MARKERS OF DEFINITIVE ENDODERM

• US 20090220959A1
• Filed: 11/13/2006
• Published: 09/03/2009
• Est. Priority Date: 12/23/2003
• Status: Active Grant

• Current Assignee: Cynthera, Inc., Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Original Assignee: Viacyte Incorporated (Vertex Pharmaceuticals Incorporated)
• Inventors: D'Amour, Kevin Allen, Baetge, Emmanuel Edward, Agulnick, Alan D.
• Granted Patent: US 8,586,357 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: A61K 35/38 Stomach; Intestine; Goblet cells; Oral mucosa; Saliva
• CPC Class Codes: A61K 35/407 Liver; Hepatocytes
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 5/0603 Embryonic cells production of embryos, nuclear transfer A01K67/027; Embryoid bodies
• CPC Class Codes: C12N 5/0617 Thyroid and parathyroid glands
• CPC Class Codes: C12N 5/0672 Stem cells; Progenitor cells; Precursor cells; Oval cells
• CPC Class Codes: C12N 5/068 Stem cells; Progenitors

Abstract

Disclosed herein are reagent-cell complexes comprising one or more definitive endoderm cells. Also described herein are compositions for detecting definitive endoderm. Method of enriching, isolating and/or purifying definitive endoderm cells are also disclosed.

Claims

1. An ex vivo reagent-cell complex comprising:

a human definitive endoderm cell expressing a marker selected from the group consisting of CMKOR1, CXCR4, GPR37, RTN4RL1, SLC5A9, SLC40A1 and TRPA1 said definitive endoderm cell being a multipotent cell that can differentiate into cells of the gut tube or organs derived therefrom; and

a reagent bound to said marker.

3. The reagent cell complex of claim 1, wherein said marker comprises a polypeptide selected from the group consisting of CXCR4, GPR37, NTN4, RTN4RL1, SLC40A1, SLCA9 and TRPA1.

4. The reagent-cell complex of claim 1, wherein said reagent comprises an antibody.

5. The reagent-cell complex of claim 1, wherein said reagent comprises a ligand for a receptor.

6. The reagent cell complex of claim 1, said reagent cell complex present in a population of human cells.

7. The reagent cell complex of claim 6, wherein at least 15% of said human cells form reagent-cell complexes.

8. The reagent cell complex of claim 6, wherein at least 40% of said human cells form reagent-cell complexes.

9. The reagent cell complex of claim 6, wherein at least 60% of said human cells form reagent-cell complexes.

10. The reagent cell complex of claim 6, wherein at least 80% of said human cells form reagent-cell complexes.

2. (canceled)

11-16. -16. (canceled)

17. A composition for the identification of definitive endoderm, said composition comprising:

a first oligonucleotide that hybridizes to a first marker, wherein said first marker is selected from the group consisting of AGPAT3, APOA2, C20orf56, C21orf129, CALCR, CCL2, CER1, CMKOR1, CRIP1, CXCR4, CXorf1, DIO3, DIO3OS, EB-1, EHHADH, ELOVL2, EPSTI1, FGF17, FLJ10970, FLJ21195, FLJ22471, FLJ23514, FOXA2, FOXQ1, GATA4, GPR37, GSC, LOC283537, MYL7, NPPB, NTN4, PRSS2, RTN4RL1, SEMA3E, SIAT8D, SLC5A9, SLC40A1, SOX17, SPOCK3, TMOD1, TRPA1, TTN, AW166727, AI821586, BF941609, AI916532, BC034407, N63706, AW772192, OCT4, AFP, Thrombomodulin (TM), SPARC and SOX7; and

a second oligonucleotide that hybridizes to a second marker, wherein said second marker is selected from the group consisting of AGPAT3, APOA2, C20orf56, C21orf129, CALCR, CCL2, CER1, CMKOR1, CRIP1, CXCR4, CXorf1, DIO3, DIO3OS, EB-1, EHHADH, ELOVL2, EPSTI1, FGF17, FLJ10970, FLJ21195, FLJ22471, FLJ23514, FOXA2, FOXQ1, GATA4, GPR37, GSC, LOC283537, MYL7, NPPB, NTN4, PRSS2, RTN4RL1, SEMA3E, SIAT8D, SLC5A9, SLC40A1, SOX17, SPOCK3, TMOD1, TRPA1, TTN, AW166727, AI821586, BF941609, AI916532, BC034407, N63706, AW772192, OCT4, AFP, Thrombomodulin (TM), SPARC and SOX7, and wherein said second marker is different from said first marker.

18. The composition of claim 17, wherein said first marker and said second marker are selected from the group consisting of AGPAT3, APOA2, C20orf56, C21orf129, CCL2, CER1, CXCR4, CXorf1, DIO3, DIO3OS, EB-1, EHHADH, ELOVL2, EPSTI1, FLJ10970, FLJ21195, FLJ22471, FLJ23514, FOXA2, GPR37, LOC283537, MYL7, NPPB, NTN4, PRSS2, RTN4RL1, SEMA3E, SIAT8D, SLC5A9, SLC40A1, SOX17, SPOCK3, TMOD1, TRPA1, TTN, AW166727, AI821586, BF941609, AI916532, BC034407, N63706, AW772192, OCT4, AFP, Thrombomodulin (TM), SPARC and SOX7.

