) culturing hESCs or hESC-derived lineage-specific cells in the presence or absence of a test compound

b) assaying hESCs or hESC-derived lineage-specific cells cultured in the presence and absence of the test compound to identify one or a plurality of cellular metabolites having a molecular weight of from about 10 to about 1500 Daltons produced by hESCs contacted with a toxic or teratogenic compound; and

c) identifying test compounds wherein hESCs or hESC-derived lineage-specific cells produce one or a plurality of cellular metabolites having a molecular weight of from about 10 to about 1500 Daltons in the presence or absence of the test compound wherein said cellular metabolites are produced by hESCs or hESC-derived lineage-specific cells contacted with a toxic or teratogenic compound.

32. A method according to claim 31, wherein at least one of the cellular metabolites is produced in greater amounts in the presence of the test compound than in the absence of the test compound.

33. A method according to claim 31, wherein at least one of the cellular metabolites is produced in greater amounts in the absence of the test compound than in the presence of the test compound.

34. A method according to claim 31, wherein the cellular metabolites are secreted or excreted from the hESCs or hESC-derived lineage-specific cells.

35. A method according to claim 31, wherein the cellular metabolite is a small molecule metabolite having a molecular weight of from about 100 to about 1000 Daltons.

36. A method according to claim 31, wherein the test compound is a toxic or teratogenic compound.

37. A method according to claim 31, wherein cellular metabolites are assayed using a physical separation method.

38. A method according to claim 37, wherein the physical separation method is liquid chromatography/electrospray ionization time of flight mass spectrometry.

39. A method according to claim 31, wherein the candidate cellular metabolites are tetrahydrofolate, dihydrofolate or other metabolites in the folate metabolic pathway, glutathione, or oxidized glutathione.

40. A method according to claim 31, wherein the candidate cellular metabolites are kynurenine, 8-methoxykynurenate, N′

-formylkynurenine 7,8-dihydro-7,8-dihydroxykynurenate 5-Hydroxytryptophan, N-acetyl-D-tryptophan, glutamate, pyroglutamic acid or other metabolites in the tryptophan or glutamate metabolic pathways, histamine, dopamine, 3,4-dihydroxybutyric acid, serotonin, gamma-aminobutyric acid (GABA) or other butyric acid species.

41. A method according to claim 31, wherein a plurality of cellular metabolites are identified.

45. A method according to claim 31, wherein the plurality of identified cellular metabolites comprise a biomarker profile characteristic of hESC response to a toxic or teratogenic compound.

51. A method of claim 31, further comprising the step of comparing at least one metabolite product having a molecular weight of from about 10 to about 1500 Daltons that is differentially produced in the cells in the presence or absence of the test compound with a biomarker profile comprising one or a plurality of cellular metabolites having a molecular weight of from about 10 to about 1500 Daltons that are differentially produced in human embryonic stem cells (hESCs) or hESC-derived lineage-specific cells contacted with a toxic compound or compounds.

54. The method of claim 31, wherein at least two cellular metabolites having a molecular weight of from about 10 to about 1500 Daltons that are differentially produced in the cells in the presence or absence of the test compound are detected.

46. A biomarker profile for identifying a test compound as a toxic compound, wherein the biomarker profile comprises one or a plurality of cellular metabolites having a molecular weight of from about 10 to about 1500 Daltons that are differentially produced in human embryonic stem cells (hESCs) or hESC-derived lineage-specific cells contacted with a toxic compound or compounds.

47. A biomarker profile of claim 46, comprising tetrahydrofolate, dihydrofolate or other metabolites in the folate metabolic pathway, glutathione, or oxidized glutathione.

48. A biomarker profile of claim 46, comprising kynurenine, 8-methoxykynurenate, N′

-formylkynurenine 7,8-dihydro-7,8-dihydroxykynurenate 5-Hydroxytryptophan, N-acetyl-D-tryptophan, glutamate, pyroglutamic acid or other metabolites in the tryptophan or glutamate metabolic pathways, histamine, dopamine, 3,4-dihydroxybutyric acid, serotonin, gamma-aminobutyric acid (GABA) or other butyric acid species.