20. The composition of claim 17, wherein said marker is a nucleic acid.

23. The composition of claim 17, wherein at least one oligonucleotide comprises a label.

24. The composition of claim 17 further comprising a polymerase.

19. (canceled)

21-22. -22. (canceled)

25-27. -27. (canceled)

28. A method for producing a cell population enriched in definitive endoderm cells, said method comprising the steps of:

providing a cell population comprising definitive endoderm cells with a reagent that binds to a marker selected from the group consisting of CALCR, CMKOR1, CXCR4, GPR37, RTN4RL1, SLC5A9, SLC40A1 and TRPA1; and

separating definitive endoderm cells bound to said reagent from cells that are not bound to said reagent, thereby producing a cell population enriched in definitive endoderm cells.

30. The method of claim 28, wherein the marker is selected from the group consisting of GPR37, NTN4, RTN4RL1, SLC40A1, SLC5A9 and TRPA1.

31. The method of claim 30, wherein said reagent comprises an antibody or a ligand for a receptor.

32. The method of claim 28 further comprising the steps of:

obtaining a cell population comprising pluripotent human cells;

providing said cell population with at least one growth factor of the TGFβ

superfamily in an amount sufficient to promote differentiation of said pluripotent cells to definitive endoderm cells; and

allowing sufficient time for definitive endoderm cells to form.

33. The method of claim 32, wherein said at least one growth factor is selected from the group consisting of Nodal, activin A, activin B and combinations thereof

37. The method of claim 32, wherein said pluripotent human cells are human embryonic stem cells.

29. (canceled)

34-36. -36. (canceled)

38-46. -46. (canceled)

47. A method of detecting definitive endoderm cells, said method comprising detecting the presence of definitive endoderm cells in a cell population by detecting expression of at least one marker selected from the group consisting of AGPAT3, APOA2, C20orf56, C21orf129, CCL2, CER1, CXorf1, DIO3, DIO3OS, EB-1, EHHADH, ELOVL2, EPSTI1, FLJ10970, FLJ21195, FLJ22471, FLJ23514, FOXA2, GPR37, LOC283537, MYL7, NPPB, NTN4, PRSS2, RTN4RL1, SEMA3E, SIAT8D, SLC5A9, SLC40A1, SPOCK3, TMOD1, TRPA1, TTN, AW166727, AI821586, BF941609, AI916532, BC034407, N63706 and AW772192 in cells of said cell population.

48. The method of claim 47, wherein said at least one marker is selected from a group consisting of GPR37, NTN4, RTN4RL1, SEMA3E, SLC40A1, SLC5A9 and TRPA1.

49. The method of claim 47 further comprising detecting expression of at least one marker selected from the group consisting of OCT4, AFP, TM, SPARC and SOX7 in cells of said cell population, wherein the expression of said at least one marker selected from the group consisting of AGPAT3, APOA2, C20orf56, C21orf129, CCL2, CER1, CXorf1, DIO3, DIO3OS, EB-1, EHHADH, ELOVL2, EPSTI1, FLJ10970, FLJ21195, FLJ22471, FLJ23514, FOXA2, GPR37, LOC283537, MYL7, NPPB, NTN4, PRSS2, RTN4RL1, SEMA3E, SIAT8D, SLC5A9, SLC40A1, SPOCK3, TMOD1, TRPA1, TTN, AW166727, AI821586, BF941609, AI916532, BC034407, N63706 and AW772192 is greater than the expression of said at least one marker selected from the group consisting of:

OCT4, AFT, TM, SPARC and SOX7 in said definitive endoderm cells.

50. The method of claim 47 further comprising detecting expression of at least one marker selected from the group consisting of SOX17 and CXCR4, and detecting expression of at least one marker from the group consisting of AGPAT3, APOA2, C20orf56, C21orf129, CCL2, CER1, CXorf1, DIO3, DIO3OS, EB-1, EHHADH, ELOVL2, EPSTI1, FLJ10970, FLJ21195, FLJ22471, FLJ23514, FOXA2, GPR37, LOC283537, MYL7, NPPB, NTN4, PRSS2, RTN4RL1, SEMA3E, SIAT8D, SLC5A9, SLC40A1, SPOCK3, TMOD1, TRPA1, TTN, AW166727, AI821586, BF941609, AI916532, BC034407, N63706 and AW772192, wherein expression of said at least one marker selected from the group consisting of SOX17 and CXCR4 and expression of said at least one marker selected from the group consisting of AGPAT3, APOA2, C20orf56, C21orf129, CCL2, CER1, CXorf1, DIO3, DIO3OS, EB-1, EHHADH, ELOVL2, EPSTI1, FLJ10970, FLJ21195, FLJ22471, FLJ23514, FOXA2, GPR37, LOC283537, MYL7, NPPB, NTN4, PRSS2, RTN4RL1, SEMA3E, SIAT8D, SLC5A9, SLC40A1, SPOCK3, TMOD1, TRPA1, TTN, AW166727, AI821586, BF941609, AI916532, BC034407, N63706 AW772192 is greater than the expression of said at least one marker selected from the group consisting of OCT4, AFP, TM, SPARC and SOX7 in said definitive endoderm cells.

51. The method of claim 47, wherein expression of said at least one marker is determined by quantitative polymerase chain reaction (Q-PCR).

52. The method of claim 47, wherein expression of said at least one marker is determined by immunocytochemistry.

53-74. -74. (canceled)